Urban and rural risks of Lyme disease in the Scottish Highlands.

BACKGROUND: This paper investigates the pattern of Lyme disease testing and infection within the Highland region of Scotland. METHODS: Data from all Highland samples tested during 2004-2006 were analysed according to result and patient's residence in relation to the eight fold Scottish Executive's urban/rural classification, and distance from woodland. RESULTS: In total, 1602 patients were tested for Lyme disease, 0.71% of the Highland population. From these, 104 (6.5%) were seropositive. There were more patients tested, and seropositive patients from rural than urban locations, 1113 vs 489, and 79 vs 25 respectively. There were also significantly more seropositive patients per patients tested from rural locations (chi2, p<0.0001). The number of patients tested and seropositive patients increased as the rural areas become more remote. The likelihood of being tested for Lyme disease also increased as the distance between a patient's residence and woodland decreased. The relative risk of being tested elevated by 74% for those patients living within 200 metres of woodland. CONCLUSIONS: Those living in the most rural areas of Highland and those living closest to woodland have an increased risk of being tested and having Lyme disease.

The contribution of arginine residues within the P6-P1 region of alpha 1-antitrypsin to its reaction with furin.

A series of mutants incorporating furin recognition sequences within the P6-P1 region of the reactive site loop of alpha(1)-antitrypsin were constructed. Variants containing different combinations of basic residues in the P1, P2, P4, and P6 positions replacing the wild type (P6)LEAIPM(P1) sequence were evaluated for their capacity to establish SDS-resistant complexes with furin, to affect association rate constants (k(ass) and k'(ass)), or to inhibit furin-dependent proteolysis of a model precursor in vivo. Each variant abolished processing of pro-von Willebrand factor in transfected hEK293 cells. The k(ass) of all variants were found to be similar (1.1-1.7 x 10(6) m(-1) s(-1)) except for one mutant, RERIRR, which had a k(ass) of 3.3 x 10(5) m(-1) s(-1). However, the stoichiometry of inhibition varied with values ranging from 2.9 to >24, indicating rapid formation of the acyl-enzyme intermediate (high k'(ass)). Moreover, those variants having high stoichiometry of inhibition values were accompanied by the rapid formation of cleaved forms of the inhibitors. The data suggest that the rate of conversion of the acyl-enzyme (EI') into the highly stable complex (EI*) was affected by replacement of specific residues within the reactive site loop. Taken together, the results reveal how furin recognition sequences within the context of the biochemical properties of serpins will play a role in the capacity of the protein to follow either the inhibitory or the substrate pathway.

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.

Evolution of serpin specificity: cooperative interactions in the reactive-site loop sequence of antithrombin specifically restrict the inhibition of activated protein C.

Protease cascades and their inhibitors are a common feature of many biological regulatory systems, and the various components of such cascades have been subjected to a long and concerted evolution. We present here evidence that in the coagulation cascade, the sequence of the protease-binding reactive-site loop of antithrombin has evolved such that the majority of its residues has been acquired not for the efficient inhibition of its target proteases, thrombin and factor Xa, but to avoid the inhibition of activated protein C (APC). We substituted residues of the reactive-site loop of antithrombin into alpha(1)-antitrypsin and tested the chimeras against thrombin, factor Xa, and APC. With respect to factor Xa and thrombin, the difference in association rate between the fastest and the slowest inhibitors was 5.5- and 88-fold, respectively. However, with respect to APC the difference was 12,500-fold. While most of the variation in the inhibition rates of thrombin could be accounted for by P2 Gly-to-Pro substitutions, for APC almost every residue had an effect on inhibition. In 22 of 25 direct comparisons of antitrypsin residues with antithrombin residues, either singly or in blocs, the antithrombin residues caused a decrease in the rate of inhibition of APC. The antithrombin residue Asn393, at position P'3, emerged as particularly important for avoiding the inhibition of APC, however, its 190-fold effect was seen only when in conjunction with antithrombin P7 to P'2 residues. Cooperative effects among residues of the reactive-site loop thus emerged as critical for restricting the activity of this sequence against APC.

Alpha 1-antitrypsin polymerisation can occur by both loop A and C sheet mechanisms.

A number of disease states are attributable to alpha1-antitrypsin polymerisation within the endoplasmic reticulum of hepatocytes and subsequent plasma deficiency. Two distinct mechanisms describing the process of alpha1-antitrypsin polymerisation have been proposed, the loop A-sheet and C-sheet mechanisms. We report fluorescence studies using alpha1-antitrypsin covalently modified with pyrene maleimide. These results in conjunction with detailed molecular modelling studies, show that alpha1-antitrypsin is capable of undergoing both loop A-sheet and loop C-sheet mechanisms of polymerisation, depending upon the in vitro buffer conditions.

Effects of dietary taurine on auditory function in full-term infants.

Three groups of neonates fed taurine supplemented infant formula, non-supplemented infant formula or breast milk, respectively, were studied from birth to 12 weeks of age. In addition to the measurement of whole blood taurine content, auditory function was monitored using auditory brainstem responses (ABRs) and transient evoked otoacoustic emissions (TEOAEs). The results showed a significant reduction in whole blood taurine concentration in the non-supplemented formula group. In addition, there was a significant drop in whole blood taurine levels in all 3 groups over the first four weeks of life. ABR wave latencies were significantly shorter in the non-supplemented group, with wave V showing the greatest reductions. Falling taurine levels after full-term birth may aid synaptic maturation/efficiency within the auditory system. TEOAE responses were significantly larger over the low to mid frequencies in the breast fed group suggesting improved middle ear function.

Serpin-like properties of alpha1-antitrypsin Portland towards furin convertase.

Recent studies have demonstrated that a serpin variant, alpha1-antitrypsin Portland (AT-PDX), can inhibit the mammalian convertase furin. Here, we examine the mechanism by which this inhibition takes place. We find that furin, which does not belong to the trypsin-like serine protease family, the usual targets of serpins, forms an SDS-heat denaturation-resistant complex with AT-PDX both in vitro and in vivo. AT-PDX inhibited furin with an association rate constant (k(ass)) of 1.5 x 10(6) M(-1) s(-1) which is similar to k(ass) values reported for serpins with trypsin-like enzymes. These results illustrate that AT can be modified to act essentially as a suicide inhibitor of furin, an enzyme of the subtilase superfamily of serine proteases.

Inhibitory mechanism of serpins. Mobility of the C-terminal region of the reactive-site loop.

The reactive-site loops of serpins are characterized by a defined mobility where the loop adopts a new secondary structure as an essential part of the inhibitory process. While the importance of mobility in the N-terminal region of the reactive-site loop has been well studied, the role of mobility in the C-terminal portion has not been investigated. The requirements for mobility of the C-terminal portion of the reactive-site loop of alpha1-antitrypsin were investigated by creating a disulfide bridge between the P'3 residue and residue 283 near the top of strand 2C; this disulfide would restrict the mobility of the C-terminal portion of the reactive-site loop by locking together strands 1 and 2 of the C beta-sheet. The engineered disulfide bond had no effect on the inhibitory activity of alpha1-antitrypsin, indicating that there is no requirement for mobility in this region of the molecule. Moreover, these results, coupled with those from molecular modeling, indicate that insertion into the A beta-sheet of the intact reactive-loop beyond P12 is not rate-limiting for the formation of the stable complex. The engineered disulfide bond should also prove useful in the creation of more stable serpin variants; for example, such a bond in plasminogen activator inhibitor-1 would prevent it from becoming latent by locking strand 1C onto the C beta-sheet.

Importance of the release of strand 1C to the polymerization mechanism of inhibitory serpins.

Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emphysema, liver cirrhosis, and angioedema. Understanding the structure of the polymers and the mechanism of polymerization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12-m34 peptide (homologous to P14-P3 of antithrombin reactive loop), representing the entire length of s4A, prevented polymerization totally. A 6-mer peptide (homologous to P14-P9 of antithrombin) not only allowed polymerization to occur, but induced it. This effect could be blocked by the addition of a 5-mer peptide with s1C sequence of antithrombin or by an unrelated peptide representing residues 26-31 of cholecystokinin. The s1C or cholecystokinin peptide alone was unable to form a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 361-->Cys, Ser 283-->Cys, was engineered for the purpose of forming a disulfide bond between s1C and s2C to prevent movement of s1C. This mutant was resistant to polymerization if the disulfide bridge was intact, but, under reducing conditions, it regained the potential to polymerize. We have also modeled long-chain serpin polymers with acceptable stereochemistry using two previously proposed loop-A-sheet and loop-C-sheet polymerization mechanisms and have shown both to be sterically feasible, as are "mixed" linear polymers. We therefore conclude that the release of strand 1C must be an element of the mechanism of serpin polymerization.

Role of the P2 residue in determining the specificity of serpins.

The importance of the P2 residue in determining serpin specificity was examined by making a series of substitutions in the P2 position of recombinant alpha 1-antichymotrypsin that contained an arginine P1 residue. The importance of the P2 residue in governing the association rate constant (Kon) of the serpin varied with the protease examined. For trypsin, the P2 residue played a relatively minor role, whereas the nature of this residue markedly influenced the rates of inhibition of thrombin, factor Xa, and APC. A 1000-fold difference in Kon values was observed between the fastest (P2 proline) and the slowest (P2 threonine) inhibitors of thrombin. Similar differences were observed with factor Xa; the best inhibitor (P2 glycine) displayed a 200-fold higher Kon value than the poorest (P2 threonine). The nature of the P2 residue also affected whether the interaction of the serpin with the protease resulted in inhibition of the protease or cleavage of the serpin; a P2 proline residue increased the rate of cleavage of alpha 1-antichymotrypsin by trypsin. By using mutants of thrombin, it was possible to show that the B-insertion loop, which partially occludes the active site, is important in determining the P2 specificity of this enzyme. Deletion of three amino acids from this loop yielded a protease (des-PPW) that became more like trypsin in its specificity. In addition, it was shown that Glu192 dramatically restricts thrombin's ability to accommodate a threonine in the P2 position. Taken together, the results demonstrated the importance of complementary interactions between the P2 residue of the serpin and the S2 binding site of the protease in regulating the specific interaction between serpin and protease.

The contribution of the conserved hinge region residues of alpha1-antitrypsin to its reaction with elastase.

The hinge region of serpins is a conserved sequence of 8 amino acids located 7 residues away from the scissile bond at P8 to P15, on the edge of the protease-binding domain. In the inhibitory serpins the P8 to P12 residues of this motif are usually small side-chain amino acids, most commonly alanine. Each of these residues in alpha1-antitrypsin was mutated to a glutamate, and the effect of a hinge-region glutamic acid substitution was found. While substitutions at positions P10 and P12 affected the inhibitory characteristics of alpha1-antitrypsin, substitutions at positions P7, P8, P9, and P11 had no effect on inhibition. Thus, the conservation of residues with small side chains at the latter positions does not appear to be related to an essential function in the inhibitory mechanism. Following the glutamate substitution at P10, alpha1-antitrypsin remained a rapid inhibitor of elastase, but the elastase--serpin complex slowly broke down to yield active elastase and cleaved alpha1-antitrypsin. The glutamate substitution at P12 caused the resultant molecule (P12 Ala-->Glu) to become a partial substrate of elastase such that four moles of inhibitor were required to inhibit one mole of enzyme, and led to a 12-fold decrease in the association rate constant. The data could be interpreted in terms of the suicide substrate inhibition model for serpin-protease interactions and allowed a further refinement of the role of the hinge region in this process.

Development of a novel recombinant serpin with potential antithrombotic properties.

Recombinant alpha 1-antitrypsin with a P1 arginine residue (Arg-alpha 1-antitrypsin) is a rapid inhibitor of both thrombin and activated protein C (APC). A series of mutants were made in an attempt to increase the specificity of this serpin for thrombin over APC. Initially, P2 and P'1 residues of Arg-alpha 1-antitrypsin were replaced in single and double mutations by the corresponding residues in antithrombin and C1 inhibitor which are very poor inhibitors of APC. No improvement in selectivity was achieved by these mutations. In fact, all P2/P'1 substitutions led to a decrease in selectivity for thrombin over APC. For example, replacement of the P2 proline of Arg-alpha 1-antitrypsin by glycine decreased the association rate constant (kass) with thrombin by 37-fold while the kass value with APC was reduced by only 16-fold. Cooperative effects were observed with the double P2 and P'1 substitutions; the mutational effects were not additive. The decrease in the kass for thrombin caused by the mutation of the P2 proline to alanine or glycine was 3-fold greater when threonine was present in the P'1 position instead of the normal serine. In contrast to the disappointing results with the P2/P'1 mutations, replacement of the P7 to P'3 residues of alpha 1-antitrypsin by those of antithrombin led to a dramatic increase in selectivity. Although this substitution only affected the kass value with thrombin by 10-fold, a 12,500-fold decrease in this value with APC was observed. Substitution of proline for the P2 glycine of this chimeric serpin increased the kass values with thrombin and APC by 7- and 90-fold, respectively. The effect of the P2 substitution was again found to depend on the sequence surrounding the residue; the change in the kass for APC caused by the P2 Pro-->Gly replacement was 6-fold larger in the chimeric serpin. Evaluation of the kass values of the chimeric serpin with a P2 proline in light of the likely rates of inhibition of thrombin and APC during antithrombotic therapy with heparin suggested that this serpin may have kinetic parameters suitable for an antithrombotic agent.

Contribution of interactions with the core domain of hirudin to the stability of its complex with thrombin.

The importance of interactions with residues 15-21 in the core domain of hirudin for the formation of the complex with thrombin has been investigated by site-directed mutagenesis. Contacts made by Leu-15 were found to be particularly important; replacement of this residue by alanine led to a decrease in the binding energy (delta delta Gbo) of 15 kJ.mol-1. Comparison with effects obtained in previous mutagenesis studies indicate that interactions with the side chain of Leu-15 contribute more to the stability of the complex than those of any other hirudin residues. Interactions with the side chains of Glu-17, Asn-20 and Val-21 also contributed significantly to binding energy; the delta delta Gbo value for these mutations was between 3 and 6 kJ.mol-1. Examination of the crystal structure of the thrombin-hirudin complex suggested the possibility that ionic interactions that would increase binding energy could be engineered by mutating Ser-19, Asn-20 and Gln-49 to acidic residues. The stability of the thrombin-hirudin complex was not, however, increased by these substitutions. The results obtained are discussed in terms of the crystal structure of the thrombin-hirudin complex.

Reduced levels of 5-HT3 receptor recognition sites in the putamen of patients with Huntington's disease.

The present study assessed 5-HT3 receptor recognition site levels in homogenates of putamen derived from patients with clinically and neurochemically diagnosed Huntington's disease or Parkinson's disease and those from age-, sex- and post-mortem delay-matched neurologically and psychiatrically normal patients to investigate the cellular location of 5-HT3 receptors in the human putamen. Specific [3H]granisetron (0.91 nM) binding (defined by ondansetron, 10 microM) was significantly reduced in putamen homogenates from eight out of ten patients with Huntington's disease compared to similar homogenates from 'control' patients (72 +/- 6 and 39 +/- 8 fmol/g wet weight, mean +/- S.E.M., n = 10 and 8, tissue from 'control' and Huntington's disease patients, respectively, P = 0.004). In contrast, specific [3H]granisetron (1.04 nM) binding levels were similar in putamen homogenates from patients with Parkinson's disease when compared to homogenates from 'control' patients. The present results indicate that at least a proportion of the 5-HT3 receptor population in the human putamen is located on neurones that have their cell bodies within this brain region and that these receptors are not primarily located on dopamine neurone terminals in the human putamen.

Effects of mutations in the hinge region of serpins.

An expression system for alpha 1-antitrypsin in Escherichia coli was developed using a T7 RNA polymerase promoter. Addition of rifampicin to inhibit the E. coli RNA polymerase after induction of the T7 RNA polymerase gene resulted in about 30% of newly synthesized protein being alpha 1-antitrypsin. This expression system was then used to examine the effect of mutations in the hinge region of alpha 1-antitrypsin on its activity. The mutations were based on ones in antithrombin III that had previously been shown to have adverse effects on activity. Mutation of Ala347 to threonine in alpha 1-antitrypsin did not affect the kinetic behavior of the protein with trypsin or human leukocyte elastase. In contrast, mutation of Gly349 to proline converted the majority of the protein into a substrate for both proteinases. The small fraction of this mutant that was active, however, had kinetic parameters that were indistinguishable from wild-type alpha 1-antitrypsin. Cleavage within the reactive-site loop of wild-type alpha 1-antitrypsin causes a conformational change in the molecules (the S-to-R transition) and results in a marked increase in heat stability. This increase in heat stability was also seen upon cleavage within the reactive-site loops of both of the alpha 1-antitrypsin mutants. The results are discussed in terms of a kinetic mechanism for serpin-proteinase interactions, in which after the formation of an initial complex the serpin partitions between the formation of a stable complex and a cleavage reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

A rapid method for the determination of taurine in biological tissue.

A rapid method for the determination of taurine is described. Based on high performance liquid chromatography (HPLC) separation, it avoids all transfers, precleaning treatments and the use of internal standards. The method consists of precolumn derivatization with O-phthaldialdehyde (OPA) followed by separation on a reversed phase styrene column using an acetonitrile-citrate buffer mixture as the eluting solvent. Naturally occurring substances, often quoted to co-migrate with taurine, are separated and the method described has been used to determine the taurine content of various tissues and blood fractions. These determinations indicate that the only satisfactory determination of blood taurine is that of whole blood.