Cost-effectiveness of routine imaging of suspected appendicitis.

Introduction The misdiagnosis of appendicitis and consequent removal of a normal appendix occurs in one in five patients in the UK. On the contrary, in healthcare systems with routine cross-sectional imaging of suspected appendicitis, the negative appendicectomy rate is around 5%. If we could reduce the rate in the UK to similar numbers, would this be cost effective? This study aimed to calculate the financial impact of negative appendicectomy at the Queen Alexandra Hospital and to explore whether a policy of routine imaging of such patients could reduce hospital costs. Materials and methods We performed a retrospective analysis of all appendicectomies over a 1-year period at our institution. Data were extracted on outcomes including appendix histology, operative time and length of stay to calculate the negative appendicectomy rate and to analyse costs. Results A total of 531 patients over 5 years of age had an appendicectomy. The negative appendicectomy rate was 22% (115/531). The additional financial costs of negative appendicectomy to the hospital during this period were £270,861. Universal imaging of all patients with right iliac fossa pain that could result in a 5% negative appendicectomy rate would cost between £67,200 and £165,600 per year but could save £33,896 (magnetic resonance imaging), £105,896 (computed tomography) or £132,296 (ultrasound) depending on imaging modality used. Conclusions Negative appendicectomy is still too frequent and results in additional financial burden to the health service. Routine imaging of patients with suspected appendicitis would not only reduce the negative appendicectomy rate but could lead to cost savings and a better service for our patients.

Identification and characterization of a misfolded monomeric serpin formed at physiological temperature.

The native serpin state is kinetically trapped. However, under mildly destabilizing conditions, the conformational landscape changes, and a number of nonnative conformations with increased stability can be readily formed. The ability to undergo structural change is due to intrinsic strain within the serpin's tertiary fold, which is utilized for proteinase inhibition but renders the protein susceptible to aberrant folding and self-association. The relationship between these various conformations is poorly understood. Antichymotrypsin (ACT) is an inhibitory serpin that readily forms a number of inactive conformations, induced via either environmental stress or interaction with proteinases. Here we have used a variety of biophysical and structural techniques to characterize the relationship between some of these conformations. Incubation of ACT at physiological temperature results in the formation of a range of conformations, including both polymer and misfolded monomer. The ability to populate these nonnative states and the native conformation reflects an energy landscape that is very sensitive to the solution conditions. X-ray crystallography reveals that the misfolded monomeric conformation is in the delta conformation. Further polymerization and seeding experiments show that the delta conformation is an end point in the misfolding pathway of ACT and not an on-pathway intermediate formed during polymerization. The observation that ACT readily forms this inactive conformation at physiological temperature and pH suggests that it may have a role in both health and disease.

Towards the treatment of polyglutamine diseases: the modulatory role of protein context.

Protein aggregation is a key mechanism involved in neurodegeneration associated with Alzheimer's, Parkinson's and Huntington's diseases. Nine diseases (including Huntington's) arise from polyglutamine (polyQ) expansion above a repeat threshold of approximately 37 glutamines, and neuronal toxicity correlates with the process of protein aggregation. The similar toxic gain-of-function mechanism of the nine diseases supports the hypothesis that disease onset and progression is dependent upon polyQ expansion. However, there is an increasing body of literature demonstrating that the protein context of the polyQ tract has an essential role in the disease process. The composition of regions flanking repeats can alter the biochemical and biophysical properties of the polyQ region. A number of the disease proteins are proteolytically cleaved, with release of the polyQ-containing fragment initiating aggregation. Interactions of flanking domains with other molecules can also influence aggregation and cellular localization, which are critical factors for toxicity. More recently, there is evidence that domains flanking the polyQ tract can also aggregate independent of the polyQ tract, and that this significantly alters the rate at which the polyQ regions form fibrillar aggregates and the properties of these aggregates. In this review we consider the role of protein context in modulating the polyQ diseases and the therapeutic potential of targeting non-polyQ protein properties.

Relapsing hypocupraemic myelopathy requiring high-dose oral copper replacement.

Adult-onset copper deficiency with neurological manifestations is a newly recognised syndrome. Long-term oral copper replacement therapy has been the mainstay of treatment in the literature. A case of relapsing hypocupraemic myelopathy responsive to increased doses of copper replacement is reported. Standard doses of copper may not be sufficient for all patients.

CNS demyelination associated with copper deficiency and hyperzincemia.

CNS demyelination is not a previously reported feature of acquired copper deficiency. The authors report two patients with idiopathic hypocupremia and hyperzincemia, hematologic changes of copper deficiency, and extensive CNS demyelination. Hematologic recovery followed copper supplementation, both initially and after relapse off copper therapy, while serum zinc levels remained high and the neurologic abnormalities only stabilized.

Dissection of protease-activated receptor-1-dependent and -independent responses to thrombin in skeletal myoblasts.

Thrombin exerts a number of effects on skeletal myoblasts in vitro. It stimulates proliferation and intracellular calcium mobilization and inhibits differentiation and apoptosis induced by serum deprivation in these cells. Many cellular responses to thrombin are mediated by protease-activated receptor-1 (PAR-1). Expression of PAR-1 is present in mononuclear myoblasts in vitro, but repressed when fusion occurs to form myotubes. In the current study, we used PAR-1-null mice to determine which of thrombin's effects on myoblasts are mediated by PAR-1. Thrombin inhibited fusion almost as effectively in cultures prepared from the muscle of PAR-1-null myoblasts as in cultures prepared from wild-type mice. Apoptosis was inhibited as effectively in PAR-1-null myoblasts as in wild-type myoblasts. These effects in PAR-1-null myoblasts were mediated by a secreted inhibitor of apoptosis and fusion, as demonstrated previously for normal rat myoblasts. Thrombin failed to induce an intracellular calcium response in PAR-1-null myoblast cultures, although these cells were able to mobilize intracellular calcium in response to activation of other receptors. PAR-1-null myoblasts also failed to proliferate in response to thrombin. These results demonstrate that thrombin's effects on myoblast apoptosis and fusion are not mediated by PAR-1 and that PAR-1 is the only thrombin receptor capable of inducing proliferation and calcium mobilization in neonatal mouse myoblasts.

Probing the equilibrium denaturation of the serpin alpha(1)-antitrypsin with single tryptophan mutants; evidence for structure in the urea unfolded state.

The native conformation of proteins in the serpin superfamily is metastable. In order to understand why serpins attain the native state instead of more stable conformations we have begun investigations into the equilibrium-unfolding of alpha(1)-antitrypsin. alpha(1)-Antitrypsin contains two tryptophan residues, Trp194 and Trp238, situated on the A and B beta-sheets, respectively. Site-directed mutagenesis was used to construct two single-tryptophan variants. Both variants were fully active and had similar secondary structure and stabilities to alpha(1)-antitrypsin. The denaturation of alpha(1)-antitrypsin and its variants was extremely similar when followed by far-UV CD, indicating the presence of a single intermediate. Fluorescence analysis of the unfolding behavior of each single tryptophan variant indicated that the sole tryptophan residue reported the structural changes within its immediate environment. These data suggest that the A beta-sheet is expanded in the intermediate state whilst no structural change around the B beta-sheet has occurred. In the urea-induced unfolded state, Trp238 does not become fully solvated, suggesting the persistence of structure around this residue. The implications of these data on the folding, misfolding and function of the serpin superfamily are discussed.

The role of strand 1 of the C beta-sheet in the structure and function of alpha(1)-antitrypsin.

Serpins inhibit cognate serine proteases involved in a number of important processes including blood coagulation and inflammation. Consequently, loss of serpin function or stability results in a number of disease states. Many of the naturally occurring mutations leading to disease are located within strand 1 of the C beta-sheet of the serpin. To ascertain the structural and functional importance of each residue in this strand, which constitutes the so-called distal hinge of the reactive center loop of the serpin, an alanine scanning study was carried out on recombinant alpha(1)-antitrypsin Pittsburgh mutant (P1 = Arg). Mutation of the P10' position had no effect on its inhibitory properties towards thrombin. Mutations to residues P7' and P9' caused these serpins to have an increased tendency to act as substrates rather than inhibitors, while mutations at P6' and P8' positions caused the serpin to behave almost entirely as a substrate. Mutations at the P6' and P8' residues of the C beta-sheet, which are buried in the hydrophobic core in the native structure, caused the serpin to become highly unstable and polymerize much more readily. Thus, P6' and P8' mutants of alpha(1)-antitrypsin had melting temperatures 14 degrees lower than wild-type alpha(1)-antitrypsin. These results indicate the importance of maintaining the anchoring of the distal hinge to both the inhibitory mechanism and stability of serpins, the inhibitory mechanism being particularly sensitive to any perturbations in this region. The results of this study allow more informed analysis of the effects of mutations found at these positions in disease-associated serpin variants.

Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins.

BACKGROUND: Peptostreptococcus magnus protein L (PpL) is a multidomain, bacterial surface protein whose presence correlates with virulence. It consists of up to five homologous immunoglobulin binding domains that interact with the variable (VL) regions of kappa light chains found on two thirds of mammalian antibodies. RESULTS: We refined the crystal structure of the complex between a human antibody Fab fragment (2A2) and a single PpL domain (61 residues) to 2.7 A. The asymmetric unit contains two Fab molecules sandwiching a single PpL domain, which contacts similar VL framework regions of two light chains via independent interfaces. The residues contacted on VL are remote from the hypervariable loops. One PpL-Vkappa interface agrees with previous biochemical data, while the second is novel. Site-directed mutagenesis and analytical-centrifugation studies suggest that the two PpL binding sites have markedly different affinities for VL. The PpL residues in both interactions are well conserved among different Peptostreptococcus magnus strains. The Fab contact positions identified in the complex explain the high specificity of PpL for antibodies with kappa rather than lambda chains. CONCLUSIONS: The PpL-Fab complex shows the first interaction of a bacterial virulence factor with a Fab light chain outside the conventional combining site. Structural comparison with two other bacterial proteins interacting with the Fab heavy chain shows that PpL, structurally homologous to streptococcal SpG domains, shares with the latter a similar binding mode. These two bacterial surface proteins interact with their respective immunoglobulin regions through a similar beta zipper interaction.

Prevention of polymerization of M and Z alpha1-Antitrypsin (alpha1-AT) with trimethylamine N-oxide. Implications for the treatment of alpha1-at deficiency.

alpha1-Antitrypsin (alpha1-AT) is the most abundant circulating proteinase inhibitor. The Z variant results in profound plasma deficiency as the mutant polymerizes within hepatocytes. The retained polymers are associated with cirrhosis, and the lack of circulating protein predisposes to early onset emphysema. We have investigated the role of the naturally occurring solute trimethylamine N-oxide (TMAO) in modulating the polymerization of normal M and disease-associated Z alpha1-AT. TMAO stabilized both M and Z alpha1-AT in an active conformation against heat-induced polymerization. Spectroscopic analysis demonstrated that this was due to inhibition of the conversion of the native state to a polymerogenic intermediate. However, TMAO did not aid the refolding of denatured alpha1-AT to a native conformation; instead, it enhanced polymerization. These data show that TMAO can be used to control the conformational transitions of folded alpha1-AT but that it is ineffective in promoting folding of the polypeptide chain within the secretory pathway.

Intrinsic fluorescence changes and rapid kinetics of proteinase deformation during serpin inhibition.

The X-ray crystal structure of the serpin-proteinase complex suggested that the serpin deformed the proteinase thereby inactivating the molecule. Using a variant of alpha(1)-antitrypsin in which both tryptophan residues have been replaced by phenylalanine, we have shown that the proteinase becomes partially unfolded during serpin inhibition. The tryptophan free variant, alpha(1)-antitrypsin((FF)), is fully active as an inhibitor of thrombin. Thrombin has a fluorescence emission maximum of 340 nm which blue shifts to 346 nm, concomitant with a 40% increase in intensity, upon formation of the serpin-proteinase complex indicative of substantial conformational change within the proteinase. Stopped-flow analysis of the fluorescence changes within the proteinase indicated a two-step mechanism. A fast bimolecular reaction with a rate constant of 2.8x10(6) M(-1) s(-1) is followed by a slow unimolecular process with a rate of 0.26 s(-1) that is independent of concentration. We propose that the first rate is formation of an initial complex which is then followed by a slower process involving the partial unfolding of the proteinase during its translocation to the opposite pole of the serpin.

Structure of a serpin-enzyme complex probed by cysteine substitutions and fluorescence spectroscopy.

The x-ray crystal structure of the serpin-proteinase complex is yet to be determined. In this study we have investigated the conformational changes that take place within antitrypsin during complex formation with catalytically inactive (thrombin(S195A)) and active thrombin. Three variants of antitrypsin Pittsburgh (an effective thrombin inhibitor), each containing a unique cysteine residue (Cys(232), Cys(P3'), and Cys(313)) were covalently modified with the fluorescence probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine. The presence of the fluorescent label did not affect the structure or inhibitory activity of the serpin. We monitored the changes in the fluorescence emission spectra of each labeled serpin in the native and cleaved state, and in complex with active and inactive thrombin. These data show that the serpin undergoes conformational change upon forming a complex with either active or inactive proteinase. Steady-state fluorescence quenching measurements using potassium iodide were used to further probe the nature and extent of this conformational change. A pronounced conformational change is observed upon locking with an active proteinase; however, our data reveal that docking with the inactive proteinase thrombin(S195A) is also able to induce a conformational change in the serpin.

Studies on a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus: the role of tyrosine-53 in the reaction with human IgG.

Chemical modification experiments with tetranitromethane (TNM) have been used to investigate the role of tyrosine residues in the formation of the complex between PpL (the single Ig-binding domain of protein L, isolated from P. magnus strain 3316) and the kappa light chain (kappa-chain). Reaction of PpL with TNM causes the modification of 1.9 equiv. of tyrosine (Tyr(51) and Tyr(53)) and results in an approx. 140-fold decrease in affinity for human IgG. Similar experiments with mutated PpL proteins suggest that nitration predominantly inactivates the protein by modification of Tyr(53). Reduction of the nitrotyrosine groups to aminotyrosine by incubation with sodium hydrosulphite does not restore high affinity for IgG. Modification of kappa-chain by TNM resulted in the nitration of 3.1+/-0.09 tyrosine residues. When the PpL-kappa-chain complex was incubated with TNM, 4.1+/-0.04 tyrosine residues were nitrated, indicating that one tyrosine residue previously modified by the reagent was protected from TNM when the proteins are in complex with each other. The K(d) for the equilibrium between PpL, human IgG and their complex has been shown by ELISA to be 112+/-20 nM. A similar value (153+/-33 nM) was obtained for the complex formed between IgG and the Tyr(64)-->Trp mutant (Y64W). However, the K(d) values for the equilibria involving the PpL mutants Y53F and Y53F,Y64W were found to be 3.2+/-0.2 and 4.6+/-1 microM respectively. These suggest that the phenol group of Tyr(53) in PpL is important to the stability of the PpL-kappa-chain complex.

Osmolytes as modulators of conformational changes in serpins.

Protein misfolding and aggregation play an integral role in many diseases. The misfolding of the serpin (SERine Proteinase INhibitor) alpha1-antitrypsin results in the accumulation of insoluble polymers within hepatocytes and alpha1-antitrypsin deficiency in plasma, predisposing patients to liver cirrhosis and emphysema. We have examined the effect of three naturally occurring osmolytes, sarcosine, glycine betaine and trimethylamine N-oxide, on conformational changes in alpha1-antitrypsin. All three solutes protected native alpha1-antitrypsin against thermally induced polymerisation and inactivation in a concentration-dependent manner. Further spectroscopic analysis showed that sarcosine stabilises the native conformation of alpha1-antitrypsin, thus hindering its conversion to an intermediate state and subsequent polymerisation. On refolding in the presence of sarcosine, alpha1-antitrypsin formed a heterogeneous population, with increasing proportions of molecules adopting an inactive conformation in higher concentrations of the osmolyte. These data show that sarcosine can be used to prevent abnormal structural changes in native alpha1-antitrypsin, but is ineffective in facilitating the correct folding of the protein. The implications of these results in the context of conformational changes and states adopted by alpha1-antitrypsin are discussed.

Iron deficiency due to excessive therapeutic phlebotomy in hemochromatosis.

Thirteen adults (eight men, five women) with hemochromatosis had undergone routine iron depletion therapy but while on maintenance phlebotomies developed iron deficiency which persisted for 25 +/- 13 (mean +/- 1 SD) months before diagnosis. All had symptoms and signs of iron deficiency. Levels of transferrin saturation were 10% +/- 5% (1 SD), and serum ferritin concentrations were 8 +/- 3 ng/mL. Eleven had anemia; eight had hypochromia and microcytosis. Bone marrow specimens obtained in five patients revealed no stainable iron. Medical records indicated that parameters of body iron status were infrequently or incorrectly used for adjusting the frequency of phlebotomies. Two patients developed iron deficiency due to additional blood loss from esophageal varices and bilateral hip replacement, respectively. Ten of the patients were treated with ferrous sulfate, 325 mg daily, for 2-6 weeks when anemia was corrected. In patients who were not given iron, anemia and microcytosis recovered in 8-24 months. We conclude that (i) sustained iron deficiency in hemochromatosis patients should be prevented by monitoring hemoglobin levels and serum ferritin; and (ii) hemoglobin concentrations and values of mean corpuscular hemoglobin may be higher in iron-deficient persons with hemochromatosis than in individuals without hemochromatosis. Symptomatic iron deficiency in hemochromatosis patients may be treated safely with a brief course of ferrous sulfate. Recovery is slower when iron is not given. However, iron supplementation is unnecessary and not recommended for the mild, self-limited anemia and decreased serum iron and ferritin concentrations encountered after initial iron depletion therapy for hemochromatosis.

Protease-activated receptor-2 mediates proliferative responses in skeletal myoblasts.

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved by proteases within the N terminus, exposing a new tethered ligand that binds and activates the receptor. Activators of PAR-2 include trypsin and mast cell tryptase. Skeletal myoblasts are known to express PAR-1, a thrombin receptor. The current study was undertaken to determine whether myoblasts express PAR-2. Primary neonatal rat and mouse skeletal myoblast cultures were shown to express PAR-2 in polymerase chain reaction and immunocytochemical studies. Expression of PAR-2 was also demonstrated by immunohistochemistry in developing mouse skeletal muscle in vivo. Trypsin or a synthetic peptide corresponding to the rat PAR-2 tethered ligand caused a dose-dependent elevation in intracellular calcium in cultured rat myoblasts, with an EC(50) of 13 nM or 56 microM, respectively. Studies aimed at identifying the function of PAR-2 in myoblasts demonstrated no effect of the receptor-activating peptide on survival or fusion in serum-deprived myoblasts. The PAR-2-activating peptide did, however, stimulate proliferation of serum-deprived myoblasts. These results demonstrate that skeletal muscle cells express PAR-2, activation of which leads to stimulation of myoblast proliferation.

Development of a ubiquitin transfer assay for high throughput screening by fluorescence resonance energy transfer.

An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu(3+), and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc). When Rsc is ubiquitinated, Eu(3+) and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wavelength of APC, almost entirely free of nonspecific fluorescence from Eu(3+) and APC. The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 microg/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening.

The inositol polyphosphate 5-phosphatases and the apurinic/apyrimidinic base excision repair endonucleases share a common mechanism for catalysis.

Inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from the inositol ring of phosphatidylinositol-derived signaling molecules; however, the mechanism of catalysis is only partially characterized. These enzymes play critical roles in regulating cell growth, apoptosis, intracellular calcium oscillations, and post-synaptic vesicular trafficking. The UCLA fold recognition server (threader) predicted that the conserved 300-amino acid catalytic domain, common to all 5-phosphatases, adopts the fold of the apurinic/apyrimidinic (AP) base excision repair endonucleases. PSI-BLAST searches of GENPEPT, using the amino acid sequence of AP endonuclease exonuclease III, identified all members of the 5-phosphatase family with highly significant scores. A sequence alignment between exonuclease III and all known 5-phosphatases revealed six highly conserved motifs containing residues that corresponded to the catalytic residues in the AP endonucleases. Mutation of each of these residues to alanine in the mammalian 43-kDa, or yeast Inp52p 5-phosphatase, resulted in complete loss of enzyme activity. We predict the 5-phosphatase enzymes share a similar mechanism of catalysis to the AP endonucleases, consistent with other common functional similarities such as an absolute requirement for magnesium for activity. Based on this analysis, functional roles have been assigned to conserved residues in all 5-phosphatase enzymes.

The citrate ion increases the conformational stability of alpha(1)-antitrypsin.

Sodium citrate has previously been shown to convert native alpha(1)-antitrypsin into the inactive latent state and cause alpha(1)-antitrypsin to polymerize via the C-sheet pathway instead of the more common A-sheet pathway. In order to begin to understand these dramatic effects, we have examined the influence of low concentrations of sodium citrate upon the structure, stability and function of alpha(1)-antitrypsin. In 0.5 M citrate, the midpoint of guanidine hydrochloride-induced unfolding was increased by 1.8 M and the rate of heat inactivation was decreased approximately 30-fold compared with Tris or phosphate buffer. alpha(1)-Antitrypsin was fully active in the presence of a range of citrate concentrations (0. 1-0.5 M), forming a stable 1:1 complex with chymotrypsin. The association rate constant between alpha(1)-antitrypsin and chymotrypsin was decreased with increasing citrate concentration. Fluorescence and circular dichroism spectroscopy demonstrated no significant changes in the tertiary structure due to the presence of citrate. However, the insertion rate of exogenous reactive-center loop peptide increased with increasing citrate concentration, indicating some structural changes in the A beta-sheet region. Taken together, these data suggest that in the presence of 0.5 M citrate alpha(1)-antitrypsin adopts a highly stable but active conformation.