Bovine muc1 inhibits binding of enteric bacteria to caco-2 cells

Inhibition of bacterial adhesion to intestinal epithelial receptors by the consumption of natural food components is an attractive strategy for the prevention of microbial related gastrointestinal illness. We hypothesised that Muc1, a highly glycosylated mucin present in cows’ milk, may be one such food component. Purified bovine Muc1 was tested for its ability to inhibit binding of common enteric bacterial pathogens to Caco-2 cells grown in vitro. Muc1 caused dose-dependent binding inhibition of Escherichia coli, Salmonella enterica serovar Typhimurium (S. Typhimurium), Staphylococcus aureus and Bacillus subtilis. This inhibition was more pronounced for the Gram negative compared with Gram positive bacteria. It was also demonstrated that Muc1, immobilised on a membrane, bound all these bacterial species in a dose-dependent manner, although there was greater interaction with the Gram negative bacteria. A range of monosaccharides, representative of the Muc1 oligosaccharide composition, were tested for their ability to prevent binding of E. coli and S. Typhimurium to Caco-2 cells. Inhibition was structure dependent with sialic acid, L(-) fucose and D(+) mannose significantly inhibiting binding of both Gram negative species. N-acetylglucosamine and N-acetylgalactosamine significantly inhibited binding of E. coli whilst galactose, one of the most abundant Muc1 monosaccharides, showed the strongest inhibition against S. Typhimurium. Treatment with sialidase significantly decreased the inhibitory properties of Muc1, demonstrating the importance of sialic acid in adhesion inhibition. It is concluded that bovine Muc1 prevents binding of bacteria to human intestinal cells and may have a role in preventing the binding of common enteropathogenic bacteria to human intestinal epithelial surfaces.

An aqueous Chlorella extract inhibits IL-5 production by mast cells in vitro and reduces ovalbumin-induced eosinophil infiltration in the airway in mice in vivo

An aqueous extract of the edible microalga, Chlorella pyrenoidosa (CP) (1), has recently been tested for its immunomodulatory effects in a human clinical trial. Here, the CP extract was dialyzed and fractionated using Sephadex G 100 chromatography. The effects of a dialyzed aqueous CP extract, fraction 2, on mast cell mediator release in vitro and ovalbumin-induced allergic airway inflammation in vivo were examined. In vitro, treatment of mouse bone marrow-derived mast cells with 2 for 18 h significantly inhibited antigen (trinitrophenyl-BSA)-induced IL-5 production. In vivo, treatment of mice with 2 during ovalbumin sensitization and stimulation process significantly reduced eosinophil and neutrophil infiltration in the airways. Moreover, fractions obtained by size exclusion chromatography of 2 inhibited IgE-dependent cytokine GM-CSF production from human cord blood-derived mast cells. Taken together, these results suggest that 2 is composed of biopolymers with anti-allergic potential.

Characterisation of nickel(II) extraction by 2-hydroxy-5-nonylacetophenone oxime (LIX 84) in a micellar phase

The properties of the nickel(II)/2-hydroxy-5-nonylacetophenone oxime (HNAPO), an active ingredient in LIX 84, extraction system were characterised in a micellar system. The extinction coefficient, λ_max of HNAPO (316 nm) and the Ni²⁺ complex (387 nm) in a neutral micellar system, poly dispersed octa-ethyleneglycol mono-n-dodecyl ether (G₁₂A₈) were determined as 3100 and 3500 M⁻¹ cm⁻¹, respectively. HNAPO was found to have a neutral micellar phase and bulk aqueous phase pK_a of 11.5 and 12.5, respectively. The extraction equilibrium constant, K_ex, was determined to be 10^−8.0, and the deviation from theory observed at high pH can be accounted for by consideration of the competition for nickel(II) ions by hydroxide ions and HNAPO. A micellar phase of octa-ethyleneglycol mono-n-dodecyl ether (C₁₂E₈) was determined to be an appropriate model of the free oil/water interface from the solubilised location of HNAPO. Utilising the interfacial probe, 4-heptadecyl-7-hydroxy coumarin (HHC) allowed the determination of the electrostatic surface potential of mixed micelles of G₁₂A₈ and sodium dodecyl sulphate (SDS) or dodecyl trimethyl ammonium chloride (DTAC). The electrostatic surface potential was a linear function of the number of additional surfactant monomers within the G₁₂A₈ micelle, for the concentration range studied. For G₁₂A₈/DTAC mixed micelles, the surface potential was given by +1.1 mV per DTAC molecule per micelle, and for G₁₂A₈/SDS mixed micelles the relationship was −1.4 mV per SDS molecule per micelle.

Host manipulation by cancer cells: Expectations, facts, and therapeutic implications.

Similar to parasites, cancer cells depend on their hosts for sustenance, proliferation and reproduction, exploiting the hosts for energy and resources, and thereby impairing their health and fitness. Because of this lifestyle similarity, it is predicted that cancer cells could, like numerous parasitic organisms, evolve the capacity to manipulate the phenotype of their hosts to increase their own fitness. We claim that the extent of this phenomenon and its therapeutic implications are, however, underappreciated. Here, we review and discuss what can be regarded as cases of host manipulation in the context of cancer development and progression. We elaborate on how acknowledging the applicability of these principles can offer novel therapeutic and preventive strategies. The manipulation of host phenotype by cancer cells is one more reason to adopt a Darwinian approach in cancer research.

Stimulation of cytokine production in human peripheral blood mononuclear cells by an aqueous Chlorella extract

CPE is an aqueous extract of the edible micro alga Chlorella pyrenoidosa, which has been shown to have immunostimulatory effects in vivo. In the present study, CPE was evaluated for an ability to stimulate cytokine production by human peripheral blood mononuclear cells (PBMC). PBMC from healthy individuals were treated ex vivo for 24 hours with 1, 10 and 100 μg/mL CPE. This resulted in a marked increase in the level of IL-10, a regulatory cytokine, and strong stimulation of the T-helper-1 (Th1) cell cytokines, IFN-γ and TNF-α. In contrast, stimulation of representative T-helper-2 (Th2) cell cytokines, IL-4 and IL-13, was minor. CPE (1, 10 or 100 μg/mL) did not cause a proliferation of human PBMC suggesting that enhanced secretion of cytokines was not secondary to an increase in cell number. We conclude that CPE stimulation of human PBMC induces a Th1-patterned cytokine response and a strong anti-inflammatory regulatory cytokine response, observations that await confirmation in vivo.

Synthesis and preliminary investigations into novel 1,2,3-triazole-derived androgen receptor antagonists inspired by bicalutamide.

A versatile and high yielding synthesis of novel androgen receptor (AR) antagonists is presented. Using this methodology, six 1,4-substituted-1,2,3-triazole derived bicalutamide mimics were synthesised in five steps and in isolated overall yields from 41% to 85%. Evaluation of these compounds for their anti-proliferative properties against androgen dependent (LNCaP) and independent (PC-3) cells showed promising IC50 values of 34-45 μM and 29-151 μM, respectively. The data suggest that the latter compounds may be an excellent starting point for the development of prostate cancer therapeutics for both androgen dependent and independent forms of this disease. Docking of these compounds (each enantiomer) in silico into the T877A mutated androgen receptor, as possessed by LNCaP cells, was also undertaken.

Prevention of type 2 diabetes by lifestyle intervention in an Australian primary health care setting : Greater Green Triangle (GGT) Diabetes Prevention Project

Background
Randomised controlled trials demonstrate a 60% reduction in type 2 diabetes incidence through lifestyle modification programmes. The aim of this study is to determine whether such programmes are feasible in primary health care.

Methods
An intervention study including 237 individuals 40–75 years of age with moderate or high risk of developing type 2 diabetes. A structured group programme with six 90 minute sessions delivered during an eight month period by trained nurses in Australian primary health care in 2004–2006. Main outcome measures taken at baseline, three, and 12 months included weight, height, waist circumference, fasting plasma glucose and lipids, plasma glucose two hours after oral glucose challenge, blood pressure, measures of psychological distress and general health outcomes. To test differences between baseline and follow-up, paired t-tests and Wilcoxon rank sum tests were performed.

Results
At twelve months participants' mean weight reduced by 2.52 kg (95% confidence interval 1.85 to 3.19) and waist circumference by 4.17 cm (3.48 to 4.87). Mean fasting glucose reduced by 0.14 mmol/l (0.07 to 0.20), plasma glucose two hours after oral glucose challenge by 0.58 mmol/l (0.36 to 0.79), total cholesterol by 0.29 mmol/l (0.18 to 0.40), low density lipoprotein cholesterol by 0.25 mmol/l (0.16 to 0.34), triglycerides by 0.15 mmol/l (0.05 to 0.24) and diastolic blood pressure by 2.14 mmHg (0.94 to 3.33). Significant improvements were also found in most psychological measures.

Conclusion
This study provides evidence that a type 2 diabetes prevention programme using lifestyle intervention is feasible in primary health care settings, with reductions in risk factors approaching those observed in clinical trials.

Trial Number
Current Controlled Trials ISRCTN38031372

A non-radioactive method for measuring Cu uptake in HepG2 cells

At present, all data on Cu uptake and metabolism have been derived from radioactive uptake experiments. These experiments are limited by the availability of the radioactive isotopes ⁶⁴Cu or ⁶⁷Cu, and their short half-life (12.5 and 62 h, respectively). In this paper, we investigate an alternative method to study the uptake of Cu with natural isotopes in HepG2 cells, a liver cell line used extensively to study Cu metabolism. In nature, Cu occurs as two stable isotopes, ⁶³Cu and ⁶⁵Cu (⁶³Cu/⁶⁵Cu = 2.23). This ratio can be measured accurately using inductively coupled plasma mass spectrometry (ICP-MS). In initial experiments, we attempted to measure the time course of Cu uptake using ⁶⁵Cu. The change in the ⁶³Cu/⁶⁵Cu ratio, however, was too small to allow measurement of Cu uptake by the cells. To overcome this difficulty, the natural ⁶³Cu/⁶⁵Cu ratio in HepG2 cells was altered using long-term incubation with ⁶³Cu. This had a significant effect on Cu concentration in HepG2 cells, changing it from 81.9 ± 9.46 pmol μg DNA⁻¹ (week 1) to 155 ± 8.63 pmol μg DNA⁻¹ (week 2) and stabilising at 171 ± 4.82 pmol μg DNA⁻¹ (week 3). After three weeks of culture with 2 μM ⁶³Cu the ⁶³Cu/⁶⁵Cu changed from 2.18 ± 0.05 to 15.3 ± 1.01. Cu uptake was then investigated as before using ⁶⁵Cu. Uptake was linear over 60 min, temperature dependent and consistent with previous kinetics data. These observations suggest that stable isotope ICP-MS provides an alternative technique for the study of Cu uptake by HepG2 cells.

Bioactivating the surfaces of titanium by sol-gel process

In the present study, titanium (Ti) samples were surface-modified by titania (TiO₂), silica (SiO₂) and hydroxyapatite (HA) coatings using a sol-gel process. The bioactivity of the film-coated Ti samples was investigated by cell attachment and morphology study using human osteoblast-like SaOS-2 cells. Results of the cell attachment indicated that the densities of cell attachment on the surfaces of Ti samples were significantly increased by film coatings; the density of cell attachment on HA film-coated surface was higher than those on TiO₂ and SiO₂ film-coated surfaces. Cell morphology study showed that the cells attached, spread and grew well on the three kinds of film-coated surfaces. It can be concluded that the three kinds of film coatings can bioactivate the surfaces of Ti samples effectively. Overall, Ti sample with HA film-coated surface exhibited the best bioactivity.

Chemotoxicity of doxorubicin and surface expression of P-glycoprotein (MDR1) is regulated by the Pseudomonas aeruginosa toxin Cif

P-glycoprotein (Pgp), a member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily, is a major drug efflux pump expressed in normal tissues, and is overexpressed in many human cancers. Overexpression of Pgp results in reduced intracellular drug concentration and cytotoxicity of chemotherapeutic drugs and is thought to contribute to multidrug resistance of cancer cells. The involvement of Pgp in clinical drug resistance has led to a search for molecules that block Pgp transporter activity to improve the efficacy and pharmacokinetics of therapeutic agents. We have recently identified and characterized a secreted toxin from Pseudomonas aeruginosa, designated cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif). Cif reduces the apical membrane abundance of CFTR, also an ABC transporter, and inhibits the CFTR-mediated chloride ion secretion by human airway and kidney epithelial cells. We report presently that Cif also inhibits the apical membrane abundance of Pgp in kidney, airway, and intestinal epithelial cells but has no effect on plasma membrane abundance of multidrug resistance protein 1 or 2. Cif increased the drug sensitivity to doxorubicin in kidney cells expressing Pgp by 10-fold and increased the cellular accumulation of daunorubicin by 2-fold. Thus our studies show that Cif increases the sensitivity of Pgp-overexpressing cells to doxorubicin, consistent with the hypothesis that Cif affects Pgp functional expression. These results suggest that Cif may be useful to develop a new class of specific inhibitors of Pgp aimed at increasing the sensitivity of tumors to chemotherapeutic drugs, and at improving the bioavailability of Pgp transport substrates.

hZip1 (hSLC39A1) regulates zinc homoeostasis in gut epithelial cells

Zinc is an essential trace element required for enzyme catalysis, gene regulation and signal transduction. Zinc absorption takes place in the small intestine, however, the mechanisms by which cells accumulate zinc are not entirely clear. Zip1 (SLC39A1) is a predicted transmembrane protein that is postulated, but not conclusively proven to mediate zinc influx in gut cells. The aim of this study was to investigate a role for hZip1 in mediating zinc uptake in human enterocytes. Both hZip1 mRNA and protein were detected in human intestinal tissue. In non-differentiated Caco-2 human gut cells, hZip1 was partially localised to the endoplasmic reticulum. In contrast, in differentiated Caco-2 cells cultured in extracellular matrix, the hZip1 protein was located in proximity to the apical microvilli. Lack of surface antibody binding and internalisation indicated that hZip1 was not present on the plasma membrane. Functional studies to establish a role for hZip1 in cellular zinc accumulation were carried out using 65Zn. In Caco-2 cells harbouring an hZip1 overexpression construct, cellular zinc accumulation was enhanced relative to the control. Conversely, Caco-2 cells with an hZip1 siRNA construct showed reduced zinc accumulation. In summary, we show that the Caco-2 cell differentiation endorses targeting of hZip1 to a region near the apical domain. Given the absence of hZip1 at the apical plasma membrane, we propose that hZip1 may act as an intracellular sensor to regulate zinc homoeostasis in human gut cells.

Synergistic effects of IAP inhibitor LCL161 and paclitaxel on hepatocellular carcinoma cells

Inhibitor of Apoptosis Proteins (IAPs) are key regulators of apoptosis in hepatocellular carcinoma (HCC) and their expression is negatively correlated with patient survival. LCL161 is a small molecule inhibitor of IAPs that has potent antitumour activity in a range of solid tumours. In HCC, response to LCL161 therapy has shown to be mediated by Bcl-2 expression. In this study, we aim to determine whether LCL161 has any therapeutic potential in HCC. Protein expression was determined by Western blot. Cell proliferation was determined by Cell Proliferation ELISA and BrdU colorimetric assays. Apoptosis was determined by Annexin V assay. Cell cycle analysis was performed by staining cells with propidium iodide and analysed in a FACScan. Automated Cell Counter and phase contrast microscopy were used to determine the cell viability. We have found that LCL161 targets (cIAP1, cIAP2 and XIAP) were up-regulated in HCC tumours. Both high Bcl-2 expressing HuH7 cells and low Bcl-2 expressing SNU423 cells showed strong resistance to LCL161 therapy with significant effects on both apoptosis and cell viability only evident at LCL161 concentrations of ⩾100μM. At these doses there was significant inhibition of IAP targets, however there was also significant inhibition of off-target proteins including pERK and pJNK suggesting apoptosis caused by drug toxicity. However, when used in combination with paclitaxel in HuH7 and SNU423 cells, LCL161 had significant antiproliferative effects at doses as low as 2μM and this was independent of Bcl-2 expression. Thus, LCL161 may be a useful agent in combination with paclitaxel to treat liver tumours.

Fe-bLf nanoformulation targets survivin to kill colon cancer stem cells and maintains absorption of iron, calcium and zinc

To validate the anticancer efficacy of alginate-enclosed, chitosan-conjugated, calcium phosphate, iron-saturated bovine lactoferrin (Fe-bLf) nanocarriers/nanocapsules (NCs) with improved sustained release and ability to induce apoptosis by downregulating survivin, as well as cancer stem cells.

New 2,N6-Disubstituted adenosines: Potent and selective A1 adenosine receptor agonists

A number of adenosine analogues substituted in the 2- and N⁶-positions were synthesized and evaluated for affinity, functional potency and intrinsic activity at the A₁ and A_2A adenosine receptors (AR). Three classes of N⁶-substituents were tested; norbornen-2-yl (series 1), norborn-2-yl (series 2) and 5,6-epoxynorborn-2-yl (series 3). The halogens; fluoro, bromo, and iodo were evaluated as C-2 substituents. All compounds showed relatively high affinity (nanomolar) for the A₁AR and high potency for inhibiting (−)isoproterenol-stimulated cAMP accumulation in hamster smooth muscle DDT1 MF-2 cells with the 2-fluoro derivatives from each series having the highest affinity. All of the derivatives showed the same intrinsic activity as CPA. At the A2AAR, all of the derivatives showed relatively low affinity and potency (micromolar) for stimulating cAMP accumulation in rat pheochromocytoma PC-12 cells. The intrinsic activity of the derivatives compared to CGS 21680 was dependent upon the halogen substituent in the C-2 position with most showing partial agonist activity. Of particular interest is 2-iodo-N⁶-(2S-endo-norborn-2-yl)adenosine (5e), which is over 100-fold selective for the A₁AR, is a full agonist at this receptor subtype and has no detectable agonist activity at the A_2AAR.

Predictors of wellbeing experienced by Australian police officers

The aim of this study was to assess the ability of the Demand-Control-Support (DCS) model as well as the psychological contract model (PCM) to predict the wellbeing experienced by 2,566 Australian police officers. While the level of explained variance attributed to the PCM was substantially less than the DCS, measures of contract breach and organisational fairness still captured significant portions of intrinsic and extrinsic job satisfaction. Overall, the results of this study suggest that both the DCS and the PCM should figure prominently in strategies aimed at reducing or preventing police stress.