Evaluation of chromogenic technologies for use in Australian potable water

Aims: To compare the use of MI agar, Membrane Lactose Glucoronide Agar (MLGA), CM1046 agar and Colilert^®-18 (Defined Substrate Technology^®, IDEXX Laboratories Pty. Ltd., Sydney) on Australian potable water. Methods and Results: Both potable (n = 369) and nonpotable waters (n = 35) were analysed by membrane filtration using chromogenic agars as well as Colilert^®-18 over a period of 12 months. Recoveries of stressed organisms on these chromogenic media were also investigated. Agar-based chromogenic technologies compared favourably to Colilert^®-18 for chlorinated waters, but there are possible limitations when using these agars for chloraminated waters. Additionally, the breakthrough of problematic organisms, especially oxidase positive organisms, may lead to misrepresentation or over-estimation of E. coli and total coliforms, particularly on MLGA and CM1046. The recovery of stressed organisms was favoured in the Colilert^®-18 system when compared to chromogenic agars. Conclusions: MI agar performed better than the other chromogenic agars with respect to recovery and colour identification and discrimination of organisms, and compared favourably with Colilert^®-18. The use of chromogenic agars in chloraminated waters should be done cautiously. Significance and Impact of the Study: This study provides comparison data for laboratories looking to adopt chromogenic technologies, and is especially important for Australian laboratories wanting to uptake the use of MI agar (as used in USEPA method 1604) for routine use and for gaining accreditation. Additionally, to the best of our knowledge, this is the first reported evaluation of these agars in chloraminated waters and is especially timely as the use of this disinfection agent is increasing.     

Emerging technologies for the detection and genetic characterization of protozoan parasites

The development and adaptation of new technologies for the genetic characterization and identification of parasites continue to accelerate, providing an increasing number of research and analytical tools. We review emerging technologies that have applications in this area, including real-time PCR and microarrays, and discuss the fundamental principles of some of these technologies and how they are applied to characterize parasites. We give special consideration to the application of genetic data to biological questions, where selection of the most appropriate technique depends on the biological question posed by the investigator.     

Undersulfated, low-molecular-weight glycol-split heparin as an antiangiogenic VEGF antagonist

Vascular endothelial growth factor (VEGF) represents a target for antiangiogenic therapies in a wide spectrum of diseases, including cancer. As a novel strategy to generate nonanticoagulant antiangiogenic substances exploiting binding to VEGF while preventing receptor engagement, we assessed the VEGF-antagonist activity of a low-molecular-weight (LMW) compound (ST2184, Mw = 5800) generated by depolymerization of an undersulfated glycol-split heparin derivative. The parental compound was obtained by introducing regular sulfation gaps along the prevalently N-sulfated heparin regions, followed by glycol-splitting of all nonsulfated uronic acid residues (approximately 50% of total uronic acid residues). ST2184 was endowed with a negligible anticoagulant activity after S.C. injection in mice. ST2184 binds VEGF165 as evaluated by its capacity to retard 125I-VEGF165 electrophoretic migration in a gel mobility shift assay and to prevent VEGF165 interaction with heparin immobilized onto a BIAcore sensor chip. Unlike heparin, ST2184 was unable to present 125I-VEGF165 to its high-affinity receptors in endothelial cells and inhibited VEGF165-induced neovascularization in the chick embryo chorioallantoic membrane. Undersulfated, LMW glycol-split heparins may therefore provide the basis for the design of novel nonanticoagulant angiostatic compounds.     

Water soluble prodrug of a COX-2 selective inhibitor suitable for intravenous administration in models of cerebral ischemia

A water soluble choline prodrug (17) of a COX-2 selective inhibitor (16) suitable for intravenous dosing in models of cerebral ischemia has been developed. Constant infusion studies using 17 demonstrate that extrapolated brain levels of 16 may be maintained for over 24h in rats.     

Shared and non-shared antigens from three different extracts of the metacestode of Echinococcus granulosus

Hydatid cyst fluid (HCF), somatic antigens (S-Ag) and excretory-secretory products (ES-Ag) of Echinococcus granulosus protoscoleces are used as the main antigenic sources for immunodiagnosis of human and dog echinococcosis. In order to determine their non-shared as well as their shared antigenic components, these extracts were studied by ELISA-inhibition and immunoblot-inhibition. Assays were carried out using homologous rabbit polyclonal antisera, human sera from individuals with surgically confirmed hydatidosis, and sera from dogs naturally infected with E. granulosus. High levels of cross-reactivity were observed for all antigenic extracts, but especially for ES-Ag and S-Ag. Canine antibodies evidenced lesser avidity for their specific antigens than antibodies from human origin. The major antigenic components shared by HCF, S-Ag, and ES-Ag have apparent molecular masses of 4-6, 20-24, 52, 80, and 100-104 kDa, including doublets of 41/45, 54/57, and 65/68 kDa. Non-shared polypeptides of each antigenic extract of E. granulosus were identified, having apparent masses of 108 and 78 kDa for HCF, of 124, 94, 83, and 75 kDa for S-Ag, and of 89, 66, 42, 39, 37, and 35 kDa for ES-Ag.     

Environmental temperature controls Cryptosporidium oocyst metabolic rate and associated retention of infectivity

Cryptosporidium is a significant cause of water-borne enteric disease throughout the world and represents a challenge to the water industry and a threat to public health. In this study we report the use of a cell culture-TaqMan PCR assay to measure oocyst inactivation rates in reagent-grade and environmental waters over a range of temperatures. While oocysts incubated at 4 degrees C and 15 degrees C remained infective over the 12-week holding period, we observed a 4 log(10) reduction in infectivity for both 20 and 25 degrees C incubation treatments at 12 and 8 weeks, respectively, for all water types examined, a faster rate of inactivation for oocysts than previously reported. This temperature-dependent inactivation was further investigated using a simple and rapid ATP assay described herein. Time course experiments performed in reagent-grade water at incubation temperatures of 4, 15, 20, 25, 30, and 37 degrees C identified a close relationship between oocyst infectivity and oocyst ATP content, demonstrating that temperature inactivation at higher temperatures is a function of increased oocyst metabolic activity. While water quality did not affect oocyst inactivation, biological antagonism appears to be a key factor affecting oocyst removal from environmental waters. Both the cell culture-TaqMan PCR assay and the ATP assay provide a sensitive and quantitative method for the determination of environmental oocyst inactivation, providing an alternative to the more costly and time-consuming mouse infection assay. The findings presented here relating temperature to oocyst inactivation provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in water.     

Hyposmolarity-induced ErbB4 phosphorylation and its influence on the non-receptor tyrosine kinase network response in cultured cerebellar granule neurons

Exposure of cultured cerebellar granule neurons (24 h serum-starved) during 3 min to 30% hyposmotic medium activated the tyrosine kinase receptor ErbB4 in the absence of its ligand. Hyposmolarity also activated the non-receptor tyrosine kinases, Src, focal adhesion kinase (FAK), extracellular signal-regulated protein kinase (ERK)1/2, and the tyrosine kinase target phosphatidyl-inositol-3-kinase (PI3K). The hyposmotic-induced activation of these kinases required the prior phosphorylation of ErbB4 as shown by the effect of ErbB4 blockade with AG213 reducing by 85-95% the phosphorylation of FAK and ERK1/2, by 74% and 36% that of PI3K and Src, respectively. These results suggest a key role of ErbB4 as a signal integrator of events associated with hyposmolarity. PI3K seems to be an important connecting element in the signaling network evoked by the hyposmolarity/ErbB4 activation as: (i) the p85 regulatory subunit of PI3K co-immunoprecipitates with ErbB4 and with FAK; (ii) PI3K blockade with wortmannin reduced the hyposmotic activation of FAK (90%) and ERK1/2 (84-91%). Inhibition of Src with PP2 reduced ErbB4 phosphorylation and inhibited the subsequent cytosolic kinase activation with the same potency as ErbB4 blockade. These results point to Src and ErbB4 and as early targets of the hyposmotic stimulus and osmosignaling. The functional significance for cell volume regulation of the ErbB4-Src-PI3K signaling cascade is indicated by the 48-66% decrease of the hyposmotic taurine efflux observed by inhibition of these kinases.     

Novel adipocyte lines from brown fat: a model system for the study of differentiation, energy metabolism, and insulin action

Adipose tissue has emerged as an important endocrine regulator of glucose metabolism and energy homeostasis. By virtue of the mitochondrial protein uncoupling protein-1 (UCP-1), brown fat additionally plays a unique role in thermoregulation. Interest has focused on this tissue not only as a target for pharmacotherapy of obesity and insulin resistance but also as an endocrine tissue with leptin secretion and high insulin sensitivity. Most studies of adipocytes have been limited either to primary cell culture or to a small number of established cell lines. Recently, we have generated immortalized brown adipocyte cell lines from single newborn mice of different knockout mouse models. These cell lines retain the main characteristics of primary cells including UCP-1 expression. They display sensitive and diverse metabolic responses to insulin and adrenergic stimulation and have proven to be useful in the characterization of UCP regulation and the role of key insulin signaling elements for insulin action. Here, we outline common approaches to the generation of adipose tissue cell lines. Furthermore, we propose that the novel technique of generating brown adipocyte lines from a single newborn mouse will be instrumental in gaining further insight into the role of a broad range of signaling molecules in adipose tissue biology and in the pathogenesis of insulin resistance.     

Molecular cloning and functional expression in bacteria of the potassium transporters CnHAK1 and CnHAK2 of the seagrass Cymodocea nodosa

The cDNAs CnHAK1 and CnHAK2, encoding K⁺ transporters, were amplified from the leaves of the seagrass Cymodocea nodosa. None of these transporters suppressed the K⁺ deficiency of a Saccharomyces cerevisiae mutant, but both suppressed the equivalent defect of an Escherichia coli mutant. Overexpression of the transporter CnHAK1, but not CnHAK2, mediated very rapid K⁺ or Rb⁺ influxes in the E. coli mutant. The concentration dependence of these influxes demonstrated that CnHAK1 is a low-affinity K⁺ transporter, which does not discriminate between K⁺ and Rb⁺. CnHAK1 expressed in E. coli worked in reverse when the external K⁺ concentrations were low, and we established the condition of a simple functional test of K⁺ loss for transporters of the Kup-HAK family. In comparison with its homologue barley transporter HvHAK2, CnHAK1 was insensitive to Na⁺.