Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells

Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INF alpha and INF gamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry.

Young's modulus varies with differential orientation of keratin in feathers

Feathers are composed of a structure that, whilst being very light, is able to withstand the large aerodynamic forces exerted upon them during flight. To explore the contribution of molecular orientation to feather keratin mechanical properties, we have examined the nanoscopic organisation of the keratin molecules by X-ray diffraction techniques and have confirmed a link between this and the Young's modulus of the feather rachis. Our results indicate that along the rachis length, from calamus to tip, the keratin molecules become more aligned than at the calamus before returning to a state of higher mis-orientation towards the tip of the rachis. We have also confirmed the general trend of increasing Young's modulus with distance along the rachis. Furthermore, we report a distinct difference in the patterns of orientation of beta-keratin in the feathers of flying and flightless birds. The trend for increased modulus along the feathers of volant birds is absent in the flightless ostrich.

Effect of oxidized low-density lipoprotein on differential gene expression in primary human endothelial cells

Oxidative modification of low-density lipoprotein (LDL) plays an important role in the initiation and progression of atherosclerosis. It has been proposed that the biological action of oxidized LDL (ox-LDL) may be partially attributed to its effect on a shift of the pattern of gene expression in endothelial cells. To examine the transcriptional response to ox-LDL, we applied cDNA array technology to cultured primary human endothelial cells challenged with oxidized human LDL. A twofold or greater difference in the expression of a particular gene was considered a significant difference in transcript abundance. Seventy-eight of the 588 genes analyzed were differentially expressed in response to the treatment. Ox-LDL significantly affected the expression of genes encoding for transcription factors, cell receptors, growth factors, adhesion molecules, extracellular matrix proteins, and enzymes involved in cholesterol metabolism. The alteration of the expression pattern of several genes was substantiated post hoc using RT-PCR. The experimental strategy identified several novel ox-LDL-sensitive genes associated with a "response to injury" providing a conceptual background to be utilized for future studies addressing the molecular basis of the early stages of atherogenesis.

Dietary vitamin E modulates differential gene expression in the rat hippocampus: potential implications for its neuroprotective properties

A wide range of cell culture, animal and human epidemiological studies are suggestive of a role of vitamin E (VE) in brain function and in the prevention of neurodegeneration. However, the underlying molecular mechanisms remain largely unknown. In the current investigation Affymetrix gene chip technology was utilised to establish the impact of chronic VE deficiency on hippocampal genes expression. Male albino rats were fed either a VE deficient or standard diet (60 mg/kg feed) for a period of 9 months. Rats were sacrificed, the hippocampus removed and genes expression established in individual animals. VE deficiency showed to have a strong impact on genes expression in the hippocampus. An important number of genes found to be regulated by VE was associated with hormones and hormone metabolism, nerve growth factor, apoptosis, dopaminergic neurotransmission, and clearance of amyloid-beta and advanced glycated endproducts. In particular, VE strongly affected the expression of an array of genes encoding for proteins directly or indirectly involved in the clearance of amyloid beta, changes which are consistent with a protective effect of VE on Alzheimer's disease progression.

Dietary alpha-tocopherol affects differential gene expression in rat testes

Gene-chip technology was employed to study the effect of dietary vitamin E (VE) on gene expression in rat testes. Male albino rats were fed with either a diet deficient in VE or a standard diet containing VE. Differential gene expression was monitored at five individual time-points over a period of 14 months with all animals individually pro. led. Low VE intake resulted in the consistent upregulation of 7-dehydrocholesterol reductase and GATA binding protein 4, both involved in testosterone synthesis. Cyclin D3, important in cell cycle progression and Wilms tumor 1, related to cancer development, were also up-regulated in the vitamin E deficient animals. This study demonstrates that low dietary VE intake has long-term effects on gene expression in the testes. Our data provides insights into the possible molecular mechanisms underlying the beneficial effects of vitamin E on the male reproductive organ.

Effect of Ginkgo biloba (EGb 761®) on differential gene expression

Supplementation of diets with plant extracts such as ginkgo biloba extract (EGb 761®) (definition see editorial) for health and prevention of degenerative diseases is popular. However, it is often difficult to analyse the biological activities of plant extracts due to their complex nature and the possible synergistic and/or antagonistic effects of their components. Genome-wide expression monitoring with high-density oligonucleotide arrays provides one way to examine the molecular targets of plant extracts and may prove a useful tool in evaluating their therapeutic claims. Here, we will briefly describe some of our work on the effect of EGb 761® on differential gene expression in relation to its potential anti-carcinogenic, photoprotective and neuromodulatory properties.

Differential uptake of subfractions of triglyceride-rich lipoproteins by THP-1 macrophages

It is well known that raised plasma triglycerides (TG) are positively linked to the development of coronary heart disease. However, triglycerides circulate in a range of distinct lipoprotein subtractions and the relative atherogenicity of these subtractions is not clear. In this study, three fractions of triglyceride rich lipoprotein (TRL) were isolated from normolipidaemic males according to their differing Svedberg flotation (S-f) rates: chylomicron (CM, S-f > 400), very low-density lipoprotein (VLDL)-1 (S-f 60-400) and VLDL-2 (S-f 20-60). These fractions were incubated with THP-1 monocyte-derived macrophages for determination of cholesterol and TG accumulation, in the presence and absence of the lipoprotein lipase (LPL) inhibitor orlistat. Expression of LDL receptor related protein (LRP) and apolipoprotein B48 receptor (apoB48R) was also examined in both differentiating monocytes, and monocyte-derived macrophages, incubated with TRL. VLDL-I caused a significantly greater accumulation of TG within macrophages compared to VLDL-2. Binding studies also tended to show a greater preference for VLDL-1. No change in expression of LRP or apoB48R was observed in fully differentiated macrophages incubated with VLDL-1, VLDL-2 or CM, although a greater expression of LRP mRNA was observed in differentiating monocytes exposed to VLDL-1, compared to those incubated with CM or VLDL-2. TG loading in response to all three TRL fractions was blocked by orlistat, suggesting that it is likely that the major pathway for uptake of TG was hydrolysis by LPL. Calculations suggested that direct uptake of particles accounts for between 12 and 25% of total TAG uptake. In conclusion, THP monocyte-derived macrophages demonstrate a preference for VLDL-1, both through the LPL pathway and by direct uptake of whole particles. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Differential effects of apolipoprotein E3 and E4 on markers of oxidative status in macrophages

ApoE is secreted by macrophages at the lesion site of the atherosclerotic plaque, where it is thought to play a protective role against atherosclerosis independently of its effects on lipid metabolism. Of the three common isoforms for apoE, apoE4 is associated with higher risk of cardiovascular disease (CVD). In vitro studies have shown that recombinant apoE may act as an antioxidant in an isoform-dependent manner (E2 > E3 > E4). The oxidative status of the macrophages plays a key role in the process of atherosclerosis. In the present study the possible differential actions of apoE3 and apoE4 on several parameters of oxidative status were determined in stably transfected murine macrophages (RAW 2647-apoE3 and apoE4). No differences between genotypes were observed after peroxide challenge in either protection against cytotoxicity or in cell membrane oxidation, and modest differences were observed in the non-enzymatic antioxidants (glutathione and a-tocopherol) in apoE3 v. apoE4 macrophages. Importantly, cells secreting apoE4 showed increased membrane oxidation under basal conditions, and produced more NO and superoxide anion radicals than the apoE3 macrophages after stimulation. The present data suggest that apoE genotype influences the oxidative status of macrophages, and this could partly contribute to the higher CVD risk observed in apoE4 carriers.

Plant- and marine-derived n-3 polyunsaturated fatty acids have differential effects on fasting and postprandial blood lipid concentrations and on the susceptibility of LDL to oxidative modification in moderately hyperlipidemic subjects

Background: Dietary a-linolenic acid (ALA) can be converted to long-chain n-3 polyunsaturated fatty acids (PUFAs) in humans and may reproduce some of the beneficial effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on cardiovascular disease risk factors. Objective: This study aimed to compare the effects of increased dietary intakes of ALA and EPA+DHA on a range of atherogenic risk factors. Design: This was a placebo-controlled, parallel study involving 150 moderately hyperlipidemic subjects randomly assigned to 1 of 5 interventions: 0.8 or 1.7 g EPA+DHA/d, 4.5 or 9.5 g ALA/d, or an n-6 PUFA control for 6 mo. Fatty acids were incorporated into 25 g of fat spread and 3 capsules to be consumed daily. Results: The change in fasting or postprandial lipid, glucose, or insulin concentrations or in blood pressure was not significantly different after any of the n-3 PUFA interventions compared with the n-6 PUFA control. The mean (+/-SEM) change in fasting triacylglycerols after the 1.7-g/d EPA+DHA intervention (-7.7 +/- 4.99%) was significantly (P < 0.05) different from the change after the 9.5-g/d ALA intervention (10.9 +/- 4.5%). The ex vivo susceptibility of LDL to oxidation was higher after the 1.7-g/d EPA+DHA intervention than after the control and ALA interventions (P < 0.05). There was no significant change in plasma a-tocopherol concentrations or in whole plasma antioxidant status in any of the groups. Conclusion: At estimated biologically equivalent intakes, dietary ALA and EPA+DHA have different physiologic effects.

Differential induction of apoptosis in human colonic carcinoma cells (Caco-2) by Atopobium, and commensal, probiotic and enteropathogenic bacteria: mediation by the mitochondrial pathway

The induction of apoptosis in mammalian cells by bacteria is well reported. This process may assist infection by pathogens whereas for non-pathogens apoptosis induction within carcinoma cells protects against colon cancer. Here, apoptosis induction by a major new gut bacterium, Atopobium minutum, was compared with induction by commensal (Escherichia coli K-12 strains), probiotic (Lactobacillus rhamnosus, Bifidobacterium latis) and pathogenic (E. coli: EPEC and VTEC) gut bacteria within the colon cancer cell line, Caco-2. The results show a major apoptotic effect for the pathogens, mild effects for the probiotic strains and A. minutum, but no effect for commensal E. coli. The mild apoptotic effects observed are consistent with the beneficial roles of probotics in protection against colon cancer and suggest, for the first time, that A. minutum possesses similar advantageous, anti-cancerous activity. Although bacterial infection increased Caco-2 membrane FAS levels, caspase-8 was not activated indicating that apoptosis is FAS independent. Instead, in all cases, apoptosis was induced through the mitochondrial pathway as indicated by BAX translocation, cytorchrome c release, and caspase-9 and -3 cleavage. This suggests that an intracellular stimulus initiates the observed apoptosis responses.

The differential tissue distribution of the citrus flavanone naringenin following gastric instillation

Citrus flavonoids have been investigated for their biological activity, with both anti-inflammatory and -carcinogenic effects being reported. However, little information is known on the bioavailability of these compounds in vivo. The objectives of this study were to determine the tissue distribution of naringenin after gastric gavage of [H-3]-naringenin to rats. Unlabelled naringenin was also used to quantify the levels of naringenin and its major metabolites in tissues and eliminated in the urine and faeces. Significant radioactivity was detected in the plasma as well as all tissues examined 2 h post-gavage. After 18 h, higher levels of radioactivity were retained in plasma and tissues (55% of the administered radioactivity). Investigation of the nature of metabolites, using unlabelled naringenin, revealed that the glucuronides were the major components in plasma, tissues and urine, in addition to the colonic metabolite 3-(4- hydroxyphenyl) propionic acid, detected in the urine. The aglycone was the form extensively retained in tissues after 18 h post-gavage. Total identified metabolites detected after 18 h in most tissues were only 1-5% of the levels detected after 2 h. However, the brain, lungs and heart retained 27, 20 and 11%, respectively, relative to the total metabolites detected at 2 h. While radioactive detection suggests increased levels of breakdown products of naringenin after 18 h versus 2 h, the products identified using unlabelled naringenin are not consistent with this, suggesting that a predominant proportion of the naringenin breakdown products at 18 h are retained as smaller decomposition molecules which cannot yet be identified.

Differential expression of mRNAs encoding co-receptor (betaglycan) and activin type-I preovulatory and prehierarchical follicles of the putative inhibin and type-II receptors in the laying hen ovary

Ovarian follicle development is primarily regulated by an interplay between the pituitary gonadotrophins, LH and FSH, and ovary-derived steroids. Increasing evidence implicates regulatory roles of transforming growth factor-beta (TGF beta) superfamily members, including inhibins and activins. The aim of this study was to identify the expression of mRNAs encoding key receptors of the inhibin/activin system in ovarian follicles ranging from 4 mm in diameter to the dominant F1 follicle (similar to 40 turn). Ovaries were collected (n=16) from inid-sequence hens maintained on a long-day photoschedule (16h of light:8 h of darkness). All follicles removed were dissected into individual granulosa and thecal layers. RNA was extracted and cDNA synthesized. Real-time quantitative PCR was used to quantify the expression of niRNA encoding betaglycan, activin receptor (ActR) subtypes (type-I, -IIA and -IIB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); receptor expression data were normalized to GAPDH expression. Detectable levels of ActRI, -IIA and -IIB and the inhibin co-receptor (betaglycan) expression were found in all granulosa and thecal layers analysed. Granulosa ActRI mRNA peaked (P < 0(.)05) in 8-9(.)9 mm follicles, whereas ActRIIA rose significantly from 6-7(.)9 mm to 8-9(.)9 nun, before filling to F3/2; levels then rose sharply (3-fold) to F1 levels. Granulosa betaglycan niRNA expression rose 3-fold from 4-5(.)9 min to 8-9(.)9 mm, before falling 4-fold to F3/2; levels then rose sharply (4-fold) to F1 levels. ActRIIB levels did not vary significantly during follicular development. Thecal ActRI mRNA expression was similar from 4-7(.)9 mm then decreased significantly to a nadir at the F4 position, before increasing 2-fold to the F1 (P < 0(.)05). Although thecal ActRIIB and -IIA expression did not vary significantly from 4 nim to F3, ActRIIB expression increased significantly (2-fold) from F3 to F1 and ActIIA, increased 22-fold from F2 to F1 (P < 0(.)05). Thecal betaglycan fell to a nadir at F6 after follicle selection; levels then increased significantly to F2, before filling similar to 50% in the F I. In all follicles studied expression of betaglycan and ActRI (granulosa: 1-0(.)65, P < 0-001, n=144/group; theca: r=0(.)49, P < 0-001, n=144/group) was well correlated. No significant correlations were identified between betaglycan and ActRIIA or -IIB. Considering all follicles analysed, granulosa mRNA expression of betaglycan, ActRI ActRIIA and ActRIIB were all significantly lower than in corresponding thecal tissue (betaglycan, 11(.)4-fold; ActRIIB, 5(.)1-fold; ActR(.) 3-8-fold: ActRIIA, 2(.)8-fold). The co-localization of type-I and -II activin receptors and betaglycan on granulosa and thecal cells are consistent with a local auto/paracrine role of inhibins and activins in modulating ovarian follicle development, selection and progression in the domestic fowl.

A differential thermal expansion approach to crystal structure determination from powder diffraction data

Differential thermal expansion over the range 90-210 K has been applied successfully to determine the crystal structure of chlorothiazide from synchrotron powder diffraction data using direct methods. Key to the success of the approach is the use of a multi-data-set Pawley refinement to extract a set of reflection intensities that is more 'single-crystal-like' than those extracted from a single data set. The improvement in reflection intensity estimates is quantified by comparison with reference single-crystal intensities. (C) 2008 International Union of Crystallography Printed in Singapore - all rights reserved

Differential cognitive effects of Ginkgo biloba after acute and chronic treatment in healthy young volunteers

Acute doses of Ginkgo biloba have been shown to improve attention and memory in young, healthy participants, but there has been a lack of investigation into possible effects on executive function. In addition, only one study has investigated the effects of chronic treatment in young volunteers. This study was conducted to compare the effects of ginkgo after acute and chronic treatment on tests of attention, memory and executive function in healthy university students. Using a placebo-controlled double-blind design, in experiment 1, 52 students were randomly allocated to receive a single dose of ginkgo (120 mg, n=26) or placebo (n=26), and were tested 4h later. In experiment 2, 40 students were randomly allocated to receive ginkgo (120 mg/day; n=20) or placebo (n=20) for a 6-week period and were tested at baseline and after 6 weeks of treatment. In both experiments, participants underwent tests of sustained attention, episodic and working memory, mental flexibility and planning, and completed mood rating scales. The acute dose of ginkgo significantly improved performance on the sustained-attention task and pattern-recognition memory task; however, there were no effects on working memory, planning, mental flexibility or mood. After 6 weeks of treatment, there were no significant effects of ginkgo on mood or any of the cognitive tests. In line with the literature, after acute administration ginkgo improved performance in tests of attention and memory. However, there were no effects after 6 weeks, suggesting that tolerance develops to the effects in young, healthy participants.

Estimation of Hidden Markov Models parameters using differential evolution

Hidden Markov Models (HMMs) have been successfully applied to different modelling and classification problems from different areas over the recent years. An important step in using HMMs is the initialisation of the parameters of the model as the subsequent learning of HMM’s parameters will be dependent on these values. This initialisation should take into account the knowledge about the addressed problem and also optimisation techniques to estimate the best initial parameters given a cost function, and consequently, to estimate the best log-likelihood. This paper proposes the initialisation of Hidden Markov Models parameters using the optimisation algorithm Differential Evolution with the aim to obtain the best log-likelihood.