Microstructural modification of pure Mg for improving mechanical and biocorrosion properties.

In this study, the effect of microstructural modification on mechanical properties and biocorrosion resistance of pure Mg was investigated for tailoring a load-bearing orthopedic biodegradable implant material. This was performed utilizing the friction stir processing (FSP) in 1-3 passes to refine the grain size. Microstructure was examined in an optical microscope and scanning electron microscope with an electron backscatter diffraction unit. X-ray diffraction method was used to identify the texture. Mechanical properties were measured by microhardness and tensile testing. Electrochemical impedance spectroscopy was applied to evaluate corrosion behavior. The results indicate that even applying a single pass of FSP refined the grain size significantly. Increasing the number of FSP passes further refined the structure, increased the mechanical strength and intensified the dominating basal texture. The best combination of mechanical properties and corrosion resistance were achieved after three FSP passes. In this case, the yield strength was about six times higher than that of the as-cast Mg and the corrosion resistance was also improved compared to that in the as-cast condition.

Effect of nano-SiC incorporation on mechanical properties of micro and nano-structured Ni-Co electrodeposits.

Ni-Co/SiC nano-composites were electrodeposited from modified Watts bath containing SiC particles with 50 nm average size, SDS as surfactant and saccharin as grain refiner in appropriate amounts. The effect of grain size and nano-particle incorporation on microstructure and mechanical properties of electrodeposits was investigated. The grain size of the deposits was calculated from X-ray diffraction (XRD) patterns using Williamson-Hall equation and surface morphology of the coatings was studied by means of scanning electron microscopy (SEM). The mechanical properties of electrodeposits were investigated by Vickers microhardness and tensile tests. The results indicated that incorporation of SiC nano-particles increased the microhardness and yield strength of micro-grained deposits due to the change in crystallographic texture, decrease in grain size as well as dispersion hardening. But dispersion hardening mechanism could not be confirmed in nano-structured deposits. Moreover, the increase in the concentration of uniformly dispersed SiC particles initially improved and then decreased the elongation to failure of coarse-grained deposits while a continuous increase was observed for nano-structured deposits.

Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts.

MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V(L)) domain (kappa chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 A resolution by molecular replacement with an R(cryst) of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the beta-sheet of the V(L) domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

Crystal structure of Escherichia coli UvrB C-terminal domain, and a model for UvrB-uvrC interaction.

A crystal structure of the C-terminal domain of Escherichia coli UvrB (UvrB') has been solved to 3.0 A resolution. The domain adopts a helix-loop-helix fold which is stabilised by the packing of hydrophobic side-chains between helices. From the UvrB' fold, a model for a domain of UvrC (UvrC') that has high sequence homology with UvrB' has been made. In the crystal, a dimerisation of UvrB domains is seen involving specific hydrophobic and salt bridge interactions between residues in and close to the loop region of the domain. It is proposed that a homologous mode of interaction may occur between UvrB and UvrC. This interaction is likely to be flexible, potentially spanning > 50 A.

Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: crystallization and preliminary X-ray diffraction analysis of the enzyme.

Wild-type and site-specific mutants C166S and C166A (Cys-166-->Ser and Cys-166-->Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a=b=c=84 A; alpha=beta=gamma=75 degrees) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility.

Crystal structure of the zinc-dependent beta-lactamase from Bacillus cereus at 1.9 A resolution: binuclear active site with features of a mononuclear enzyme.

The structure of the zinc-dependent beta-lactamase II from Bacillus cereus has been determined at 1.9 A resolution in a crystal form with two molecules in the asymmetric unit and 400 waters (space group P3121; Rcryst = 20.8%). The active site contains two zinc ions: Zn1 is tightly coordinated by His86, His88, and His149, while Zn2 is loosely coordinated by Asp90, Cys168, and His210. A water molecule (W1) lies between the two zinc ions but is significantly closer to Zn1 and at a distance of only 1.9 A is effectively a hydroxide moiety and a potential, preactivated nucleophile. In fact, Asp90 bridges W1 to Zn2, and its location is thus distinct from that of the bridging water molecules in the binuclear zinc peptidases or other binuclear zinc hydrolases. Modeling of penicillin, cephalosporin, and carbapenem binding shows that all are readily accommodated within the shallow active site cleft of the enzyme, and the Zn1-bound hydroxide is ideally located for nucleophilic attack at the beta-lactam carbonyl. This enzyme also functions with only one zinc ion present. The Zn1-Zn2 distances differ in the two independent molecules in the crystal (3.9 and 4.4 A), yet the Zn1-W1 distances are both 1.9 A, arguing against involvement of Zn2 in W1 activation. The role of Zn2 is unclear, but the B. cereus enzyme may be an evolutionary intermediate between the mono- and bizinc metallo-beta-lactamases. The broad specificity of this enzyme, together with the increasing prevalence of zinc-dependent metallo-beta-lactamases, poses a real clinical threat, and this structure provides a basis for understanding its mechanism and designing inhibitors.

The structure of a human rheumatoid factor bound to IgG Fc.

This is the first crystal structure analysis of a complex between an autoantibody and its autoantigen, and it reveals a mode of interaction never before seen in an antibody-antigen complex. Not only are there relatively few antibody contact residues, contributing perhaps to its very low affinity, but these residues are to be found on only one side of the potential combining site surface. Indeed, so many CDR residues are not involved in Fc binding, including those in the central region of the combining site, that it is easy to envisage that this RF may have another, entirely different, specificity. The antibody may therefore have originated in response to another, as yet unidentified, antigen, and the reactivity with IgG Fc may be an unfortunate cross-reactivity. Certainly some of the CDR residues which do interact with IgG Fc are germline encoded, but significantly one of only two residues in the light chain, Pro56, which makes many contacts with Fc, is a somatic mutation. Since this mutation would appear to make a significant contribution to the binding affinity, it is therefore evidence for an antigen driven response to the IgG Fc in the generation of this autoantibody. The Fc epitope recognised by RF-AN is strikingly similar to the binding sites for the bacterial binding proteins A and G, but the significance of this is not clear. What is clear however is that the epitope does not include any part of the Fc carbohydrate residues, although the structure of the complex does reveal that there is an alteration in the carbohydrate conformation when the galactose residues are absent. Loss of the interaction between the terminal galactose residue on the alpha (1-6) linked branch and the C gamma 2 domain appears to allow the carbohydrate chains to become mobile, at the same time exposing a predominantly hydrophobic patch on the C gamma 2 surface. Accessibility to either the agalactosyl carbohydrate chains or the newly exposed residues may account for the enhanced reactivity for G0-IgG that has been reported for certain RFs, and such an epitope need not be very different to that recognised by RF-AN. In order to understand more completely the effect of the presence or absence of the terminal galactose residue, the fully galactosylated glycoform of Fc must be studied for comparison; this work is underway. It is also important now to study a RF which is known to sense this difference in oligosaccharide composition, and also to study RFs of higher affinity, of the IgG class, and from the synovium. RF-AN was the first RF to be immortalised as a cell line, and in many ways it is a typical RF (in terms of specificity, relationship to germline sequence and affinity), but we must now establish whether the novel structural features revealed in this analysis are indeed typical of other RFs. Only when comparisons can be made between RFs of different origin and with contrasting functional properties will we begin to understand what constitutes a pathogenic RF, and the mechanism by which such auto-reactive antibodies are generated.

Structure of human IgM rheumatoid factor Fab bound to its autoantigen IgG Fc reveals a novel topology of antibody-antigen interaction.

Rheumatoid factors are the characteristic autoantibodies of rheumatoid arthritis, which bind to the Fc regions of IgG molecules. Here we report the crystal structure of the Fab fragment of a patient-derived IgM rheumatoid factor (RF-AN) complexed with human IgG4 Fc, at 3.2 A resolution. This is the first structure of an autoantibody-autoantigen complex. The epitope recognised in IgG Fc includes the C gamma 2/C gamma 3 cleft region, and overlaps the binding sites of bacterial Fc-binding proteins. The antibody residues involved in autorecognition are all located at the edge of the conventional combining site surface, leaving much of the latter available, potentially, for recognition of a different antigen. Since an important contact residue is somatic mutation, the structure implicates antigen-driven selection, following somatic mutation of germline genes, in the production of pathogenic rheumatoid factors.

Purification and crystallisation of the autoantigen thyroid peroxidase from human Graves' thyroid tissue.

Milligram quantities of the human membrane autoantigen thyroid peroxidase (TPO) have been purified to a high degree of homogeneity by a combination of detergent solubilisation, monoclonal antibody affinity, and ion exchange chromatography, from pooled Graves' disease thyroid glands. The purified TPO of greater than 90% purity was enzymatically active as judged by its ability to oxidise guaiacol. Crystals of TPO have been grown from solutions of the protein solubilised in sodium deoxycholate, in the presence of ammonium sulphate. The crystals exhibited birefringence under polarised light, indicative of molecular order. Crystallisation of this large, membrane autoantigen represents the first step in delineating the complete three-dimensional structure of a human autoantigen involved in destructive thyroiditis.

Crystallization of a complex between the Fab fragment of a human immunoglobulin M (IgM) rheumatoid factor (RF-AN) and the Fc fragment of human IgG4.

Rheumatoid factors (RF) are the characteristic autoantibodies found in patients with rheumatoid arthritis. They recognize epitopes in the Fc region of immunoglobulin G (IgG) and are often of the IgM isotype. In order to analyse the nature of RF-Fc interactions, we have crystallized a complex between the Fab fragment of a human monoclonal IgM rheumatoid factor (RF-AN) and the Fc fragment of human IgG4. The stoichiometry of the complex within the crystals was found to be 2:1 Fab:Fc. The crystals diffracted X-rays to 0.3 nm resolution, and the space group was C2, with cell dimensions a = 16.03 nm, b = 8.19 nm, c = 6.42 nm, beta = 98.3 degrees. We have also determined the sequence of the variable region of the RF-AN light chain, not hitherto reported. This belongs to the V lambda III-a subgroup and is closely related to the germline gene Humlv318, from which it differs in three amino acid residues. This is the first reported crystallized complex between a human autoantibody and its autoantigen.

Crystallization and X-ray analysis of a single fab binding domain from protein L of Peptostreptococcus magnus.

Protein L is a multidomain cell wall constituent of certain strains of Peptostreptococcus magnus which binds to the variable domain of immunoglobulin kappa-light chains. A single immunoglobulin-binding domain of M(r) = 9000 from this protein has been isolated and crystallized. The crystals are of space group P4(2)2(1)2, with cell dimensions a = b = 66.9 A, c = 68.3 A, and diffract to at least 2.2 A resolution. The asymmetric unit of the crystal contains two molecules of the protein L domain, related by a noncrystallographic 2-fold axis, as revealed by a self-rotation function calculated with native diffraction data.

Crystallization and preliminary X-ray analysis of the Fab fragment of a human monoclonal IgM rheumatoid factor (2A2).

Crystals of the Fab fragment of a human monoclonal IgM rheumatoid factor have been obtained and are suitable for X-ray structure determination. This molecule, derived from the synovial B cells of a patient with rheumatoid arthritis, is an autoantibody with specificity for IgG Fc. The crystals have space group P2(1), cell dimensions a = 69.0 A, b = 76.6 A, c = 98.8 A and beta = 90.6 degrees, and diffract to a resolution of at least 2.8 A.

The role of cAMP and calcium in the stimulation of proliferation of immature erythroblasts by erythropoietin.

The hypothesis that cAMP or calcium are the second messengers of erythropoietin (Epo) was tested on fractionated, Epo-responsive immature erythroblasts from anemic rabbit bone marrow by examining whether the proliferative effects of the hormone could be mimicked by agents that increase the intracellular concentration of cAMP or Ca2+. None of the compounds tested (including 10(-6)-10(-4) M db-cAMP, forskolin, isoprenaline or 10(-7)-10(-6) M of the calcium ionophore A23187) alone or in combination could either initiate or potentiate the mitogenic action of the hormone. Furthermore, addition of 0.2 U/ml erythropoietin produced no permanent or transient increase in the uptake of 45Ca2+ by erythroblasts at 37 degrees C. However, cells cultured with imidazole or cordycepin (which reduce the level of intracellular cAMP), or with the calcium chelator EGTA, or the drugs verapamil or TMB-8 (which interfere with the utilization of extracellular or intracellular calcium) showed a decreased stimulation of DNA synthesis by Epo. Finally, the tumour promoter phorbol ester TPA could partially mimic the action of Epo when added to cultures containing more immature progenitor cells. We conclude then that an artificial increase in the cytoplasmic concentration of either cAMP or Ca2+ is not sufficient to elicit the proliferation of Epo-responsive cells.

The 66 kDa component of eukaryotic initiation factor 3 interacts with globin mRNA and 18 S rRNA in preinitiation complexes.

The 66 kDa protein present in a complex with globin mRNA and 18 S rRNA [(1984) Eur. J. Biochem. 143, 27-33] has been reincorporated into functional eukaryotic initiation factor 3 (eIF-3) under conditions of protein synthesis. Additionally, two-dimensional polyacrylamide gel electrophoresis has been used to demonstrate the identity of the 66 kDa protein with the 66 kDa subunit of eIF-3.

The effect of haemin on RNA synthesis and stability in differentiating rabbit erythroblasts.

Haemin accelerates the maturation of erythroid cells but whether this is the result of increased globin gene transcription or processing and stabilization of globin mRNA is not clear. The effect of haemin on the synthesis and stability of non-globin messengers is also unknown. We examined the changes that occur in RNA metabolism when anaemic rabbit bone marrow erythroblasts, fractionated into immature and mature fractions, are cultured with 20 microM or 50 microM haemin for brief periods (5-8 h). With both cell types haemin increases the incorporation of [3H]uridine into newly synthesized RNA, particularly into the poly(A)-rich fraction which can increase threefold. Haemin also increases the synthesis of globin mRNA (up to 500% absolutely and 50% relative to the synthesis of total RNA) in the immature, but not in the mature, cells. These results suggest that haemin increases the transcription of both globin and non-globin mRNAs and that the relative increase of each depends on the stage of erythroid cell development. When the [3H]RNA from prelabelled cells was chased in the presence of haemin (with or without actinomycin D) the proportion of 3H remaining in globin mRNA increased in the immature, but not in the mature, cells. These changes in the relative concentration of globin mRNA were also shown by the translation of extracted bone marrow RNAs in a nuclease-treated reticulocyte lysate. We conclude that a secondary effect of haemin is on RNA stability and that it enhances the accumulation of globin mRNA by both molecular and cellular mechanisms.

Regulation of erythroid cell differentiation by haemin.

The differentiation of immature erythroblasts, isolated from anaemic rabbit bone marrow by density centrifugation to bovine serum albumin gradients, is accelerated by the addition of 10(-5)-10(-4) M haemin to the culture medium. Both the proportion of benzidine-positive cells and the synthesis of haemoglobin relative to the total protein were increased, whereas cell growth and DNA synthesis were decreased. Some of these changes were detected within 4 h and were maximal after 18-40 h. It is suggested that haem may have a physiological role in regulating in vivo erythropoiesis during haemolysis by accelerating terminal erythroid cell differentiation.

Regulation of protein synthesis in reticulocyte lysates. Characterization of the inhibitor generated in the postribosomal supernatant by heating at 44 degrees C.

Heating of a rabbit reticulocyte lysate at 44 degrees C, in the presence of optimal concentrations of haemin, results in an inhibition of protein synthesis and in the appearance of an inhibitory activity in the postribosomal supernatant. These effects of supraoptimal heating are similar to those observed in lysates and supernatants incubated at physiological temperatures in the absence of haemin. In this paper we examined whether the haem-regulated inhibitor produced by treating postribosomal supernatant with N-ethylmaleimide and the inhibitor generated by heating at 44 degrees C in the presence of haemin are the same molecular entity. Both inhibitors behaved similarly through a partial purification consisting of 50% (NH4)2SO4 precipitation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography, and had the same pattern of polypeptides after polyacrylamide gel electrophoresis. However, when incubated with an antiserum to the haem-regulated inhibitor the activity of the 44 degrees C heated supernatant was not neutralized, whereas that of its more purified fractions was. This apparent contradiction was shown to be due to an interference of the immune serum assay by the levels of proinhibitor and haemoglobin present in the crude supernatant. Further experiments, with extensively diluted 44 degrees C supernatants or with isolated proinhibitor subsequently heated, are consistent with the conclusion that both heating at supraoptimal temperatures and incubating in the absence of haem finally activate the same inhibitor.