A turning point: Head and neck cancer patients' exercise preferences and barriers before and after participation in an exercise intervention.

This study examined the exercise barriers and preferences of head and neck cancer (HNC) survivors in relation to exercise experience. Participants (n = 22; 46.8% response rate) completed retrospective self-report questionnaires on demographic and medical information, exercise barriers and preferences. A subset of participants then completed semi-structured interviews (n = 18). Participants had previously engaged in the ENHANCE trial during, or immediately following, radiation treatment, an average of 22.1 ± 5.8 months before. Retrospective questionnaires revealed that before ENHANCE participation, lack of interest and time were the primary exercise barriers. After participation, there was a significant decrease in typical barriers including lack of interest (p = .008), exercise not a priority (p = .039) and exercise not in routine (p = .004). Number of barriers experienced after ENHANCE participation was negatively correlated with age, quality of life and minutes of resistance exercise training per week. After ENHANCE participation, significant increases were found in preference for exercising at a cancer centre (p = .031) and with other cancer survivors (p = .016). Four higher order themes emerged inductively from interview data analysis pertaining to preferences (i.e., class format) and three higher order themes regarding barriers (physical, psychological and external). By investigating participants' perspectives after ENHANCE participation, key factors for effective HNC exercise programme design were identified.

Prediction of adherence to a gluten-free diet using protection motivation theory among adults with coeliac disease.

BACKGROUND: Coeliac disease is a chronic autoimmune disease that requires strict adherence to a gluten-free diet. However, strict adherence to a gluten-free diet is difficult, with findings from a recent review suggesting that up to 42% of individuals with coeliac disease do not eat a strict gluten-free diet. METHODS: The present study aimed to examine psychosocial predictors of adherence (purposeful and accidental) to a gluten-free diet among adults with coeliac disease over a 1-month period. In this longitudinal study, 212 North American adults with coeliac disease completed online questionnaires at two time points, baseline and 1 month later. RESULTS: The results revealed that intentions partially mediated the effects of symptom severity, self-regulatory efficacy, planning and knowledge on purposeful gluten consumption. Intentions did not mediate the effects of severity, response cost, self-regulatory efficacy, planning and knowledge for accidental gluten consumption but, interestingly, self-regulatory efficacy directly predicted fewer accidental incidents of gluten-consumption. CONCLUSIONS: These findings delineate the differential psychological processes in understanding accidental and purposeful gluten consumption among adults with coeliac disease and emphasise the importance of bolstering self-regulatory efficacy beliefs to prevent accidental and purposeful consumption of gluten.

Motives for adherence to a gluten-free diet: a qualitative investigation involving adults with coeliac disease.

BACKGROUND: Currently , the only treatment for coeliac disease is life long adherence to a strict gluten-free diet. Strict adherence to a gluten-free diet is challenging, with recent reports suggesting that adherence rates range from 42% to 91%. The present study aimed to: (i) identify motives for adhering to a gluten-free diet and (ii) explore factors implicated in adherence and non-adherence behaviour in terms of accidental and purposeful gluten consumption among adults with coeliac disease. METHODS: Two hundred and three adults with coeliac disease completed an online questionnaire. Using a qualitative design, relationships were examined between reported adherence and motivation to follow a gluten-free diet, as well as the onset, duration and severity of symptoms. RESULTS: Feelings of desperation ('hitting rock bottom') and needing to gain or lose weight were associated with the strictest adherence to a gluten-free diet. Participants who accidentally consumed gluten over the past week developed symptoms the most quickly and reported the most pain over the past 6 months. Participants who consumed gluten on purpose over the past week reported a shorter duration of symptoms and less pain over the past 6 months. CONCLUSIONS: Hitting rock bottom and needing to gain or lose weight were factors associated with the strictest adherence, when considered in the context of both accidental and purposeful gluten consumption. Future research is warranted to develop resources to help people with coeliac disease follow a strict gluten-free diet.

Purification and identification of a 7.6-kDa protein in media conditioned by superinvasive cancer cells.

BACKGROUND: Selection of the human drug sensitive and invasive cell line (MDA-MB-435S-F) with the chemotherapeutic agent paclitaxel, resulted in the development of drug resistant cell lines displaying enhanced invasion-related characteristics. MATERIALS AND METHODS: Serum-free conditioned media from the human cancer drug-sensitive and invasive cell line (MDA-MB-435S-F) and its paclitaxel-resistant superinvasive variant (MDA-MB-435S-F/Taxol10p4pSI) were analyzed using Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). RESULTS: A differentially expressed protein was observed at 7.6 kDa, which was 4-fold up-regulated in MDA-MB-435S-F/Taxol10p4pSI. The differentially expressed protein was identified using matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS), as a fragment of bovine transferrin. The transferrin receptor was also found to be overexpressed in the superinvasive cell line. CONCLUSION: Cleavage of serum proteins such as transferrin could provide a valuable source of markers for malignant tumours and could also play a role in aspects of cancer pathogenesis, such as tumour cachexia.

Purification of a diagnostic, secreted cysteine protease-like protein from the hookworm Ancylostoma caninum.

The enteric infection of humans with the canine hookworm Ancylostoma caninum varies in its clinical presentation, ranging from asymptomatic to eosinophilic gastroenteritis requiring surgical intervention. Infections are not patent, but can be diagnosed immunologically by detecting antibodies to an immunodominant secreted hookworm protein termed Ac68. To characterise Ac68, we purified the native protein from A. caninum excretory/secretory products using size exclusion followed by anion exchange chromatography. The epitopes in the purified protein recognised by human infection sera were shown to be proteins and not carbohydrates. The N-terminal amino acid sequence of the purified Ac68 was determined and six of the 11 residues obtained were shared with a previously characterised cysteine protease of A. caninum, AcCP1.

Molecular modeling and substrate specificity of discrete cruzipain-like and cathepsin L-like cysteine proteinases of the human blood fluke Schistosoma mansoni.

Adult Schistosoma mansoni blood flukes express two discrete cysteine proteinases, SmCL1 and SmCL2, both of which are related to the cathepsin L-like enzymes of the C1 family of peptidases. Our previous phylogenetic analysis indicated that SmCL1 is more closely related to cruzipain from the parasitic protozoa Trypanosoma cruzi than to human cathepsin L, whereas the converse situation applies with SmCL2. To characterize their catalytic subsites and substrate specificities, we have now developed three-dimensional (3D) homology models of SmCL1 and SmCL2 using the structure of cruzipain and cathepsin L. Eisenberg analysis of the 3D models revealed self-compatibility scores of 90.1 and 96.1 out of a possible score of 97.6 for SmCL1 and SmCL2, respectively, verifying the accuracy and utility of the models. Substrate preferences of recombinant SmCL1 and SmCL2 at positions P3, P2, and P1 conformed to the substrate specificity predicted by the models. In particular, SmCL1 and SmCL2 both exhibited high affinity (k(cat)/K(m)) for substrates with hydrophobic residues at P2 including Z-Leu-Arg-NHMec (773.4 and 548.5 mM(-1) s(-1), respectively), Boc-Val-Leu-Lys-NHMec (116.8 and 306.5 mM(-1) s(-1)), and Z-Phe-Arg-NHMec (38.9 and 113.4 mM(-1) s(-1)). SmCL1 exhibited only a low affinity for the cathepsin B diagnostic substrate Z-Arg-Arg-NHMec while SmCL2 failed to cleave this substrate. The substrate specificities of SmCL1 and SmCL2 were clearly differentiated with H-Leu-Val-Tyr-NHMec and Suc-Leu-Tyr-NHMec since SmCL1 cleaved both efficiently (k(cat)/K(m) values of 51.9 and 41.1 mM(-1) s(-1), respectively), whereas SmCL2 cleaved neither. The 3D models revealed that this difference in specificity was due to restrictions imposed on the S3 subsite of SmCL2 as a result of insertion of two amino acids vicinal to residue 60.

Schistosoma mansoni: differential expression of cathepsins L1 and L2 suggests discrete biological functions for each enzyme.

Schistosoma mansoni cathepsins L1 (SmCL1) and L2 (SmCL2) were expressed as active recombinant proteinases in Saccharomyces cerevisiae. The recombinant enzymes exhibited substrate preferences characteristic of cathepsin-L-like cysteine proteinases. However, the enzymes differed in their substrate specificities; SmCL1 cleaved Boc-Val-Leu-Lys-NHMec with a higher efficiency than it cleaved Z-Phe-Arg-NHMec, whereas the opposite was true for SmCL2. The enzymes also differed in their pH profiles of activity; SmCL1 exhibited a broad pH profile with an optimum of pH 6. 5, while SmCL2 was active only in the acidic pH range with an optimum of 5.35. Immunoblot and RT-PCR analyses revealed that the native forms of both SmCL1 and SmCL2 are expressed in male and female worms, but at higher levels in adult female compared to male schistosomes. Additionally, both enzymes were observed in the excretory/secretory products of adult worms. The RT-PCR analysis indicated that neither enzyme is expressed in S. mansoni eggs or in miracidia, suggesting that the cathepsin-L-like activity that has been previously reported to be expressed in these stages may be the product of another gene(s). Cercariae do not express SmCL2, but appear to express SmCL1 in its inactive precursor form. Together with the findings of previous immunolocalization and phylogenetic analyses, the results reported here demonstrate that SmCL1 and SmCL2 are distinct cathepsin cysteine proteinases and strongly suggest that they play discrete biological roles.

Immune responses of cattle to experimental anti-Fasciola hepatica vaccines.

Fasciola hepatica infection of cattle and sheep is an important cause of clinical disease and production losses, and is controlled at present by a combination of chemotherapy and management measures. However, the prospects for the control of F. hepatica infection by vaccination are good, and we have previously shown substantial protection of cattle against experimental challenge infection following immunisation with a combination of the purified fluke-derived enzymes cathepsin L1 (CATL 1), cathepsin L2 (CATL 2) and fluke-derived Hb fraction (FHB). This and other recent studies have also demonstrated fundamental differences between protective and non-protective immune responses to liver fluke infection. In this present study we have further analysed the response of animals to liver fluke challenge following experimental vaccination. Calves were vaccinated with either CATL 2 plus FHB, or CATL 1 plus CATL 2. Partial protection against challenge infection was achieved in both vaccinated groups, with the greatest level of protection (55 per cent reduction in fluke burdens) recorded in the group vaccinated with CATL 1 plus CATL 2. This latter group also showed the greater level of lymphocyte proliferation and the greater production of gamma-INF in response to stimulation with fluke antigen in vitro following challenge. These results are significant in our attempts to characterise the elements within the immune response to vaccination which are protective.

Short report: Immunodiagnosis of human fascioliasis using recombinant Fasciola hepatica cathepsin L1 cysteine proteinase.

Our laboratory recently developed a diagnostic test (ELISA) for human fascioliasis based on the detection of serum IgG4 antibodies reactive with Fasciola hepatica cathepsin L1 (CL1). In the present study, we have used recombinant CL1, generated by functional expression of the cDNA in Saccharomyces cerevisiae, in this immunodiagnostic test and compared its performance with native CL1. Sera obtained from 64 individuals living in Cutusuma village in the northern Altiplano of Bolivia, a region with a high prevalence of human fascioliasis, were analyzed by the IgG4-ELISA. A highly statistically significant correlation (r2 = 0.751, P < 0.001) was demonstrated between the absorbances obtained using the recombinant and native proteins. These assays showed that 38 (59%) of the individuals tested were seropositive for fascioliasis, whereas only 26 of them were coprologically positive for F. hepatica eggs. All seronegative patients were also coprologically negative. Serum from individuals infected with schistosomiasis mansoni, cysticercosis, hydatidosis, and Chagas disease did not contain antibodies reactive with the recombinant or native CL1. Therefore, recombinant CL1 shows excellent potential for the development of the first standardized assay for the sensitive and specific diagnosis of human fascioliasis. Finally, our data supports earlier reports on the high prevalence of human fascioliasis in the Bolivian Altiplano, which collectively suggest that the disease has been endemic there for more than a decade.

Recombinant expression and localization of Schistosoma mansoni cathepsin L1 support its role in the degradation of host hemoglobin.

Cysteine proteinases expressed by schistosomes appear to play key roles in the digestion of host hemoglobin, the principal source of amino acid nutrients utilized by these parasites. We have shown previously that the predominant cysteine proteinase activity in soluble extracts and excretory/secretory (ES) products of adults of Schistosoma mansoni and S. japonicum is cathepsin L-like in its substrate specificity. However, biochemical analysis of the cathepsin L activity in extracts and ES products of schistosomes has been complicated by the presence of at least two distinct forms of schistosome cathepsin L, termed SmCL1 and SmCL2. We now report the purification and enzyme characteristics of active, recombinant SmCL1 which was obtained by transforming Saccharomyces cerevisiae with an expression plasmid encoding the preproenzyme of SmCL1. Recombinant SmCL1 was secreted by the transformed yeast into the culture media from which it was purified by gel filtration and ion-exchange chromatography. The purified enzyme exhibited substrate specificity against synthetic peptidyl substrates (e.g., Boc-Val-Leu-Lys-NHMec and Z-Phe-Arg-NHMec; kcat/Km = 17.25 and 6.24 mM-1 s-1, respectively) and against gelatin and hemoglobin, characteristic of cathepsin L. Immunoblot analysis using antiserum raised against recombinant SmCL1 demonstrated that native SmCL1 of 33 kDa was present in ES products and soluble extracts of S. mansoni. Using this antiserum and thin tissue sections, we localized the native SmCL1 to the gastrodermis and to the tegument of adult schistosomes. Recombinant SmCL1 was capable of degrading human hemoglobin at pH 4.0 to 4.5 but not higher, suggesting that denaturation of hemoglobin by low pH, as found in the cecum of the adult schistosome, may be necessary for its catalysis by cathepsin L and other gut-associated proteinases. Together, these results support a role for SmCL1 in the degradation of host hemoglobin within the gut of the schistosome.

Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expression in Saccharomyces cerevisiae.

Cathepsin L2 is a major cysteine proteinase secreted by adult Fasciola hepatica. The enzyme differs from other reported cathepsin Ls in that it can cleave peptide substrates that contain proline in the P2 position. A cDNA was isolated from an expression library by immunoscreening with antiserum prepared against purified native cathepsin L2. This cDNA was sequenced and shown to encode a complete preprocathepsin L proteinase. Functionally active recombinant cathepsin L proteinase was expressed and secreted by Saccharomyces cerevisiae transformed with the cDNA. The recombinant enzyme was purified from large-scale fermentation broths using ultrafiltration and gel filtration chromatography on Sephacryl S200 HR columns. NH2-terminal amino acid sequencing showed that the cleavage point for activation of the recombinant pro-enzyme is identical to that of the F. hepatica-produced cathepsin L2. The mature active recombinant proteinase behaved similarly to the native enzyme when analysed by SDS-PAGE, immunoblotting and zymography and also cleaved peptides containing proline in the P2 position. Finally, the recombinant cathepsin L2 cleaved fibrinogen to form a fibrin clot, a property we described for F. hepatica cathepsin L2.

Functional expression of Fasciola hepatica cathepsin L1 in Saccharomyces cerevisiae.

A cDNA encoding the complete precursor of a Fasciola hepatica cathepsin L protease was isolated and sequenced. Functionally active enzyme was expressed and secreted by Saccharomyces cerevisiae transformed with a plasmid carrying the complete gene. Experiments with temperature-sensitive yeast mutants showed that the enzyme is trafficked through the yeast secretory pathway. Yeast transformed with a truncated gene, which lacked the pre-peptide-encoding and most of the pro-peptide-encoding sequences, did not express funtionally active enzyme. The yeast-expressed enzyme exhibited physicochemical properties in common with the native enzyme including, pH optimum for activity, stability at 37 degrees C and ability to cleave gelatin and immunoglobulin. Enzyme kinetic data showed that the native and yeast-expressed cathepsin L1 have similar specificities for substrates with hydrophobic residues in the P2 position. This is the first report of the functional expression of a cathepsin L proteinase in S. cerevisiae that did not require the use of yeast secretory signal sequences.

Fasciola hepatica cathepsin L proteinase cleaves fibrinogen and produces a novel type of fibrin clot.

A cathepsin L proteinase secreted by the parasitic helminth Fasciola hepatica can cleave fibrinogen and produce a fibrin clot with a specific activity of 4.7 National Institutes of Health thrombin-equivalent U/mg. This is the first report of a fibrinogen-clotting activity aside that of thrombin and the snake venom proteinases, which are all serine proteinases. Clot formation by cathepsin L is not inhibited by the thrombin inhibitor hirudin or by the anti-polymerant H-Gly-Pro-Arg-Pro-OH. The enzyme exerts its activity on fibrinogen in a unique manner. Although the cleavage of fibrinogen may involve the initial removal of fibrinopeptides, additional proteolysis of the alpha, beta and gamma fibrinogen polypeptides takes place. SDS/PAGE analysis of the cathepsin-L-produced clots revealed that cleavage of the alpha polypeptide (66 kDa) precedes that of the beta (52 kDa) and gamma (46.5 kDa) polypeptides. Concurrent with the cleavage of these polypeptides is the appearance of components of 120, 100 and 25 kDa. The appearance of higher molecular-sized components in the cathepsin L clots suggests that polymerisation involves the formation of molecular interactions that are resistant to boiling in mercaptoethanol and SDS.

Simple monitoring technique for muscle flaps.

A simple monitoring technique for the detection of postoperative arterial flow failure in muscle flaps is described. The technique consists of isolating a musculocutaneous perforator on elevation of a muscle flap. This cutaneous perforator is observed for pulsation in the postoperative period. Abrupt cessation prior to 48 hours should be an indicator for prompt clinical evaluation of the muscle flap by an experienced microsurgeon to rule out arterial thrombosis.

Secretion of cysteine proteinase activity by the zoonotic hookworm Ancylostoma caninum.

The zoonotic hookworm, Ancylostoma caninum, probably induces human eosinophilic enteritis by inducing allergic responses to its secretions. This species is already known to secrete metalloproteinases, but in other parasites, cysteine proteinases are involved in pathogenesis. We studied somatic extracts of A. caninum adults and infective larvae and adult excretory/secretory (ES) antigens for cysteine proteinase activity using fluorogenic peptide substrates and by gelatin and fluorogenic substrate polyacrylamide gel electrophoresis. Proteolytic activity was observed against the cathepsins L and B-specific substrate Z-phe-arg-AMC, against the plasmin substrate Boc-val-leu-lys-AMC, and against gelatin. The Z-phe-arg-AMC-hydrolyzing activity in ES antigens and in adult extracts was enhanced up to 15-fold by the reducing agent dithiothreitol (DTT), but was totally blocked by specific inhibitors of cysteine proteinases, including the peptidyl diazomethyl ketone Z-phe-ala-CHN2,E-64, leupeptin, and N-ethylmaleimide. In a similar fashion, gelatinolytic activity in ES antigens detected using substrate gels was enhanced by the addition of reducing agents and inhibited by Z-phe-ala-CHN2 and E-64. The DTT-enhanced, Z-phe-arg-AMC-hydrolyzing activity in ES antigens was active over a wide pH range (pH 5-9). Similar cysteine proteinase activity to that detected in ES antigens was present in extracts of adult and infective larvae of A. caninum. Because the substrate Z-phe-arg-AMC was specifically hydrolyzed, and because this hydrolysis was totally blocked by cysteine proteinase-specific inhibitors, ES antigens and tissue extracts of A. caninum clearly possess cysteine proteinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterisation of a second cathepsin L proteinase secreted by the parasitic trematode Fasciola hepatica.

A 29.5-kDa cysteine proteinase was purified from medium in which mature Fasciola hepatica parasites were maintained. The N-terminal sequence (14 residues) of the purified protein is similar to known cathepsin L proteinases, including a 27-kDa cathepsin L proteinase, also secreted by this parasite, which had been isolated previously in our laboratory [Smith, A. M., Dowd, A. J., Mc Gonigle, S., Keegan, P.S., Brennan, G., Trudgett, A. & Dalton, J.P. (1993) Mol. Biochem. Parasitol. 62, 1-8]. The N-terminal sequences of the 29.5-kDa and 27-kDa cathepsin L proteinases differ only in residue number seven (arginine and proline, respectively). Immunoblot studies, using antiserum that reacts with both cathepsin L proteinases, rule out the possibility of both enzymes arising from a higher molecular sized parent molecule. The reaction kinetics of the two F. hepatica cathepsin L proteinases on a variety of peptide substrates revealed that the two enzymes differ in their substrate specificity. Five peptide substrates that are cleaved with high affinity by the 29.5-kDa cathepsin L isolated in this study are not cleaved by the previously purified 27-kDa cathepsin L. The protein-modifying reagent, tetranitromethane, affected the 29.5-kDa cathepsin L proteinase only, causing inactivation of the enzyme and changing its migration in polyacrylamide gel electrophoresis. Our studies suggest that the two F. hepatica cysteine proteinases represent two distinct subclasses within the cathepsin L class.

A dipeptidylpeptidase secreted by Fasciola hepatica.

A dipeptidylpeptidase (DPP) was isolated from Fasciola hepatica by gel-filtration and ion-exchange chromatography. The exoproteinase is secreted by newly excysted juveniles, immature and mature flukes. The liver fluke DPP is a serine proteinase of molecular weight > 200 kDa and differs from previously characterized mammalian DPPs in its substrate preference and susceptibility to inactivation by inhibitors. The parasite DPP may function in the latter stages of the proteolytic digestion of host macromolecules. In this manner, the enzyme may be important in providing the parasite with dipeptides that could be absorbed through the intestine as nutrient.

Neutralization of the activity of a Fasciola hepatica cathepsin L proteinase by anti-cathepsin L antibodies.

Fasciola hepatica secretes a cathepsin L proteinase that is suggested to play an in vivo role in immunoprotection since the enzyme can cleave host immunoglobulin. In the present report, rabbit anti-cathepsin L IgG was shown to bind to the cathepsin L enzyme and inhibit its ability to cleave IgG molecules. Cathepsin L can prevent the antibody-mediated attachment of eosinophils to newly excysted juveniles in in vitro assays; however, if anti-cathepsin L IgG are mixed with the cathepsin L prior to the addition of the enzyme to the assay, eosinophils attach to the newly excysted juveniles. Thus it is possible to prepare antibodies that can bind and disrupt the biological activity of the F. hepatica cathepsin L.

Fasciola hepatica: a secreted cathepsin L-like proteinase cleaves host immunoglobulin.

Adult Fasciola hepatica secrete a cysteine proteinase capable of cleaving host IgG close to the papain cleaving site. The proteinase was separated by size permeation chromatography. Gelatin-substrate polyacrylamide gel electrophoresis analysis revealed that the proteinase migrates as 6 proteolytic bands in the apparent molecular size range 60-90 kDa. Based on pH profiles of activity, inhibition studies using diethylpyrocarbonate and the diazomethylketone Z-phe-ala-CHN2, and characterising the substrate specificity of the enzymes using fluorogenic peptide substrates we have shown that the 60-90-kDa proteinases are cathepsin L-like proteinases.