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An 18.2 kDa protein from the liver fluke, Fasciola hepatica has been identified and characterised. The protein shows strongest sequence similarity to egg antigen proteins from Schistosoma mansoni, Schistosoma japonicum and Clonorchis sinensis. The protein is predicted to adopt a calmodulin-like fold; it thus represents the third calmodulin-like protein to be characterised in F. hepatica and has been named FhCaM3. Compared to the classical calmodulin structure there are some variations. Most noticeably, the central, linker helix is disrupted by a cysteine residue. Alkaline native gel electrophoresis showed that FhCaM3 binds calcium ions. This binding event increases the ability of the protein to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, consistent with an increase in surface hydrophobicity as seen in other calmodulins. FhCaM3 binds to the calmodulin antagonists trifluoperazine and W7, but not to the myosin regulatory light chain binding compound praziquantel. Immunolocalisation demonstrated that the protein is found in eggs and vitelline cells. Given the critical role of calcium ions in egg formation and hatching this suggests that FhCaM3 may play a role in calcium signalling in these processes. Consequently the antagonism of FhCaM3 may, potentially, offer a method for inhibiting egg production and thus reducing the spread of infection.

Altered lipopolysaccharide (LPS)-responsiveness is a key feature of acute pancreatitis (AP)-associated multiple organ failure (AP-MOF) in rats and humans.To determine the differential expression of 16 cytokines and chemokines in response to delayed LPS administration in established experimental AP-MOF in rats.In a cubic factorial group design (12 groups, n = 6 rats/group), 0, 6 and 30 microg/kg Escherichia coli 0111:B4 LPS was administered intra-arterially, 18 h into experimental AP-MOF or sham laparotomy. AP was induced by intraductal glycodeoxycholic acid and intravenous caerulein. Central venous serum concentrations of 16 cytokines and chemokines were measured by Searchlight multiplex ELISA.Four patterns were observed: (1) TNF-alpha, IL-1alpha, IL-1beta, IL-6, IFN-gamma, MCP-1, MIP-2alpha, MIP-3alpha, fractalkine and RANTES showed a diminished LPS response in AP versus sham (p0.001, ANOVA); (2) IL-2, IL-4 and GM-CSF levels were undetectable; (3) CINC-2alpha and GRO/KC showed little or no difference between AP and controls, and (4) IL-10 concentrations after 0 and 6 microg/kg, but not 30 microg/kg LPS injection were significantly higher in AP than controls (p0.001, ANOVA).Experimental AP-MOF in rats results in differential preservation of the cytokine and chemokine response to LPS challenge, with a predominantly regulatory expressed phenotype.

The 67LR (67 kDa laminin receptor) is a cell-surface receptor with high affinity for its primary ligand. Its role as a laminin receptor makes it an important molecule both in cell adhesion to the basement membrane and in signalling transduction following this binding event. The protein also plays critical roles in the metastasis of tumour cells. Isolation of the protein from either normal or cancerous cells results in a product with an approx. molecular mass of 67 kDa. This protein is believed to be derived from a smaller precursor, the 37LRP (37 kDa laminin receptor precursor). However, the precise mechanism by which cytoplasmic 37LRP becomes cell-membrane-embedded 67LR is unclear. The process may involve post-translational fatty acylation of the protein combined with either homo- or hetero-dimerization, possibly with a galectin-3-epitope-containing partner. Furthermore, it has become clear that acting as a receptor for laminin is not the only function of this protein. 67LR also acts as a receptor for viruses, such as Sindbis virus and dengue virus, and is involved with internalization of the prion protein. Interestingly, unmodified 37LRP is a ribosomal component and homologues of this protein are found in all five kingdoms. In addition, it appears to be strongly associated with histones in the eukaryotic cell nucleus, although the precise role of these interactions is not clear. Here we review the current understanding of the structure and function of this molecule, as well as highlighting areas requiring further research.

Triclabendazole (TCBZ) has been the drug of choice to treat liver fluke infections in livestock for > 20 years, due to its high activity against both adult and juvenile flukes. More recently, it has been used successfully to treat human cases of fascioliasis. Resistance to TCBZ first appeared in the field in Australia in the mid-1990s. Since then, resistance has been reported from a number of countries throughout Europe: Ireland, Scotland, Wales, Spain and The Netherlands. The heavy reliance on a single drug puts treatment strategies for fascioliasis at risk. Should resistance develop further, the prospect is an alarming one. This review will present an overview of progress in understanding the mechanism of resistance to TCBZ, examining possible changes in the target molecule, in drug influx/efflux mechanisms and in the metabolism of TCBZ by the fluke. The review will also consider ways to deal with resistance, covering drug-oriented options such as: the use of alternative drugs, drug combinations and the search for new compounds. © 2007 Elsevier Inc. All rights reserved.

Protein interactions play key roles throughout all subcellular compartments. In the present paper, we report the visualization of protein interactions throughout living mammalian cells using two oligomerizing MV (measles virus) transmembrane glycoproteins, the H (haemagglutinin) and the F (fusion) glycoproteins, which mediate MV entry into permissive cells. BiFC (bimolecular fluorescence complementation) has been used to examine the dimerization of these viral glycoproteins. The H glycoprotein is a type II membrane-receptor-binding homodimeric glycoprotein and the F glycoprotein is a type I disulfide-linked membrane glycoprotein which homotrimerizes. Together they co-operate to allow the enveloped virus to enter a cell by fusing the viral and cellular membranes. We generated a pair of chimaeric H glycoproteins linked to complementary fragments of EGFP (enhanced green fluorescent protein) – haptoEGFPs – which, on association, generate fluorescence. Homodimerization of H glycoproteins specifically drives this association, leading to the generation of a fluorescent signal in the ER (endoplasmic reticulum), the Golgi and at the plasma membrane. Similarly, the generation of a pair of corresponding F glycoprotein–haptoEGFP chimaeras also produced a comparable fluorescent signal. Co-expression of H and F glycoprotein chimaeras linked to complementary haptoEGFPs led to the formation of fluorescent fusion complexes at the cell surface which retained their biological activity as evidenced by cell-to-cell fusion.

BACKGROUND Two novel antiendotoxin peptides, P6 and C1, may reduce the inflammatory response. This study aimed to determine the effect of endotoxin on hepatic cytokine response and to assess P6 and C1-related attenuation of the proinflammatory response to endotoxemia, in experimental biliary obstruction. MATERIALS AND METHODS 15 Male Wistar rats were randomized to one of three groups: bile duct ligation (BDL)+P6 (n=5), BDL+C1 (n=5), and BDL+no peptide (n=5). Rats were weighed and underwent BDL surgery on day 1. On day 8, the rats were reweighed and isolated hepatic perfusion was carried out. P6 or C1 peptide (10 μmol/l) was preincubated with 300 ml of endotoxin-containing Krebs perfusate. After perfusion of 10 min with endotoxin-free perfusate, the livers were perfused for another 10 min with 300 ml of perfusate-containing endotoxin on its own or endotoxin plus peptide. This was followed by a further 100 min of perfusion with endotoxin-free perfusate. Effluent perfusate was collected at 20-min intervals for subsequent biochemical and cytokine analyses. RESULTS Perfusion with endotoxin+P6 or endotoxin+C1 resulted in no significant difference in weight loss, or interleukin-6 response compared with perfusion with endotoxin alone. However, perfusion with endotoxin+P6 or endotoxin+C1 significantly reduced the tumor necrosis factor-α response to portal endotoxemia compared with perfusion with endotoxin alone. CONCLUSION This study demonstrates that novel antiendotoxin peptides may attenuate the hepatic inflammatory response in portal endotoxemia. In obstructive jaundice, preoperative peptide administration may reduce endotoxin-related postoperative complications.

Background Multiple organ failure (MOF) is the key determinant of mortality in acute pancreatitis (AP). Mesenteric lymph cytotoxicity contributes to organ failure in experimental models of systemic inflammation. The aim of this study was to evaluate the mesenteric lymph pathway and the lymph injury proteome in experimental AP-associated MOF, and to test the hypothesis that immunoregulatory tryptophan catabolites contribute to mesenteric lymph cytotoxicity. Methods Using an experimental model of AP in rats, the humoral component of mesenteric lymph in AP was compared with that from sham-operated control animals, using in vitro and in vivo cytotoxicity assays, high-throughput proteomics and high-performance liquid chromatography. The experimental findings were corroborated in a cohort of 34 patients with AP. Results Compared with biologically inactive lymph from sham-operated rats, mesenteric lymph in AP became cytotoxic 3 h after induction. Hierarchical clustering of lymph proteomic mass spectra predicted the biological behaviour of lymph. Levels of the immunoregulatory tryptophan catabolite, 3-hydroxykynurenine, were increased in cytotoxic lymph and re-created cytotoxicity in vitro. In humans with AP, plasma kynurenine concentrations correlated in real time with MOF scores and preceded a requirement for mechanical ventilation and haemodialysis. Conclusion These results support the concept that mesenteric lymph-borne kynurenines may contribute to pancreatitis-associated MOF.

Calmodulin is a calcium ion-sensing signalling protein found in eukaryotics. Two calmodulin-like gene sequences were identified in an EST library from adult liver flukes. One codes for a protein (FhCaM1) homologous to mammalian calmodulins (98% identity), whereas the other protein (FhCaM2) has only 41% identity. These genes were cloned into expression vectors and the recombinant proteins were expressed inEscherichia coli. Gel shift assays showed that both proteins bind to calcium, magnesium and zinc ions. Homology models have been built for both proteins. As expected, FhCaM1 has a highly similar structure to other calmodulins. Although FhCaM2 has a similar fold, its surface charge is higher than FhCaM1. One of the potential metal ion-binding sites has lower metal-ion co-ordination capability, while another has an adjacent lysine residue, both of which may decrease the metal-binding affinity. These differences may reflect a specialised role for FhCaM2 in the liver fluke.

Background/Aim: During severe acute pancreatitis (AP), the liver may show an exaggerated response to the inflammatory products of gut injury transported in the portal vein. Our aim was to explore liver proinflammatory mediator production after a ‘second hit’ of portal lipopolysaccharide (LPS) during AP. Methods: Twenty-four rats underwent one of three ‘first-hit’ scenarios: (1) severe AP induced by intraductal glycodeoxycholic acid injection and intravenous caerulein infusion, (2) sham laparotomy, or (3) no first intervention. Eighteen hours later, all animals received a ‘second hit’ of portal LPS in an isolated liver perfusion system. Tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 concentrations were measured in portal and systemic serum, and in the perfusate 30 and 90 min after the ‘second hit’. Neutrophil activation by the perfusate was assayed using dihydrorhodamine-123 fluorescence. Results: We observed a six-fold increase in IL-6 concentration across the liver during AP. All livers produced TNF-α after the portal LPS challenge, but this was not exaggerated by AP. No differential neutrophil activation by the perfusate was seen. Conclusion: TNF-α, IL-1β, IL-6 and neutrophil activator production by the isolated perfused liver, in response to a ‘second hit’ of portal LPS, does not appear to be enhanced during AP.

Although it is well established that benzimidazole (BZMs) compounds exert their therapeutic effects through binding to helminth β-tubulin and thus disrupting microtubule-based processes in the parasites, the precise location of the benzimidazole-binding site on the β-tubulin molecule has yet to be determined. In the present study, we have used previous experimental data as cues to help identify this site. Firstly, benzimidazole resistance has been correlated with a phenylalanine-to-tyrosine substitution at position 200 of Haemonchus contortus β-tubulin isotype-I. Secondly, site-directed mutagenesis studies, using fungi, have shown that other residues in this region of the protein can influence the interaction of benzimidazoles with β-tubulin. However, the atomic structure of the αβ-tubulin dimer shows that residue 200 and the other implicated residues are buried within the protein. This poses the question: how might benzimidazoles interact with these apparently inaccessible residues? In the present study, we present a mechanism by which those residues generally believed to interact with benzimidazoles may become accessible to the drugs. Furthermore, by docking albendazole-sulphoxide into a modelled H. contortus β-tubulin molecule we offer a structural explanation for how the mutation conferring benzimidazole resistance in nematodes may act, as well as a possible explanation for the species-specificity of benzimidazole anthelmintics. © 2004 Elsevier Inc. All rights reserved.

HSP70 chaperones mediate protein folding by ATP-dependent interaction with short linear peptide segments that are exposed on unfolded proteins. The mode of action of the Escherichia coli homolog DnaK is representative of all HSP70 chaperones, including the endoplasmic reticulum variant BiP/GRP78. DnaK has been shown to be effective in assisting refolding of a wide variety of prokaryotic and eukaryotic proteins, including the alpha-helical homodimeric secretory cytokine interferon-gamma (IFN-gamma). We screened solid-phase peptide libraries from human and mouse IFN-gamma to identify DnaK-binding sites. Conserved DnaK-binding sites were identified in the N-terminal half of helix B and in the C-terminal half of helix C, both of which are located at the IFN-gamma dimer interface. Soluble peptides derived from helices B and C bound DnaK with high affinity in competition assays. No DnaK-binding sites were found in the loops connecting the alpha-helices. The helix C DnaK-binding site appears to be conserved in most members of the superfamily of interleukin (IL)-10-related cytokines that comprises, apart from IL-10 and IFN-gamma, a series of recently discovered small secretory proteins, including IL-19, IL-20, IL-22/IL-TIF, IL-24/MDA-7 (melanoma differentiation-associated gene), IL-26/AK155, and a number of viral IL-10 homologs. These cytokines belong to a relatively small group of homodimeric proteins with highly interdigitated interfaces that exhibit the strongly hydrophobic character of the interior core of a single-chain folded domain. We propose that binding of DnaK to helix C in the superfamily of IL-10-related cytokines may constitute the hallmark of a novel conserved regulatory mechanism in which HSP70-like chaperones assist in the formation of a hydrophobic dimeric "folding" interface.

A laminin-antagonist peptide, comprising amino acids 33-42 of murine epidermal growth factor (mEGF-(33-42)), interacts with a breast cancer- and endothelial cell-associated receptor, which is specific for the laminin B1 chain sequence, CDPGYIGSR-NH2 (Lam.B1-(925-933)), and is immunologically similar to a previously described 67-kDa laminin receptor. In whole cell receptor assays, mEGF-(33-42), Lam.B1-(925-933), and laminin all have IC50 values for displacement of 125I-laminin in the range 1-5 nM. Cell attachment to solid-phase laminin is also blocked by all three ligands, but in contrast to the receptor assays, mEGF-(33-42) or Lam.B1-(925-933), while equipotent with each other, were less effective than laminin. The concentrations of the peptides required to produce half-maximal inhibition of attachment were in the range 230-390 nM, but those for laminin were 1000-fold lower, in the range 0.2-0.3 nM. Like laminin, solid-phase mEGF-(33-42) supports cell attachment, and this ability is blocked by anti-67-kDa receptor antibodies. Modeling studies suggest that both peptides present a tyrosyl and an arginyl residue on the same face of a right-handed helical fold with elliptical cross-section.