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The technique of two-dimensional electrophoresis (2-DE) has been under investigation for its usefulness in identifying protein markers for wool quality traits in sheep. However, before this could be achieved, unique problems relating to the detection and quantitation of wool proteins needed to be overcome so that 2-DE protein maps could be examined using computational programs like Melanie II. Four protein staining regimes were examined. Colloidal Coomassie Blue G-250 was found to be superior to Coomassie Blue R-250 and gave satisfactory staining of all protein classes. Silver staining detects minor strings of keratinous proteins, but unfortunately it negatively stains intermediate filament proteins, the major high sulphur proteins (HSPs) and the high glycine tyrosine proteins and the latter two classes can only be seen by overstaining the background of the gel. In contrast, labeling reduced keratins with [ 14 C]iodoacetamide, followed by autoradiography detection, results in a protein map with low background and all protein spots stained positively. 2-DE has been used to obtain wool protein maps of Lincoln/Merino chimeric sheep to examine wool originating from two genotypes grown with different crimp frequencies within the same fleece. Between fleece, variations have also been examined. Work to date suggests that several major HSPs may be associated with the fibre curvature trait known as crimp frequency. From matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectral mapping, one of these proteins has been identified as being from the B2A family from the HSP class.

The di!erent rates of cleavage by chymosin of -casein variants has been studied. Di!erences between the C variant and the A and B variants are attributed to the direct e!ect of the Arg to His substitution at position 97 in the -casein sequence.

Aims: Limited information is available on the germination triggers for spores of nonproteolytic Clostridium botulinum. An automated system was used to study the effect of a large number of potential germinants, of temperature and pH, and aerobic and anaerobic conditions, on germination of spores of non-proteolytic Cl. botulinum types B, E and F. Methods and Results: A Bioscreen analyser was used to measure germination by decrease in optical density. Results were con®rmed by phase-contrast light microscopy. Spores of strains producing type B, E and F toxin gave similar results. Optimum germination occurred in L L-alanine/L L-lactate, L L-cysteine/L L-lactate and L L-serine/L L-lactate (50 mmol l )1 of each). A further 12 combinations of factors induced germination. Sodium bicarbonate, sodium thioglycollate and heat shock each enhanced germination, but were not essential. Germination was similar in aerobic and anaerobic conditions. The optimum pH range was 5á5±8á0, germination occurred at 1±40°C, but not at 50°C, and was optimal at 20±25°C. Conclusions: The automated system enabled a systematic study of germination requirements, and provided an insight into germination in spores of non-proteolytic Cl. botulinum. Signi®cance and Impact of the Study: The results extend understanding of germination of non-proteolytic Cl. botulinum spores, and provide a basis for improving detection of viable spores.

Comparative two-dimensional electrophoretic (2-DE) studies were performed over a time-course to examine the effect of oxidation or alkylation on the separation of wool keratin proteins. The effect of oxidation was followed by treating scoured wool fibres with increasing levels of hydrogen peroxide, ranging from 0-12 g/L, using conditions mimicking the industrial wool bleaching process. Peroxide treatment was found to have only a minor effect on the 2-DE separation of the intermediate filament protein (IFP) class. Conversely, peroxide treatment of the 24-28 kDa high sulphur protein (HSP) class, which contains up to 40 cysteine residues per protein, resulted in the gradual disappearance of the major HSP spots correlated with the appearance of a few discrete spots at lower isoelectric point (pI). This suggested that only a few specific cysteine residues were being oxidized to cysteic acid by treatment with hydrogen peroxide. Peroxide treatment also appeared to have affected a discrete number of cysteine residues among proteins in the high glycine-tyrosine protein (HGTP) class, reducing the intensity of the high pI spots, while correspondingly increasing the intensity of those at lower pI. In a separate study, wool proteins were alkylated with iodoacetamide (1 M, pH 8) for periods ranging from 10 min to 48 h. In contrast to treatment with peroxide, the pI values of the HSP spots were unaffected by alkylation, irrespective of the length of this treatment. Alkylation resulted in a shift to lower pI and a loss of resolution of individual spots in the Type I and II IFP trains, to the extent that after 24 h alkylation individual spots in these trains merged. In addition after 1 h the intensity of the high pI Type II IFPs decreased until they were no longer visible on the 2-DE map after 24 h. Similarly as alkylation time increased, the major, high pI HGTP spots decreased in intensity. In unison with their decrease, some of the lower pI spots increased in intensity, while new spots appeared at more acidic pIs. Mass spectral studies indicated that cysteine alkylation was relatively fast, with 70-95% of the cysteines in the keratin proteins being alkylated within the first 10 min, while in the case of the HGTPs there was evidence for noncysteine alkylation occurring within this period. Alkylation of proteins for periods of up to 6 h prior to electrofocusing is being promoted as a better alternative to the current 2-DE protocol of the inclusion of a reductant in the immobilized pH gradient rehydration solution. This study has clearly demonstrated that long alkylation times do not suit all protein types or classes. Abbreviations: HGTP, high glycine-tyrosine protein; HSP, high sulphur protein; IAM, iodoacetamide; IFP, intermediate filament protein; TBP, tributylphosphine 942 Proteomics 2003, 3, 942-950 Proteomics 2003, 3, 942-950

Listeria monocytogenes is an organism associated with a wide range of foods. It causes listeriosis, a severe illness that mainly affects people with weakened immune systems. Proteomic profiles of three different L. monocytogenes isolates were studied using 1D SDS PAGE, 2DE and mass spectrometry. The protein banding patterns generated by 1D SDS PAGE of three strains of L. monocytogenes were found to be similar. Visual observations from 2DE gel maps revealed that certain spots appeared to have intensity differences. Key differences in proteins synthesis of three strains of L. monocytogenes were found using the PDQest TM 2DE Analysis software. Comparison showed that the clinical isolate (strain SB92/844) had 53.4% and 53.9% protein profile similarity with dairy isolate (strain V7) and seafood isolate (SB92/870), respectively. The identity of selected protein spots was achieved using MALDI-TOF and ion trap mass spectrometry. It was found that certain identified proteins (i.e., a major c...

Comparative two-dimensional electrophoretic (2-DE) studies were performed over a time-course to examine the effect of oxidation or alkylation on the separation of wool keratin proteins. The effect of oxidation was followed by treating scoured wool fibres with increasing levels of hydrogen peroxide, ranging from 0-12 g/L, using conditions mimicking the industrial wool bleaching process. Peroxide treatment was found to have only a minor effect on the 2-DE separation of the intermediate filament protein (IFP) class. Conversely, peroxide treatment of the 24-28 kDa high sulphur protein (HSP) class, which contains up to 40 cysteine residues per protein, resulted in the gradual disappearance of the major HSP spots correlated with the appearance of a few discrete spots at lower isoelectric point (pI). This suggested that only a few specific cysteine residues were being oxidized to cysteic acid by treatment with hydrogen peroxide. Peroxide treatment also appeared to have affected a discrete number of cysteine residues among proteins in the high glycine-tyrosine protein (HGTP) class, reducing the intensity of the high pI spots, while correspondingly increasing the intensity of those at lower pI. In a separate study, wool proteins were alkylated with iodoacetamide (1 M, pH 8) for periods ranging from 10 min to 48 h. In contrast to treatment with peroxide, the pI values of the HSP spots were unaffected by alkylation, irrespective of the length of this treatment. Alkylation resulted in a shift to lower pI and a loss of resolution of individual spots in the Type I and II IFP trains, to the extent that after 24 h alkylation individual spots in these trains merged. In addition after 1 h the intensity of the high pI Type II IFPs decreased until they were no longer visible on the 2-DE map after 24 h. Similarly as alkylation time increased, the major, high pI HGTP spots decreased in intensity. In unison with their decrease, some of the lower pI spots increased in intensity, while new spots appeared at more acidic pIs. Mass spectral studies indicated that cysteine alkylation was relatively fast, with 70-95% of the cysteines in the keratin proteins being alkylated within the first 10 min, while in the case of the HGTPs there was evidence for noncysteine alkylation occurring within this period. Alkylation of proteins for periods of up to 6 h prior to electrofocusing is being promoted as a better alternative to the current 2-DE protocol of the inclusion of a reductant in the immobilized pH gradient rehydration solution. This study has clearly demonstrated that long alkylation times do not suit all protein types or classes. Abbreviations: HGTP, high glycine-tyrosine protein; HSP, high sulphur protein; IAM, iodoacetamide; IFP, intermediate filament protein; TBP, tributylphosphine 942 Proteomics 2003, 3, 942-950 Proteomics 2003, 3, 942-950

The stability of the casein micelle is dependent on the presence of k-casein ( C N ) on the surface of the micelle where it functions as an interface between the hydrophobic caseins of the micelle interior and the aqueous environment. k-Casein is also involved in thiol-catalyzed disulfide interchange reactions with the whey proteins during heat treatments and, after rennet cleavage, in the facilitation of micelle coagulation. These functions of k-CN are regulated by the three-dimensional structure of the protein on the micelle surface. The usual means of determining structure are not available for k-CN because this protein is strongly self-associating and has never been crystallized. Instead, algorithms were used to predict selected secondary structures and circular dichroism spectroscopy on k-CN and the macropeptide released by chymosin. Three peptides were synthesized to cover the chymosin-sensitive site (His 98 -Lys 111 ) , the region in the macropeptide that could be helical (Pro 130 -Ile 153 ) , and the region between. Nuclear magnetic resonance spectroscopy showed that the peptide His 98 -Lys 111 was probably a b-strand with tight turns at each end. This hypothesis was confirmed by a study of the molecular dynamics showing that the C variant of k-CN interacted less strongly with chymosin; consequently, the slow renneting time of milk that contains this protein was explainable. Both circular dichroism and nuclear magnetic resonance indicated that the peptide Pro 130 -Ile 153 was probably helical under normal physiological conditions. A preliminary study using nuclear magnetic resonance showed that the intervening peptide had no discernible secondary structure. Consequently, most of the b-sheet structure of k-CN is likely in the para-k-CN region.

Asthma is known to be a Th2 inflammatory syndrome that leads to intermittent airway obstruction. However, the mechanisms involved in development of the clinical features remain enigmatic, although genetic elements clearly are involved. Recently, based on a large genomewide screen involving families in the United Kingdom and the United States with at least two siblings with asthma, a locus was identified that encoded for a family of proteases. This group of proteins is now known as the ADAM superfamily. In this review, we discuss the ADAM superfamily and, in particular, ADAM 33, a member of a family of genes which encode a subgroup of zinc dependent metalloproteinase (metzincin). The potential for therapeutic intervention with ADAM 33 is extremely attractive and further work will not only focus on the specific domains of ADAM 33, but also the mechanisms by which they lead to bronchial hyperreactivity.

Ozone is a major air pollutant with adverse health effects which exhibit marked inter-individual variability. In mice, regions of genetic linkage with ozone-induced lung injury include the tumor necrosis factor-␣ (TNF), lymphotoxin-␣ (LTA), Toll-like receptor 4 (TLR4), superoxide dismutase (SOD2), and glutathione peroxidase (GPX1) genes. We genotyped polymorphisms in these genes in 51 individuals who had undergone ozone challenge. Mean change in FEV 1 with ozone challenge, as a percentage of baseline, was Ϫ3% in TNF Ϫ308G/A or A/A individuals, compared with Ϫ9% in G/G individuals (p ϭ 0.024). When considering TNF haplotypes, the smallest change in FEV 1 with ozone exposure was associated with the TNF haplotype comprising LTA ϩ252G/TNF Ϫ1031T/TNF Ϫ308A/TNF Ϫ238G. This association remained statistically significant after correction for age, sex, disease, and ozone concentration (p ϭ 0.047). SOD2 or GPX1 genotypes were not associated with lung function, and the TLR4 polymorphism was too infrequent to analyze. The results of this study support TNF as a genetic factor for susceptibility to ozoneinduced changes in lung function in humans, and has potential implications for stratifying health risks of air pollution.

Rationale: Asthma commonly originates in early life in association with impaired lung function, which tracks to adulthood. Objectives: Within the context of a prospective birth cohort study, we investigated the association between single nucleotide polymorphisms (SNPs) in a disintegrin and metalloprotease 33 (ADAM33) gene and early-life lung function. Methods: Children were genotyped for 17 SNPs in ADAM33. Lung function at age 3 (n ϭ 285) and 5 years (n ϭ 470) was assessed using plethysmographic measurement of specific airway resistance (sRaw). At age 5, we also measured FEV 1. SNPs were analyzed individually using logistic regression, followed by linkage disequilibrium mapping to identify the causal locus. Main Results: Carriers of the rare allele of Fϩ1 SNP had reduced lung function at age 3 years (p ϭ 0.003). When the recessive model was considered, four SNPs (Fϩ1, S1, STϩ5, V4) showed association with sRaw at age 5 years (p Ͻ 0.04). Using linkage disequilibrium mapping, we found evidence of a significant causal location between BCϩ1 and F1 SNPs, at the 5Ј end of the gene. Four SNPs were associated with lower FEV 1 (Fϩ1, Mϩ1, T1, and T2; p р 0.04). The risk of transient early wheezing more than doubled among children homozygous for the A allele of Fϩ1 (odds ratio, 2.39; 95% confidence intervals, 1.18-4.86; p ϭ 0.02), but there was no association between any SNP and allergic sensitization or physician-diagnosed asthma. Conclusions: Polymorphisms in ADAM33 predict impaired early-life lung function. The functionally relevant polymorphism is likely to be at the 5Ј end of the gene.

Background The cysteinyl-leukotriene receptor type 1 (CysLT 1) mediates the bronchoconstrictor and pro-inflammatory actions of cysteinyl-leukotrienes (LTC 4 , LTD 4 , LTE 4) in asthma and is the molecular target of the lukast class of oral anti-leukotriene drugs. We screened the CYSLTR1 gene on chromosome Xq13-21 for coding region polymorphisms, and investigated their associations with allergy and asthma. Methods Solid-phase chemical cleavage was used to screen polymorphisms in the coding region of CYSLTR1. A TaqMan allelic discrimination assay was used to genotype a 927T/C SNP and oligonucleotide ligation assays were used to genotype the previously reported 617T/C and 898G/A SNPs of CYSLTR1 in 341 asthmatic families from the UK. Associations with asthma diagnosis, atopic status, serum-specific IgE and severity of allergy and asthma were examined. Results Family-based association tests showed that the 927 T allele was associated with atopy severity, especially in female subjects, but not with asthma diagnosis or severity, atopic status, bronchial hyper-responsiveness to methacholine or forced expiratory volume in 1 s. Conclusion Mutation screening identified only one polymorphism, 927T/C, in the coding region of the CysLT 1 receptor. This polymorphsim is predictive of atopy severity, but not associated with asthma.

Endotoxin exposure may have a protective effect against asthma and atopy. An Asp299Gly polymorphism in the Toll-like receptor 4 (TLR4) gene reduces responsiveness to endotoxin. This study determined the effect of TLR4 polymorphism on the risk and severity of asthma and atopy. In all, 336 UK Caucasian families with Z2 affected sibs (physician's diagnosis of asthma and current medication use) and 179 Caucasians without asthma or a family history of asthma were genotyped using ARMS-PCR. No association of the TLR4 polymorphism was found with the risk of developing asthma, either in parent-affected sibling trios, or in case-control analyses (P40.05). In the first affected asthmatic siblings, the atopy severity score (based on size and number of positive skin-prick tests and specific IgE) was higher in those with the Asp/Gly or Gly/Gly genotypes (mean 1.8, s.d. 1.1, n¼39) compared to those with the Asp/Asp genotype (mean 1.2, s.d. 1.0, n¼279) (P¼0.003, t-test). No associations were found with total IgE, FEV 1 % predicted, slope of FEV 1 response to methacholine or asthma severity score (P40.05). This study confirms the previously observed lack of association of TLR4 polymorphisms with asthma. In contrast, the findings suggest that genetically determined hyporesponsiveness to endotoxin may increase atopy severity.

RATIONALE: Specific airway resistance (sR aw), is a sensitive and reproducible measure of lung function in young children-higher sR aw indicating poorer lung function. We investigated the relationship between single nucleotide polymorphisms (SNPs) in ADAM33 (a disintegrin and metalloprotease 33; Nature 2002;418:426-30) and sR aw at age 5 years in the setting of a prospective birth cohort study. METHODS: Children (n=504) were gentopyed for 17 SNPs spanning 11Kb of the ADAM33 gene. Lung function at age 5 years was assessed using plethysmographic measurement of sR aw at baseline and after bronchodilator (400 µg albuterol). Results were analyzed using regression analysis. RESULTS: Four SNPs showed significant association with sR aw. Children who were homozygous for the rare allele of the F+1 SNP had a significantly higher sR aw than the heterozygotes and wild type homozygotes (kPa/s, GM [95% CI] 1.26 [1.18-1.34] vs 1.15 [1.13-1.17], p=0.0008). Carriers of the rare allele for ST+5 had a lower sR aw than the wild type homozygotes (kPa/s, GM [95% CI] 1.14 [1.12-1.16] vs 1.19 [1.16-1.23], p=0.007). Children who were mutant homozygotes for V-1 (n=5) and T2 (n=4) had significantly higher sR aw values. After administration of the bronchodilator, the association between F+1, V-1 and T2 and sR aw was lost. However, for ST+5, carriers of the rare allele still had significantly lower sR aw (kPa/s GM [95%CI] 0.96 [0.94-0.98] vs 1.01 [0.98-1.04], p=0.005). CONCLUSIONS: We have confirmed an association between polymorphisms in ADAM33 and lung function in early life, suggesting a role for ADAM33 in asthma pathogenesis.

Antibiotics are a vital and commonly used therapeutic tool, but their use also results in profound changes in the intestinal microbiota that can, in turn, have significant health consequences. Understanding how the microbiota recovers after antibiotic treatment will help to devise strategies for mitigating the adverse effects of antibiotics. Using a mouse model, we have characterized the changes occurring in the intestinal microbiota immediately after five days exposure to ampicillin, and then at three and fourteen days thereafter. During the fourteen day period of antibiotic recovery, groups of mice were fed either water, cows’ milk containing high levels of IgA, or cows’ milk containing low levels of IgA as their sole source of liquid. Effects on microbiota of feeding milks for 14 days were also assessed in groups of mice that had no ampicillin exposure. Changes in microbiota were measured by high throughput sequencing of the V4 to V6 variable regions of the 16S ribosomal RNA gene...