The occurrence and significance of triploidy in the liver fluke, Fasciola hepatica.

Karyotyping of Fasciola hepatica samples from Britain and Ireland has identified a triploid isolate which is effectively aspermic, rendering it necessarily asexually reproducing. Considering the extensive presence of asexually reproducing diploid and triploid Fasciola in Asia it is suggested that facultative gynogenesis is widespread in this parasite. This has important implications for the population genetics and evolution of Fasciola, especially in relation to the development and spread of drug resistance, and must be considered in the mathematical modelling of this process.

Visualization of the earliest steps of gammadelta T cell development in the adult thymus.

The checkpoint in cell development that controls successful T cell receptor (TCR) gene rearrangements remains poorly characterized. Using mice expressing a reporter gene 'knocked into' the Tcrd constant region gene, we have characterized many of the events that mark the life of early cells in the adult thymus. We identify the developmental stage during which the Tcrd locus 'opens' in early T cell progenitors and show that a single checkpoint controls cell development during the penultimate CD4-CD8- stage. Passage through this checkpoint required the assembly of TCR heterodimers on the cell surface and signaling via the Lat adaptor protein. In addition, we show that selection triggered a phase of sustained proliferation similar to that induced by the pre-TCR.

Protein expression pattern of CDK11(p58) during testicular development in the mouse.

Protein kinases are important signalling molecules critical for normal cell growth and development. CDK11(p58) is a p34(cdc2) related protein kinase, and plays an important role in normal cell cycle progression. In this study, we mainly characterized the protein expression of CDK11(p58) during postnatal development in mouse testes and examined the cellular localization of CDK11(p58) and cyclinD3, which was associated with CDK11(p58) in mammalian cells. Western blot analysis revealed that CDK11(p58) was present in the early stages of development. It gradually increased and reached a peak in adult testes. The protein expression of CDK11(p58) was further analysed by immunohistochemistry due to its developmentally regulated expression. The variable immunostaining patterns of CDK11(p58) were visualized during different developmental periods and, in adult mouse, different stages of seminiferous tubules. CDK11(p58) expression was detected in proliferating germ cells in the early stages of developing testes. In adult testes, the protein was expressed in pachytene primary spermatocytes from stage VII to XI of spermatogenesis and in postmeiotic spermatids in all stages at different levels. The colocalization of CDK11(p58) and cyclinD3 in the adult testis was revealed by immunofluorescence analysis.

Development and validation of a lateral flow device for the detection of nicarbazin contamination in poultry feeds

Concentrations of the coccidiostat nicarbazin as low as 2 mg/kg in feed can result in violative drug residues arising in poultry liver. A lateral flow device (LFD) was developed for the detection of contaminating concentrations of nicarbazin following solvent extraction of poultry feeds. Test results, as determined by both visual and instrumental measurement, are available within minutes. For 22 feed samples, nicarbazin-free and fortified at 2 mg/kg, the % relative inhibition ranged from 0 to 45% and from 53 to 85%, respectively. Nicarbazin contamination at the critical concentration (2 mg/kg) can be determined in all cases providing the sampling is representative. A wide range of feed samples taken at a mill that incorporated nicarbazin into poultry feed were analyzed. Data generated for these samples by both the LFDs and a mass spectrometric method were compared, and a significant correlation was achieved.

Liver fluke (Fasciola hepatica)-derived peptides activate rat peritoneal mast cells

Short peptides with sequences derived from those found in the tegumental antigen of Fasciola hepatica have been synthesised. Incubation of some of these peptides with rat peritoneal mast cells resulted in the degranulation of the cells as measured by a histamine release assay. This activity was shown to be associated with the proline-lysine-proline motif, which is responsible for the induction of mast cell degranulation by the mammalian bioactive peptide substance P. Studies on the mode of action of the fluke-derived peptide indicated that it was operating through the same biochemical pathways as substance P. The implications of these findings for the development of immune responses during parasite infections are discussed. (C) 2003 Elsevier Science B.V. All rights reserved.

Relative activity of triclabendazole metabolites against the liver fluke, Fasciola hepatica

A study has been carried Out to determine the relative activity Of triclabendazole (TCBZ) and its sulphoxide (TCBZ(center dot)SO) and sulphone (TCBZ(center dot)SO(2)) metabolites against the adult stage of the liver fluke, Fasciola hepatica. Flukes were incubated for 24 h in vitro in 15 mu g/ml of each of the compounds and prepared for scanning and transmission electron microscopy.

Researcher and Employee: Reflections on Reflective Practice in Rural Development Research

This article describes an ethnographic study that was used to critically assess the links between rural development policy and practice. It does so from the novel perspective of the researcher as an employee in the organisation where the ethnography study was conducted. The article argues that this distinctive position gives rise to specific methodological issues. Particular attention is paid in the analysis to marginalised issues in reflexive practice literature, namely, the structural context. In so doing this research places at centre stage the importance of reflexivity in the field of rural sociology, an area in which to date it has had limited acceptance.

Antitumour prodrug development using cytochrome P450 (CYP) mediated activation

An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme, or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed, including identification of prodrugs activated by tumour-specific polymorphic CYPs, use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxic tumour regions. The last approach is best exemplified by AQ4N, a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite, AQ4 (T50/80 K-m = 26.7 mu M, V-max = 0.43 mu M/mg protein/min; RIF-1 K-m = 33.5 mu M, V-max = 0.42 mu M/mg protein/min) via AQM, a mono-N-oxide intermediate (T50/80 K-m = 37.5 mu M; V-max = 1.4 mu M/mg protein/min; RIF-1 K-m = 37.5 mu M; V-max = 1.2 mu M/mg protein/min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3-fold over a 12 h period, after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely, cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air, despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4, but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (K-m = 4 mu M, V-max = 3.5 pmol/mg protein/min) and normal kidney (K-m = 4 mu M, V-max = 4.0 pmol/mg protein/min), both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species, the process requires low oxygen conditions. Hence, AQ4N metabolism should be restricted to hypoxic tumour cells. The isoform selectivity of AQ4N reduction, in addition to its air sensitivity, indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active-site-bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.

Spatial characterization of salt accumulation in early stage limestone weathering using probe permeametry

This research characterizes the weathering of natural building stone using an unsteady-state portable probe permeameter. Variations between the permeability properties of fresh rock and the same rocks after the early stages of a salt weathering simulation are used to examine the effects of salt accumulation on spatial variations in surface rock permeability properties in two limestones from Spain. The Fraga and Tudela limestones are from the Ebro basin and are of Miocene age. Both stone types figure largely in the architectural heritage of Spain and, in common with many other building limestones, they are prone to physical damage from salt crystallization in pore spaces. To examine feedbacks associated with salt accumulation during the early stages of this weathering process, samples of the two stone types were subjected to simulated salt weathering under laboratory conditions using magnesium sulphate and sodium chloride at concentrations of 5% and 15%. Permeability mapping and statistical analysis (aspatial statistics and spatial prediction) before and after salt accumulation are used to assess changes in the spatial variability of permeability and to correlate these changes with salt movement, porosity change, potential rock deterioration and textural characteristics. Statistical analyses of small-scale permeability measurements are used to evaluate the drivers for decay and hence aid the prediction of the weathering behaviour of the two limestones.

The early-stage risk capital market in Scotland since 2000: issues of scale, characteristics and market efficiency

In this paper we provide a detailed profile and analysis of the regional risk capital market in Scotland, using an innovative methodology and specially developed databases which cover risk capital investment in young companies in the periods 2000–04 and 2005–07. This identifies the investment activity of all actors in the market and provides estimates of the total flow of risk capital investment into early-stage Scottish companies over the period. The paper concludes by drawing out the implications for policy makers (providing a more robust evidence base for the development, implementation and monitoring of policy) and for academic researchers (on the methodologies for estimating market scale and efficiency).

Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha

Phosphoinositide 3-kinases produce 3'-phosphorylated phosphoinositides that act as second messengers to recruit other signalling proteins to the membrane(1). Pi3ks are activated by many extracellular stimuli and have been implicated in a variety of cellular responses(1). The Pi3k gene family is complex and the physiological roles of different classes and isoforms are not clear. The gene Pik3r1 encodes three proteins (p85 alpha, p55 alpha and p50 alpha) that serve as regulatory subunits of class I-A Pi3ks (ref. 2). Mice lacking only the p85a isoform are viable but display hypoglycaemia and increased insulin sensitivity correlating with upregulation of the p55 alpha and p50 alpha variants(3). Here we report that loss of all protein products of Pik3r1 results in perinatal lethality. We observed, among other abnormalities, extensive hepatocyte necrosis and chylous ascites, We also noted enlarged skeletal muscle fibres, brown fat necrosis and calcification of cardiac tissue. In liver and muscle, loss of the major regulatory isoform caused a great decrease in expression and activity of class I-A Pi3k catalytic subunits: nevertheless, homozygous mice still displayed hypoglycaemia, lower insulin levels and increased glucose tolerance. Our findings reveal that p55 alpha and/or p50 alpha are required for survival, but not for development of hypoglycaemia, in mice lacking p85 alpha.

Estimation of survival rate and extinction probability for stage-structured populations with overlapping life stages

The development of methods providing reliable estimates of demographic parameters (e. g., survival rates, fecundity) for wild populations is essential to better understand the ecology and conservation requirements of individual species. A number of methods exist for estimating the demographics of stage-structured populations, but inherent mathematical complexity often limits their uptake by conservation practitioners. Estimating survival rates for pond-breeding amphibians is further complicated by their complex migratory and reproductive behaviours, often resulting in nonobservable states and successive cohorts of eggs and tadpoles. Here we used comprehensive data on 11 distinct breeding toad populations (Bufo calamita) to clarify and assess the suitability of a relatively simple method [the Kiritani-Nakasuji-Manly (KNM) method] to estimate the survival rates of stage-structured populations with overlapping life stages. The study shows that the KNM method is robust and provides realistic estimates of amphibian egg and larval survival rates for species in which breeding can occur as a single pulse or over a period of several weeks. The study also provides estimates of fecundity for seven distinct toad populations and indicates that it is essential to use reliable estimates of fecundity to limit the risk of under- or overestimating the survival rates when using the KNM method. Survival and fecundity rates for B. calamita populations were then used to define population matrices and make a limited exploration of their growth and viability. The findings of the study recently led to the implementation of practical conservation measures at the sites where populations were most vulnerable to extinction. © 2010 The Society of Population Ecology and Springer.

Reducing the future threat from (liver) fluke: realistic prospect or quixotic fantasy?

The liver fluke remains an economically significant parasite of livestock and is emerging as an important zoonotic infection of humans. The incidence of the disease has increased in the last few years, as a possible consequence of changes to the World's climate. Future predictions suggest that this trend is likely to continue. Allied to the changing pattern of disease, reports of resistance to triclabendazole (TCBZ) have appeared in the literature, although they do not all represent genuine cases of resistance. Nevertheless, any reports of resistance are a concern, because triclabendazole is the only drug that has high activity against the migratory and damaging juvenile stages of infection. How to deal with the twin problems (of increasing incidence and drug resistance) is the overall theme of the session on “Trematodes: Fasciola hepatica epidemiology and control” and of this review to introduce the session.

Greater knowledge of fluke epidemiology and population genetics will highlight those regions where surveillance is most required and indicate how quickly resistant populations of fluke may arise. Models of disease risk are becoming increasingly sophisticated and precise, with more refined data analysis programmes and Geographic Information Systems (GIS) data. Recent improvements have been made in our understanding of the action of triclabendazole and the ways in which flukes have become resistant to it. While microtubules are the most likely target for drug action, tubulin mutations do not seem to be involved in the resistance mechanism. Rather, upregulation of drug uptake and metabolism processes appear to be more important and the data relating to them will be discussed. The information may help in the design of new treatment strategies or pinpoint potential molecular markers for monitoring fluke populations. Advances in the identification of novel targets for drugs and vaccines will be made by the various “-omics” technologies that are now being applied to Fasciola. A major area of concern in the current control of fasciolosis is the lack of reliable tests for the diagnosis of drug (TCBZ) resistance. This has led to inaccurate reports of resistance, which is hindering successful disease management, as farmers may be encouraged to switch to less effective drugs. Progress with the development of a number of new diagnostic tests will be reviewed.

Secreted cysteine proteases of the carcinogenic liver fluke, Opisthorchis viverrini: Regulation of cathepsin F activation by autocatalysis and trans-processing by cathepsin B

Opisthorchis viverrini is an important helminth pathogen of humans that is endemic in Thailand and Laos. Adult flukes reside within host bile ducts and feed on epithelial tissue and blood cells. Chronic opisthorchiasis is associated with severe hepatobiliary diseases such as cholangiocarcinoma. Here we report that adult O. viverrini secrete two major cysteine proteases: cathepsin F (Ov-CF-1) and cathepsin B1 (Ov-CB-1). Ov-CF-1 is secreted as an inactive zymogen that autocatalytically processes and activates to a mature enzyme at pH 4.5 via an intermolecular cleavage at the prosegment-mature domain junction. Ov-CB-1 is also secreted as a zymogen but, in contrast to Ov-CF-1, is fully active against peptide and macromolecular substrates despite retaining the N-terminal prosegment. The active Ov-CB-1 zymogen was capable of trans-activating Ov-CF-1 by proteolytic removal of its prosegment at pH 5.5, a pH at which the Ov-CF-1 zymogen cannot autocatalytically activate. Both cathepsins hydrolyse human haemoglobin but their combined action more efficiently degrades haemoglobin to smaller peptides than each enzyme alone. Ov-CF-1 degraded extracellular matrix proteins more effectively than Ov-CB-1 at physiological pH. We propose that Ov-CB-1 regulates Ov-CF-1 activity and that both enzymes work together to degrade host tissue contributing to the development of liver fluke-associated cholangiocarcinoma.

“An Examination of the Key Factor of Influence In the Development Process of Credit Union Industries,”

The aim of this paper is to analyse credit union industries within a development framework. Explicit consideration is given to credit union industries in four countries – Great Britain, Ireland, New Zealand and the United States. It is argued that in terms of a developmental typology the credit union industry in Great Britain is at a nascent stage of development, the industries in Ireland and New Zealand are at a transition stage while the US credit union industry is mature in nature. In progression between stages the analysis considers the influence of factors such as situational leadership, the complexion of trade associations, professionalisation, regulatory and legislative initiatives and technology. The analysis concluded that while there was a substantial commonality of experience, there were also significant differences in the impact of these factors. This consequently encouraged the recognition of the existence of ‘a variety of the species’ in respect of credit union development.