A novel calmodulin-like protein from the liver fluke, Fasciola hepatica

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.

Chymotrypsin-like serine proteinases are involved in the maintenance of cell viability

An increasing number of studies have implicated serine proteinases in the development of apoptosis. In this study, we assessed the ability of a set of highly specific irreversible inhibitors (activity probes), incorporating an a-amino alkane diphenyl phosphonate moiety, to modulate cell death. In an initial assessment of the cellular toxicity of these activity probes, we discovered that one example, N-a-tetramethylrhodamine phenylalanine diphenylphosphonate {TMR-Phe^P(OPh)₂} caused a concentration-dependent decrease in the viability of HeLa and U251 mg cells. This reduced cell viability was associated with a time-dependent increase in caspase-3 activity, PARP cleavage and phosphatidylserine translocation, establishing apoptosis as the mechanism of cell death. SDS-PAGE analysis of cell lysates prepared from the HeLa cells treated with TMR-Phe^P(OPh)₂, revealed the presence of a fluorescent band of molecular weight 58 kDa. Given that we have previously reported on the use of this type of activity probe to reveal active proteolytic species, we believe that we have identified a chymotrypsin-like serine proteinase activity integral to the maintenance of cell viability.

Cloning of cDNAs and molecular characterisation of C-type lectin-like proteins from snake venoms

C-type lectin-like proteins (CTLPs) isolated from snake venoms are the largest and most complex non-mammalian vertebrate C-type lectin-like domain family. In the present study, we simultaneously amplified four cDNAs encoding different types of CTLP subunits from the venoms of two different species of snakes by RT-PCR with a single sense primer and a nested universal primer - two CTLP subunit-encoding cDNAs were cloned from Deinagkistrodon acutus venom and two from Agkistrodon halys Pallas venom. All four cloned CTLP subunits exhibited typical motifs in their corresponding domain regions but with relatively-low sequence similarities to each other. Compared with previously-published CTLPs, the four cloned CTLPs subunits showed slight variations in the calcium-binding sites and the disulphide bonding patterns. To our knowledge, these data constitute the first example of co-expression of CTLP platelet glycoprotein Ib-binding subunits and coagulation factors in Agkistrodon halys Pallas venom.

A prospective randomised trial of a "test and treat" policy versus endoscopy based management in young Helicobacter pylori positive patients with ulcer-like dyspepsia, referred to a hospital clinic

Management of dyspepsia remains a controversial area. Although the European Helicobacter pylori study group has advised empirical eradication therapy without oesophagogastroduodenoscopy (OGD) in young H pylori positive dyspeptic patients who do not exhibit alarm symptoms, this strategy has not been subjected to clinical trial.

Plasma concentrations of glucagon-like peptide-2 in adult patients with treated and untreated coeliac disease

Coeliac disease is a common chronic inflammatory enteropathy characterized by villous atrophy and crypt hyperplasia in the small intestine. The mechanism of the intestinal damage in coeliac disease remains unclear. Glucagon-like peptide (GLP)-2 is an enterotrophic peptide that causes crypt hyperplasia and intestinal cell proliferation. We postulate that GLP-2 may be involved in the mucosal changes found in coeliac disease.

Recurrence of Urothelial Carcinoma of the Bladder: A Role for Insulin-Like Growth Factor-II Loss of Imprinting and Cytoplasmic E-Cadherin Immunolocalization

Purpose:This study documents the frequency of insulin-like growth factor-II (IGF-II) loss of imprinting (LOI) in a series of 87 bladder tissues. E-cadherin (CDH1) immunolocalization was also investigated due to the known redistribution of this adherence protein to the cytoplasm following exogenous exposure to IGF-II.
Experimental Design: Informative IGF-II cases were identified following DNA-PCR amplification and subsequent sequencing of the transcribable ApaI RFLP in exon 9 of IGF-II. Similar approaches using primer-specific cDNA templates identified the imprinting status of IGF-II in these informative cases. CDH1cellular localization was assessed on a tissue microarray platform of 114 urothelial carcinoma of the bladder (UCB) cases (70 pTanoninvasive and 44 pT1laminapropria invasive) using the commercially available Novocastra antibody.
Results: IGF-IILOI was evident in 7 of17 (41%) UCB tumors and 4 of11 (36%) tumor-associated normal urothelial samples.Two of four pT1grade 3 tumors, the subject of much debate concerning their suitability for radical cystectomy, showed LOI at the IGF-II locus. In those tumors showing IGF-II LOI, 4 of 7 (57%) displayed concomitant CDH1cytoplasmic staining. In contrast, only 3 of 10 (30%) IGF-IImaintenance ofimprinting tumorshad concomitant CDH1cytoplasmiclocalization. UCB cell lines displaying cytoplasmic CDH1immunolocalization expressed significantly higher levels of IGF-II (CAL29, HT1376, and RT112) compared with RT4, a cell line displaying crisp membranous CDH1staining. Finally, cytoplasmic CDH1staining was an independent predictor of a shorter time to recurrence independent of tumor grade and stage.
Conclusions: We suggest that CDH1 cytoplasmic immunolocalization as a result of increased IGF-II levels identifies those nonmuscle invasive presentations most likely to recur and therefore might benefit from more radical nonconserving bladder surgery