Pharmacokinetics of cimifugin in rat plasma after oral administration of the extract of Saposhnikovia divaricatae root : Determination of cimifugin by high performance liquid chromatography coupled with solid phase extraction

High performance liquid chromatography (HPLC) coupled with the solid phase extraction method was developed for determining cimifugin (a coumarin derivative; one of Saposhnikovia divaricatae's constituents) in rat plasma after oral administration of Saposhnikovia divaricatae extract (SDE), and the pharmacokinetics of cimifugin either in SDE or as a single compound was investigated. The HPLC analysis was performed on a commercially available column (4.6 mm x 200 mm, 5 pm) with the isocratic elution of solvent A (Methanol) and solvent B (Water) (A:B=60:40) and the detection wavelength was set at 250 nm. The calibration curve was linear over the range of 0.100-10.040 microg/mL. The limit of detection was 30 ng/mL. At the rat plasma concentrations of 0.402, 4.016, 10.040 microg/mL, the intra-day precision was 6.21%, 3.98%, and 2.23%; the inter-day precision was 7.59%, 4.26%, and 2.09%, respectively. The absolute recovery was 76.58%, 76.61%, and 77.67%, respectively. When the dosage of SDE was equal to the pure compound calculated by the amount of cimifugin, it was found to have two maximum peaks while the pure compound only showed one peak in the plasma concentration-time curve. The pharmacokinetic characteristics of SDE showed the superiority of the extract and the properties of traditional Chinese medicine.

Induction of antioxidative Nrf2 gene transcription by coffee in humans : depending on genotype?

The Nrf2/ARE pathway is a major cellular defense mechanism that prevents damage by reactive oxygen species through induction of antioxidative phase II enzymes. However, the activity of the Nrf2/ARE system is not uniform with variability in response presumed to be dependent on the Nrf2 genotype. We recently completed a pilot human coffee intervention trial with healthy humans, where large interindividual differences in the antioxidative response to the study coffee were examined. Here, we address the question whether differences in the modulation of Nrf2 gene transcription, assessed as an induction of Nrf2 gene transcription by Q-PCR, might be correlated with specific Nrf2 genotypes. To date, nine single nucleotide polymorphisms (SNPs) have been identified in the Nrf2 (NFE2L2) gene. Two of these, the -617C/A and -651G/A SNPs are located within the promoter region and have previously been reported to influence the activity of the Nrf2/ARE pathway by reducing Nrf2 transcriptional activity. Sequencing of the critical Nrf2 gene promoter region not only confirmed the existence of these SNPs within the participants of the trial at the expected frequency (33% carrying the -617C/A, 17% the -651G/A and 56% the -653A/G SNP) but also indicated reduced Nrf2 gene transcription associated with a normal diet if the SNPs at position -617, -651 or -653 were present. Of note, the data also indicated the study coffee increased Nrf2 gene transcription even in SNP carriers. This further highlights the relevance of genotype-dependent induction of Nrf2 gene transcription that appears to be largely influenced by dietary factors.

A rapid and sensitive UPLC-ESI MS method for analysis of isofraxidin, a natural antistress compound, and its metabolites in rat plasma

Isofraxidin is one of the main bioactive constituents in the root of Acanthopanax senticosus, which has antifatigue, antistress, and immuno-accommondating effects. In this study, an ultraperformance LC (UPLC)-ESI MS method was developed for analyzing isofraxidin and its metabolites in rat plasma. The analysis was performed on a UPLC coupled with ESI MS (quadropole MS tandem TOF MS). The lower LOD (LLOD) for isofraxidin was 0.25 ng/mL, the intraday precision was less than 10%, the interday precision was less than 10%, and the extraction recovery was more than 80%. Isofraxidin and two metabolites (M1 and M2) were detected in rat plasma after oral administration of isofraxidin, and the molecular polarities of M1 and M2 were both increased compared to isofraxidin. The metabolites were identified as 5,6-dihydroxyl-7-methoxycoumarin and 5-hydroxyl-6,7-dimethoxycoumarin when subjected to parent ion spectra, product ion spectra, and extract mass and element composition analyses.

18FDG uptake during induction chemoradiation for oesophageal cancer fails to predict histomorphological tumour response

To determine whether [18F]-fluorodeoxyglucose-positron emission tomography (FDG-PET) could predict the pathological response in oesophageal cancer after only the first week of neoadjuvant chemoradiation. Thirty-two patients with localised oesophageal cancer had a pretreatment PET scan and a repeat after the first week of chemoradiation. The change in mean maximum standardised uptake value (SUV) and volume of metabolically active tissue (MTV) was compared with the tumour regression grade (TRG) in the final histology. Those who achieved a TRG of 1 and 2 were deemed responders and 3-5 nonresponders. In the responders (28%), the SUV fell from 12.6 (±6.3) to 8.1 (±2.9) after 1 week of chemoradiation (P = 0.070). In nonresponders (72%), the results were 9.7 (±5.4) and 7.1 (±3.8), respectively (P = 0.003). The MTV in responders fell from 36.6 (±22.7) to 22.3 (±10.4) cm3 (P = 0.180), while in nonresponders, this fell from 35.9 (±36.7) to 31.9 (±52.7) cm3 (P = 0.405). There were no significant differences between responders and nonresponders. The hypothesis that early repeat FDG-PET scanning may predict histomorphologic response was not proven. This may reflect an inflammatory effect of radiation that obscures tumour-specific metabolic changes at this time. This assessment may have limited application in predicting response to multimodal regimens for oesophageal cancer. © 2006 Cancer Research UK.

Galectin-1 : a link between tumor hypoxia and tumor immune privilege

Purpose: To identify a 15-KDa novel hypoxia-induced secreted protein in head and neck squamous cell carcinomas (HNSCC) and to determine its role in malignant progression. Methods: We used surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and tandem MS to identify a novel hypoxia-induced secreted protein in FaDu cells. We used immunoblots, real-time polymerase chain reaction (PCR), and enzyme-linked immunoabsorbent assay to confirm the hypoxic induction of this secreted protein as galectin-1 in cell lines and xenografts. We stained tumor tissues from 101 HNSCC patients for galectin-1, CA IX (carbonic anhydrase IX, a hypoxia marker) and CDS (a T-cell marker). Expression of these markers was correlated to each other and to treatment outcomes. Results: SELDI-TOF studies yielded a hypoxia-induced peak at 15 kDa that proved to be galectin-1 by MS analysis. Immunoblots and PCR studies confirmed increased galectin-1 expression by hypoxia in several cancer cell lines. Plasma levels of galectin-1 were higher in tumor-bearing severe combined immunodeficiency (SCID) mice breathing 10% O 2 compared with mice breathing room air. In HNSCC patients, there was a significant correlation between galectin-1 and CA IX staining (P = .01) and a strong inverse correlation between galectin-1 and CDS staining (P = .01). Expression of galectin-1 and CDS were significant predictors for overall survival on multivariate analysis. Conclusion: Galectin-1 is a novel hypoxia-regulated protein and a prognostic marker in HNSCC. This study presents a new mechanism on how hypoxia can affect the malignant progression and therapeutic response of solid tumors by regulating the secretion of proteins that modulate immune privilege. © 2005 by American Society of Clinical Oncology.

Increase in prevalence of obesity and diabetes and decrease in plasma cholesterol in a central Australian Aboriginal community

Objective: To document change in prevalence of obesity, diabetes and other cardiovascular diease (CVD) risk factors, and trends in dietary macronutrient intake, over an eight-year period in a rural Aboriginal community in central Australia. Design: Sequential cross-sectional community surveys in 1987, 1991 and 1995. Subjects: All adults (15 years and over) in the community were invited to participate. In 1987, 1991 and 1995, 335 (87% of eligible adults), 331 (76%) and 304 (68%), respectively, were surveyed. Main outcome measures: Body mass index and waist : hip ratio; blood glucose level and glucose tolerance; fasting total and high density lipoprotein (HDL) cholesterol and triglyceride levels; and apparent dietary intake (estimated by the store turnover method). Intervention: A community-based nutrition awareness and healthy lifestyle program, 1988-1990. Results: At the eight-year follow-up, the odds ratios (95% CIs) for CVD risk factors relative to baseline were obesity, 1.84 (1.28-2.66); diabetes, 1.83 (1.11-3.03); hypercholesterolaemia, 0.29 (0.20-0.42); and dyslipidaemia (high triglyceride plus low HDL cholesterol level), 4.54 (2.84-7.29). In younger women (15-24 years), there was a trebling in obesity prevalence and a four- to fivefold increase in diabetes prevalence. Store turnover data suggested a relative reduction in the consumption of refined carbohydrates and saturated fats. Conclusion: Interventions targeting nutritional factors alone are unlikely to greatly alter trends towards increasing prevalences of obesity and diabetes. In communities where healthy food choices are limited, the role of regular physical activity in improving metabolic fitness may also need to be emphasised.

Sterilizing tissue-materials using pulsed power plasma

This paper investigates the potential of pulsed power to sterilize hard and soft tissues and its impact on their physico-mechanical properties. It hypothesizes that pulsed plasma can sterilize both vascular and avascular tissues and the transitive layers in between without deleterious effects on their functional characteristics. Cartilage/bone laminate was chosen as a model to demonstrate the concept, treated at low temperature, at atmospheric pressure, in short durations and in buffered environment using a purposed-built pulsed power unit. Input voltage and time of exposure were assigned as controlling parameters in a full factorial design of experiment to determine physical and mechanical alteration pre- and post-treatment. The results demonstrated that, discharges of 11 kV sterilized samples in 45 s, reducing intrinsic elastic modules from 1.4 ± 0.9 to 0.9 ± 0.6 MPa. There was a decrease of 14.1 % in stiffness and 27.8 % in elastic-strain energy for the top quartile. Mechanical impairment was directly proportional to input voltage (P value < 0.05). Bacterial inactivation was proportional to treatment time for input voltages above 32 V (P < 0.001; R Sq = 0.98). Thermal analysis revealed that helix-coil transition decelerated with exposure time and collagen fibrils were destabilized as denaturation enthalpy reduced by 200 μV. We concluded by presenting a safe operating threshold for pulsed power plasma as a feasible protocol for effective sterilization of connective tissues with varying level of loss in mechanical robustness which we argue to be acceptable in certain medical and tissue engineering application.

Effects of dissolved oxygen availability and culture biomass at induction upon the intracellular expression of monoamine oxidase by recombinant E. coli in fed batch bioprocesses

The effects of oxygen availability and induction culture biomass upon production of an industrially important monoamine oxidase (MAO) were investigated in fed-batch cultures of a recombinant E. coli. For each induction cell biomass 2 different oxygenation methods were used, aeration and oxygen enriched air. Induction at higher biomass levels increased the culture demand for oxygen, leading to fermentative metabolism and accumulation of high levels of acetate in the aerated cultures. Paradoxically, despite an almost eight fold increase in acetate accumulation to levels widely reported to be highly detrimental to protein production, when induction wet cell weight (WCW) rose from 100% to 137.5%, MAO specific activity in these aerated processes showed a 3 fold increase. By contrast, for oxygenated cultures induced at WCW's 100% and 137.5% specific activity levels were broadly similar, but fell rapidly after the maxima were reached. Induction at high biomass levels (WCW 175%) led to very low levels of specific MAO activity relative to induction at lower WCW's in both aerated and oxygenated cultures. Oxygen enrichment of these cultures was a useful strategy for boosting specific growth rates, but did not have positive effects upon specific enzyme activity. Based upon our findings, consideration of the amino acid composition of MAO and previous studies on related enzymes, we propose that this effect is due to oxidative damage to the MAO enzyme itself during these highly aerobic processes. Thus, the optimal process for MAO production is aerated, not oxygenated, and induced at moderate cell density, and clearly represents a compromise between oxygen supply effects on specific growth rate/induction cell density, acetate accumulation, and high specific MAO activity. This work shows that the negative effects of oxygen previously reported in free enzyme preparations, are not limited to these acellular environments but are also discernible in the sheltered environment of the cytosol of E. coli cells.

Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent

Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial-mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of calcium-signaling pathways controlling EMT induction in cancer cells may therefore be an important therapeutic strategy for preventing metastases.

PPARγ-independent induction of growth arrest and apoptosis in prostate and bladder carcinoma

Background Although PPARγ antagonists have shown considerable pre-clinical efficacy, recent studies suggest PPARγ ligands induce PPARγ-independent effects. There is a need to better define such effects to permit rational utilization of these agents. Methods We have studied the effects of a range of endogenous and synthetic PPARγ ligands on proliferation, growth arrest (FACS analysis) and apoptosis (caspase-3/7 activation and DNA fragmentation) in multiple prostate carcinoma cell lines (DU145, PC-3 and LNCaP) and in a series of cell lines modelling metastatic transitional cell carcinoma of the bladder (TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2). Results 15-deoxy-prostaglandin J2 (15dPGJ2), troglitazone (TGZ) and to a lesser extent ciglitazone exhibited inhibitory effects on cell number; the selective PPARγ antagonist GW9662 did not reverse these effects. Rosiglitazone and pioglitazone had no effect on proliferation. In addition, TGZ induced G0/G1 growth arrest whilst 15dPGJ2 induced apoptosis. Conclusion Troglitazone and 15dPGJ2 inhibit growth of prostate and bladder carcinoma cell lines through different mechanisms and the effects of both agents are PPARγ-independent.

Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction : a model for cross-modulation

Background A feature of epithelial to mesenchymal transition (EMT) relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST) induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC. Methods PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome. Results When PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse correlation with lower expression values being predictive of increased risk. Conclusion ST in combination with EGF directed a greater EMT via actin depolymerisation and focal contact size reduction, resulting in a loosening of cell-ECM attachment along with Snail1-Zeb1/δEF1 induction. This appeared fundamentally different to the EGF-induced EMT, highlighting the multiple pathways which can regulate EMT. Our findings add support for a functional role for Snail1 in invasive breast cancer.

Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors

Background Breast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts. Methods: We utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection. Results We found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of α-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active β-catenin, localized to the cell junctions in control cells/ cells in NMF-conditioned medium, to inactive β-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium. Conclusion We found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.

Mitigation of distorted and unbalanced stator voltage of stand-alone doubly fed induction generators using repetitive control technique

Unbalanced or non-linear loads result in distorted stator currents and electromagnetic torque pulsations in stand-alone doubly fed induction generators (DFIGs). This study proposes the use of a proportional-integral repetitive control (PIRC) scheme so as to mitigate the levels of harmonic and unbalance at the stator terminals of the DFIG. The PIRC is structurally simpler and requires much less computation than existing methods. Analysis of the PIRC operation and the methodology to determine the control parameters is included. Simulation study as well as laboratory test measurements demonstrate clearly the effectiveness of the proposed PIRC control scheme.

Modeling and analysis of a novel variable-speed cage induction generator

This paper introduces a novel cage induction generator and presents a mathematical model, through which its behavior can be accurately predicted. The proposed generator system employs a three-phase cage induction machine and generates single-phase and constant-frequency electricity at varying rotor speeds without an intermediate inverter stage. The technique uses any one of the three stator phases of the machine as the excitation winding and the remaining two phases, which are connected in series, as the power winding. The two-series-connected-and-one-isolated (TSCAOI) phase winding configuration magnetically decouples the two sets of windings, enabling independent control. Electricity is generated through the power winding at both sub- and super-synchronous speeds with appropriate excitation to the isolated single winding at any frequency of generation. A dynamic mathematical model, which accurately predicts the behavior of the proposed generator, is also presented and implemented in MATLAB/Simulink. Experimental results of a 2-kW prototype generator under various operating conditions are presented, together with theoretical results, to demonstrate the viability of the TSCAOI power generation. The proposed generator is simple and capable of both storage and retrieval of energy through its excitation winding and is expected to be suitable for applications, such as small wind turbines and microhydro systems.

Plasma lipid and lipoprotein responses during exercise

The purpose of this study was to examine the effect of prolonged exercise oil plasma lipid and lipoprotein concentrations and to identify caloric time-points where changes occurred. Eleven active male Subjects ran oil a treadmill at 70%,, of maximal fitness (VO2max) and expended 6 278.7 kilojoules (Kj) energy (1500 kcal). Blood samples were obtained at the 4185.8 Kj (1000 kcal) time-point during exercise and at each additional 418.6 Kj (100 kcal) expenditure until 6278.7 Kj was expended. After correcting for plasma volume changes, decreases in low-density lipoprotein cholesterol (LDL-C) were observed during exercise at time-points corresponding to 4604.4 and 5441.5 Kj (1100 and 1300 kcal) of energy expenditure, and immediately after exercise. Total cholesterol concentrations decreased significantly at exercise kilojoule expenditures of 4604.4, 5441.5 and 5860.1 (1100, 1300 and 1400 kcal). There were also exercise induced increases in high-density lipoprotein cholesterol (HDL-C) and HDL2-C concentrations immediately after exercise. Although acute lipid and lipoprotein changes are typically reported in the days following exercise, the Current data indicate that some lipoprotein concentrations change during acute exercise. Our data suggest that a threshold of exercise may be necessary to change lipoproteins during exercise. Future work Should identify potential mechanisms (lipoprotein lipase, cholesterol ester transport protein, LDL uptake) that alter lipoprotein concentrations during prolonged exercise.