Comparison of a novel form of zinc with zinc oxide bolus licensed for prevention of facial eczema, and interaction with copper oxide in sheep.

To evaluate a novel elemental zinc bolus compared with a registered positive control zinc oxide bolus and assess serum zinc concentrations following concomitant treatment with a capsule containing copper oxide needles. Forty Romney-cross ewes were randomly allocated in a 2 × 2 factorial design study. On Day 0, 20 ewes received novel boluses containing elemental zinc (Investigational Veterinary Product, IVP) while 20 received a zinc oxide bolus (control; CP). Half the animals in each zinc treatment group (n = 10) were treated with a copper oxide needle capsule [Copasure® - Ewe]. Weekly, from Day -7 to 56, all ewes were assessed for signs of photosensitization, and for 10 ewes from each zinc treatment groups, samples were collected for analysis of serum GGT activity, serum zinc concentrations, faecal zinc concentrations and on Days -7 and 56, liver copper concentrations. Multivariable random-effects models assessed the effects of zinc treatment, copper treatment, treatment interactions and time on all analytes. Regression models examined associations between serum and faecal zinc concentrations and GGT activity. Low spore numbers indicated low Pithomyces chartarum challenge. Serum zinc levels were significantly higher in the IVP than in the CP group [p < 0.0001] and varied by time [p < 0.001] and positively associated with faecal zinc concentration [p < 0.001]. Copper treatment did not affect serum zinc [p = 0.82] or faecal zinc [p = 0.92] concentrations. Liver copper concentrations did not differ between zinc treatment groups on Day -7 [p = 0.6] or Day 56 [p = 0.95]. Only the CP/no copper group had no increase in liver copper concentrations.

'Fit for purpose' health impact assessment: a realistic way forward.

The UK government is committed to health impact assessment (HIA) as a means of ensuring that health will be a key consideration in policy formulation and other public decision making. However there has been some debate about whether current HIA practice can reliably inform decision making. In particular consultation with stakeholders and literature reviewing, key tools used in HIA, are said to suffer from a number of conceptual and methodological problems, which can undermine the validity of the assessment. In this paper, we argue that the philosophical nature of HIA, its purpose and its contribution to the promotion of public health is still being established. We outline our own HIA practice, which is based on a broad philosophy of 'fit for purpose' i.e. what is this HIA for and what is its spatial, temporal, social and political context. We suggest that it is important to guard against unrealistic expectations and illusions of total objectivity and precision in the HIA process. HIA 'screening' is capable of delivering benefits by making policies, programmes and projects, more health conscious. Once we move beyond this basic expectation and wish to be able to make judgements about the relative health benefits of alternative courses of action, the potential resource intensiveness of the process increases considerably. Even at a high level of resource usage any conclusions reached through the HIA process will always be, in part, subjective and therefore likely to be contested. We must decide what we want, what we are prepared to legislate for and what we are prepared to pay for in the HIA process.

Linkers for improved cleavage of fusion proteins with an engineered alpha-lytic protease.

Addition of an N-terminal fusion partner can greatly aid the expression and purification of a recombinant protein in Escherichia coli. We investigated two genetically engineered proteases designed to remove the fusion partner after the protein of interest has been expressed. Recombinant human insulin-like growth factor-II (hIGF-II) has been produced from E. coli-derived fusion proteins using a novel enzymatic cleavage system that uses a mutant of alpha-lytic protease. Initially, two potential fusion protein linkers were designed, Pro-Ala-Pro-His (PAPH) and Pro-Ala-Pro-Met (PAPM), and were tested as substrates in the form of synthetic dodecapeptides. Using mass spectrometry and reverse-phase HPLC, the position of cleavage was confirmed and the kinetics of synthetic peptide cleavage were examined. Use of the linkers in hIGF-II fusion proteins produced in E. coli was then evaluated. The fusion proteins constructed consist of the first 11 amino acids of porcine growth hormone linked N-terminally to hIGF-II by six amino acids that include the dipeptide Val-Asn followed by a variable tetrapeptide protease cleavage motif. Mass spectrometry and N-terminal sequencing confirmed that proteolytic cleavage of the fusion proteins had occurred at the predicted sites. Using the fusion proteins as substrates, the cleavage of the rationally designed motifs by the alpha-lytic protease mutant was compared. The fusion protein containing the motif PAPM had a k(cat)/K(M) ratio indicating a 1.6-fold preference over the PAPH fusion protein for cleavage by this enzyme. Furthermore, when hIGF-II fusion proteins containing the designed cleavable linkers were processed with the engineered alpha-lytic protease, they gave greatly improved yields of native hIGF-II compared to an analogous fusion protein cleaved by H64A subtilisin. Comparison of the peptide and protein cleavage studies shows that the efficient proteolysis of the cleavage motifs is an inherent property of the designed sequences and is not determined by secondary or tertiary structure in the fusion proteins.

How women receiving adjuvant chemotherapy for breast cancer cope with their treatment: a risk management perspective.

Retrospective interviews were undertaken with 12 women who had received an 18-week course of adjuvant chemotherapy for positive node breast cancer 1 year previously, and who had not experienced cancer recurrence. The nonstandardized interviews covered women's preconceptions about adjuvant chemotherapy, their information needs, and the impact of treatment. The qualitative data analysis drew upon the theoretical ideas of patient career, trajectory projection and qualitative risk analysis. Some women regarded adjuvant chemotherapy as no more than an 'insurance policy'. This perception may have arisen because doctors, attempting to minimize patient anxiety, did not discuss the high risk of disease recurrence which they faced. Other women equated adjuvant with curative chemotherapy, and anticipated hair loss or almost certain death. The women tried to cope with the physical and mental suffering associated with adjuvant chemotherapy through normalizing strategies, such as keeping a brave face, maintaining previous patterns of life, looking for humour and restructuring time. However, the rapid alterations in physical and mental state resulting from cycles of adjuvant chemotherapy resulted in a 'rollercoaster' experience for women which made normalization more difficult. Health professionals caring for women who must cope with uncertain future trajectories need to manage a risk communication dilemma. A strategy of fully informing women about the risks they face may cause anxiety or depression, and even impede recovery, given the evidence for psychological influences on health outcomes. But, if women do not understand the medical thinking on which their treatment is based, their misconceptions may be equally damaging.

Probing the disulfide folding pathway of insulin-like growth factor-I.

The crucial step of folding of recombinant proteins presents serious challenges to obtaining the native structure. This problem is exemplified by insulin-like growth factor (IGF)-I which when refolded in vitro produces the native three-disulfide structure, an alternative structure with mispaired disulfide bonds and other isomeric forms. To investigate this phenomenon we have examined the refolding properties of an analog of IGF-I which contains a 13-amino acid N-terminal extension and a charge mutation at position 3 (Long-[Arg3]IGF-I). Unlike IGF-I, which yields 45% of the native structure and 24% of the alternative structure when refolded in vitro, Long-[Arg3]IGF-I yields 85% and 10% of these respective forms. To investigate the interactions that affect the refolding of Long-[Arg3]IGF-I and IGF-I, we acid-trapped folding intermediates and products for inclusion in a kinetic analysis of refolding. In addition to non-native intermediates, three native-like intermediates were identified, that appear to have a major role in the in vitro refolding pathway of Long-[Arg3]IGF-I; a single-disulfide Cys18-Cys61 intermediate, an intermediate with Cys18-Cys61 and Cys6-Cys48 disulfide bonds and another with Cys18-Cys61 and Cys47-Cys52 disulfide bonds. Furthermore, from our kinetic analysis we propose that the Cys18-Cys61, Cys6-Cys48 intermediate forms the native structure, not by the direct formation of the last (Cys47-Cys52) disulfide bond, but by rearrangement via the Cys18-Cys61 intermediate and a productive Cys18-Cys61, Cys47-Cys52 intermediate. In this pathway, the last disulfide bond to form involves Cys6 and Cys48. Finally, we apply this pathway to IGF-I and conclude that the divergence in the in vitro folding pathway of IGF-I is caused by non-native interactions involving Glu3 that stabilize the alternative structure.

Optimization of the hydroxylamine cleavage of an expressed fusion protein to produce recombinant human insulin-like growth factor (IGF)-I.

The application of gene fusion technology for the production of heterologous proteins in Escherichia coli has required the development of specific cleavage methods to separate the coexpressed fusion protein partner from the protein of interest. When hydroxylamine is used to cleave Asn-Gly fusion protein linkages, undesirable chemical modification of asparagine and glutamine amino acids can also occur. In this study, hydroxylamine cleavage conditions were modified to minimize unwanted chemical heterogeneity that occurred during the cleavage of the fusion protein [Met(1)]-pGH(1-11)-Val-Asn-IGF-I (Long-IGF-I). The cleavage reaction was shown to be dependent on the hydroxylamine concentration, temperature, and pH. Optimal cleavage conditions were identified that resulted in very low levels of chemical heterogeneity, but under these mild conditions that cleavage of the labile Asn-Gly bond was reduced. Therefore, the reaction was further modified to improve the yield of IGF-I while minimizing chemical heterogeneity. The yield of unmodified IGF-I was improved from less than 25% to greater than 70%. Analysis of the heterogeneity produced using the modified cleavage technique showed that Asn(26) was converted to a hydroxamate. This variant was characterized in refolding and biological assays where it was equivalent to IGF-I. To further assess the effectiveness of the modified cleavage technique and to evaluate the potential for process scale-up, a gram-scale cleavage reaction of Long-IGF-I was carried out. The process yielded IGF-I with a low level of chemical heterogeneity that was easily removed by ion-exchange chromatography. Moreover, this work shows that the production of unmodified IGFs using hydroxylamine cleavage of fusion proteins is facilitated using the mild cleavage reaction.

Mutations in the B-domain of insulin-like growth factor-I influence the oxidative folding to yield products with modified biological properties.

The oxidative folding of human insulin-like growth factor (IGF)-I yields two major disulphide folding isomers. In the present study, B-domain analogues of IGF-I were used to investigate the effect of mutations on the folding reaction and to investigate the functional implications of misfolding. The analogues used were substitutions of the native Glu3 by Gly or Arg, or the native Glu9 by Lys. IGF-I and these analogues were also prepared attached to a hydrophobic 13-amino-acid N-terminal extension, Met-Phe-Pro-Ala-Met-Pro-Leu-Ser-Ser-Leu-Phe-Val-Asn, referred to as 'Long-IGF-I' analogues. Each IGF was fully reduced and refolded to yield native and misfolded isomers, which were subsequently purified for biological characterization. Analysis of the folding reaction at equilibrium revealed a distribution of folding isomers characteristic for each peptide. The yield of the native disulphide folding isomer was increased for the Glu3 substitutions, but not for the Glu9 substitution. The main alternative folding isomer was present in the IGF-I analogues in reduced proportions. Except for [Gly3]IGF-I the N-terminal extension increased the yield of the native isomer which was maximal for the analogue Long-[Arg3]IGF-I. A folding intermediate for the latter analogue was isolated and partially characterized. The biological assays showed that all the main alternative isomers bound poorly to IGF-binding proteins (IGFBPs) secreted by L6 myoblasts. Moreover, these isomers bound to the type 1 IGF receptor with 0.5-25% the affinity of the native isomer. In a rat L6 myoblast protein-synthesis assay, the observed biological activity of the native and main alternative isomers was explained by their modified IGFBP- and receptor-binding properties. We propose that the N-terminal extension imparts a steric constraint at a crucial point in folding, thus allowing native disulphide bonds to form efficiently.

Purification and characterization of S1 mutants of alpha-lytic protease having altered catalytic properties.

A procedure is described for purifying alpha-lytic protease and its mutants from culture supernatants of recombinant Escherichia coli. The method affords substantial amounts (approx. 80 mg) of homogeneous enzyme. We compared the cleavage preferences of wild-type alpha-lytic protease and of mutants containing the substitutions Ala190 ("parent"), Ala190/Val192/His213/Met218 (mutant 1), Ala190/His213/Leu218 (mutant 9), and Ala190/Thr213/Leu218 (mutant 55), and for each enzyme we found broad agreement between the results obtained with synthetic ester and amide substrates. Kinetic constants were determined for the purified enzymes using selected tetrapeptide p-nitroanilide substrates. Mutant 55 had broad specificity and high activity. In terms of kcat/Km it cleaved at Met and Phe residues two to three times as effectively as the Ala190 enzyme and cleaved at Ala 7 times more effectively than the wild-type protease. The Ala190/His213 enzymes showed a preference for cleavage at His and Met residues. Not only were their kcat values for cleavage at His increased (in relation to the Ala190 enzyme) by an order of magnitude, but they also exhibited large decreases in kcat/Km for cleavage at other residues; for example, the value for cleavage at Phe was 400- to 600-fold lower. Mutant 9 cleaved a recombinant IGF-II fusion protein at a unique His residue and also at a nearby Asn residue.

Insulin-like growth factor (IGF)-II binding to IGF-binding proteins and IGF receptors is modified by deletion of the N-terminal hexapeptide or substitution of arginine for glutamate-6 in IGF-II.

Recombinant insulin-like growth factor-II (IGF-II) and two structural analogues, des(1-6)IGF-II and [Arg6]-IGF-II, were produced to investigate the role of N-terminal residues in binding to IGF-binding proteins (IGFBPs) and hence the biological properties of the modified peptides. The growth factors were modelled on two previously characterized variants of IGF-I, des(1-3)IGF-I and [Arg3]-IGF-I, which both show substantially decreased binding to IGFBPs and were expressed as fusion proteins in Escherichia coli. The biological activities of the corresponding analogues of IGF-I and IGF-II were compared in rat L6 myoblasts and H35B hepatoma cells. In the L6-myoblast protein-synthesis assay, the IGF-II analogues, des(1-6)IGF-II and [Arg6]-IGF-II, were slightly more potent than IGF-II but about 10-fold less potent than IGF-I and 100-fold less potent than the respective IGF-I analogues, des(1-3)IGF-I and [Arg3]IGF-I. In H35 hepatoma cells the anabolic response measured was the inhibition of protein breakdown, and the potency order was insulin >>> [Arg3]-IGF-I > des(1-3)IGF-I > [Arg6]-IGF-II > des(1-6)IGF-II > IGF-I > IGF-II. Binding of the IGFs and their analogues to the type 1 IGF receptor in L6 myoblasts and to the insulin receptor in H35 hepatoma cells did not fully explain the observed anabolic potency differences. Moreover, binding of all four analogues to the IGFBPs secreted by L6 myoblasts and H35B hepatoma cells was greatly decreased compared with the parent IGF. We conclude that the observed anabolic response to each IGF was determined by their relative binding to the competing cell receptor and IGFBP binding sites present.

Novel recombinant fusion protein analogues of insulin-like growth factor (IGF)-I indicate the relative importance of IGF-binding protein and receptor binding for enhanced biological potency.

An efficient expression system in Escherichia coli for several biologically active insulin-like growth factor-I (IGF-I) fusion peptide analogues is described. These novel IGF-I fusion protein analogues have properties that make them very useful reagents in the investigation of IGF-I action. The analogues comprise an IGF-I sequence and the first 11 amino acids of methionyl porcine growth hormone (pGH) and include [Met1]-pGH(1-11)-Val-Asn-IGF-I, which contains the authentic IGF-I sequence, and two analogues, [Met1]-pGH(1-11)-Val-Asn-[Gly3]-IGF-I and [Met1]-pGH(1-11)-Val-Asn-[Arg3]-IGF-I, where Glu-3 in the human IGF-I sequence has been replaced by Gly or Arg respectively. The three peptides are referred to as Long IGF-I, Long [Gly3]-IGF-I or Long [Arg3]-IGF-I depending on the IGF-I sequence present. Production of the purified fusion peptides was aided by folding the reduced and denatured fusion peptide sequence under conditions that gave very high yields of biologically active product. Introduction of a hydrophobic N-terminal extension peptide appears to facilitate the correct folding of the IGF-I analogues compared with that obtained previously when folding normal-length IGFs. The biological activities of the IGF-I fusion peptides were compared with authentic IGF-I and the truncated analogue, des(1-3)IGF-I. In L6 rat myoblasts, all the analogues were more potent than authentic IGF-I in their abilities to stimulate protein and DNA synthesis and inhibit protein breakdown. In H35 hepatoma cells, where the IGFs act through the insulin receptor, the Long IGF-I analogues maintained a similar potency relative to IGF-I as was observed in the L6 myoblasts. The order of biological potency in cell lines secreting IGF-binding proteins (IGFBPs) into the medium was Long [Arg3]-IGF-I-des(1-3)IGF-I greater than Long [Gly3]-IGF-I greater than Long IGF-I greater than IGF-I. In chicken embryo fibroblasts, a cell line that does not secrete detectable IGFBPs into the medium, Long [Arg3]-IGF-I, was less potent than IGF-I. Investigation of receptor and IGFBP association by these analogues reinforced our previous findings that N-terminal analogues of IGF-I show increased biological potency due to changes in the degree of their IGFBP interactions.

Does scrotal ultrasound reduce the need for orchidectomy in the clinically malignant testis?

Many recent studies have confirmed the ability of ultrasound to elucidate scrotal pathology, but its place in the work-up of specific testicular problems is not yet fully defined. We investigated 15 cases where a firm clinical diagnosis of testicular malignancy was made. All cases progressed to orchidectomy. Scrotal ultrasound refuted the diagnosis of malignancy in seven cases with subsequent histological proof of benignity. However, the nature and extent of the benign disease was such that, on review, orchidectomy was considered to be the most appropriate method of treating the patient. We contend that in the case of firm clinical diagnosis of testicular malignancy, an ultrasound diagnosis of benignity will not alter management, and it is therefore not an essential part of the work-up in this group of patients.