Proteome analysis for identification of target proteins of genistein in primary human endothelial cells stressed with oxidized LDL or homocysteine

Background Epidemiological studies suggest that soy consumption contributes to the prevention of coronary heart disease. The proposed anti-atherogenic effects of soy appear to be carried by the soy isoflavones with genistein as the most abundant compound. Aim of the study To identify proteins or pathways by which genistein might exert its protective activities on atherosclerosis, we analyzed the proteomic response of primary human umbilical vein endothelial cells ( HUVEC) that were exposed to the pro-atherosclerotic stressors homocysteine or oxidized low-density lipoprotein (ox-LDL). Methods HUVEC were incubated with physiological concentrations of homocysteine or ox-LDL in the absence and presence of genistein at concentrations that can be reached in human plasma by a diet rich in soy products (2.5 muM) or by pharmacological intervention ( 25 muM). Proteins from HUVEC were separated by two-dimensional polyacrylamide gel electrophoresis and those that showed altered expression level upon genistein treatment were identified by peptide mass fingerprints derived from tryptic digests of the protein spots. Results Several proteins were found to be differentially affected by genistein. The most interesting proteins that were potently decreased by homocysteine treatment were annexin V and lamin A. Annexin V is an antithrombotic molecule and mutations in nuclear lamin A have been found to result in perturbations of plasma lipids associated with hypertension. Genistein at low and high concentrations reversed the stressor-induced decrease of these anti-atherogenic proteins. Ox-LDL treatment of HUVEC resulted in an increase in ubiquitin conjugating enzyme 12, a protein involved in foam cell formation. Treatment with genistein at both doses reversed this effect. Conclusions Proteome analysis allows the identification of potential interactions of dietary components in the molecular process of atherosclerosis and consequently provides a powerful tool to define biomarkers of response.

Plasma ghrelin and oxytomodulin concentrations in lactating dairy cows receiving abomasal soybean oil, corn starch, and casein infusions

The effects of increased postruminal supply of casein, corn starch, and soybean oil on plasma concentrations of the gastrointestinal hormones ghrelin and oxyntomodulin (OXM) were investigated. Four mid-lactation Holstein cows were used in a 4×4 Latin square. Treatments were continuous abomasal infusions (23h/d) for 7 d of water, soybean oil (500g/d), corn starch (1100g/d), or casein (800g/d). Jugular vein plasma was obtained every 30min for 7h on days 1 and 7. Soybean oil and casein infusion decreased preprandial plasma ghrelin concentration by approximately 20% on both d (time-by-treatment P<0.10); however, dry matter intake (DMI) was depressed only after 7 d of oil infusion. Infusion of soybean oil, corn starch, or casein did not change the plasma OXM concentration (P>0.20). The present data indicate that plasma ghrelin concentration is depressed immediately before feeding by the postruminal infusion of soybean oil and casein, but it is not affected during the postprandial period. Plasma ghrelin concentration was not altered (P>0.20), pre- or postfeeding, by increased postruminal supply of corn starch. In addition, plasma OXM concentration did not respond (P>0.20) to postruminal nutrient infusion. In conclusion, a decrease in DMI when fat is infused could be partially explained by the decrease in prefeeding plasma ghrelin concentration, but a decrease in prefeeding plasma ghrelin concentration is not always associated with a decrease in DMI, as observed for the infusion of casein. Plasma OXM concentration was not affected by postruminal infusion of macronutrients.

Effect of abomasal glucose infusion on plasma concentrations of gut peptides in periparturient dairy cows

Six Holstein cows fitted with ruminal cannulas and permanent indwelling catheters in the portal vein, hepatic vein, mesenteric vein, and an artery were used to study the effects of abomasal glucose infusion on splanchnic plasma concentrations of gut peptides. The experimental design was a randomized block design with repeated measurements. Cows were assigned to one of 2 treatments: control or infusion of 1,500 g of glucose/d into the abomasum from the day of parturition to 29 d in milk. Cows were sampled 12 ± 6 d prepartum and at 4, 15, and 29 d in milk. Concentrations of glucose-dependent insulinotropic polypeptide, glucagon-like peptide 1(7–36) amide, and oxyntomodulin were measured in pooled samples within cow and sampling day, whereas active ghrelin was measured in samples obtained 30 min before and after feeding at 0800 h. Postpartum, dry matter intake increased at a lower rate with infusion compared with the control. Arterial, portal venous, and hepatic venous plasma concentrations of the measured gut peptides were unaffected by abomasal glucose infusion. The arterial, portal venous, and hepatic venous plasma concentrations of glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1(7–36) amide increased linearly from 12 d prepartum to 29 d postpartum. Plasma concentrations of oxyntomodulin were unaffected by day relative to parturition. Arterial and portal venous plasma concentrations of ghrelin were lower postfeeding compared with prefeeding concentrations. Arterial plasma concentrations of ghrelin were greatest prepartum and lowest at 4 d postpartum, giving a quadratic pattern of change over the transition period. Positive portal venous-arterial and hepatic venous–arterial concentration differences were observed for glucagon-like peptide 1(7–36) amide. A negative portal venous–arterial concentration difference was observed for ghrelin pre-feeding. The remaining portal venous–arterial and hepatic venous–arterial concentration differences of gut peptides did not differ from zero. In conclusion, increased postruminal glucose supply to postpartum transition dairy cows reduced feed intake relative to control cows, but did not affect arterial, portal venous, or hepatic venous plasma concentrations of gut peptide hormones. Instead, gut peptide plasma concentrations increased as lactation progressed. Thus, the lower feed intake of postpartum dairy cows receiving abomasal glucose infusion was not attributable to changes in gut peptide concentrations.

Effect of feeding fat or intrajugular infusion of glucagon-like peptide-1 and cholecystokinin on dry matter intake, digestibility, and digesta rate of passage in growing wethers

A cause and effect relationship between glucagon-like peptide 1 (7, 36) amide (GLP-1) and cholecystokinin (CCK) and DMI regulation has not been established in ruminants. Three randomized complete block experiments were conducted to determine the effect of feeding fat or infusing GLP-1 or CCK intravenously on DMI, nutrient digestibility, and Cr rate of passage (using Cr(2)O(3) as a marker) in wethers. A total of 18 Targhee × Hampshire wethers (36.5 ± 2.5 kg of BW) were used, and each experiment consisted of four 21-d periods (14 d for adaptation and 7 d for infusion and sampling). Wethers allotted to the control treatments served as the controls for all 3 experiments; experiments were performed simultaneously. The basal diet was 60% concentrate and 40% forage. In Exp. 1, treatments were the control (0% added fat) and addition of 4 or 6% Ca salts of palm oil fatty acids (DM basis). Treatments in Exp. 2 and 3 were the control and 3 jugular vein infusion dosages of GLP-1 (0.052, 0.103, or 0.155 µg•kg of BW(-1)•d(-1)) or CCK (0.069, 0.138, or 0.207 µg•kg of BW(-1)•d(-1)), respectively. Increases in plasma GLP-1 and CCK concentrations during hormone infusions were comparable with increases observed when increasing amounts of fat were fed. Feeding fat and infusion of GLP-1 tended (linear, P = 0.12; quadratic, P = 0.13) to decrease DMI. Infusion of CCK did not affect (P > 0.21) DMI. Retention time of Cr in the total gastrointestinal tract decreased (linear, P < 0.01) when fat was fed, but was not affected by GLP-1 or CCK infusion. In conclusion, jugular vein infusion produced similar plasma CCK and GLP-1 concentrations as observed when fat was fed. The effects of feeding fat on DMI may be partially regulated by plasma concentration of GLP-1, but are not likely due solely to changes in a single hormone concentration.

Final report on research project PS2010, proof of concept of automated mapping of weeds in arable fields

Many weeds occur in patches but farmers frequently spray whole fields to control the weeds in these patches. Given a geo-referenced weed map, technology exists to confine spraying to these patches. Adoption of patch spraying by arable farmers has, however, been negligible partly due to the difficulty of constructing weed maps. Building on previous DEFRA and HGCA projects, this proposal aims to develop and evaluate a machine vision system to automate the weed mapping process. The project thereby addresses the principal technical stumbling block to widespread adoption of site specific weed management (SSWM). The accuracy of weed identification by machine vision based on a single field survey may be inadequate to create herbicide application maps. We therefore propose to test the hypothesis that sufficiently accurate weed maps can be constructed by integrating information from geo-referenced images captured automatically at different times of the year during normal field activities. Accuracy of identification will also be increased by utilising a priori knowledge of weeds present in fields. To prove this concept, images will be captured from arable fields on two farms and processed offline to identify and map the weeds, focussing especially on black-grass, wild oats, barren brome, couch grass and cleavers. As advocated by Lutman et al. (2002), the approach uncouples the weed mapping and treatment processes and builds on the observation that patches of these weeds are quite stable in arable fields. There are three main aspects to the project. 1) Machine vision hardware. Hardware component parts of the system are one or more cameras connected to a single board computer (Concurrent Solutions LLC) and interfaced with an accurate Global Positioning System (GPS) supplied by Patchwork Technology. The camera(s) will take separate measurements for each of the three primary colours of visible light (red, green and blue) in each pixel. The basic proof of concept can be achieved in principle using a single camera system, but in practice systems with more than one camera may need to be installed so that larger fractions of each field can be photographed. Hardware will be reviewed regularly during the project in response to feedback from other work packages and updated as required. 2) Image capture and weed identification software. The machine vision system will be attached to toolbars of farm machinery so that images can be collected during different field operations. Images will be captured at different ground speeds, in different directions and at different crop growth stages as well as in different crop backgrounds. Having captured geo-referenced images in the field, image analysis software will be developed to identify weed species by Murray State and Reading Universities with advice from The Arable Group. A wide range of pattern recognition and in particular Bayesian Networks will be used to advance the state of the art in machine vision-based weed identification and mapping. Weed identification algorithms used by others are inadequate for this project as we intend to collect and correlate images collected at different growth stages. Plants grown for this purpose by Herbiseed will be used in the first instance. In addition, our image capture and analysis system will include plant characteristics such as leaf shape, size, vein structure, colour and textural pattern, some of which are not detectable by other machine vision systems or are omitted by their algorithms. Using such a list of features observable using our machine vision system, we will determine those that can be used to distinguish weed species of interest. 3) Weed mapping. Geo-referenced maps of weeds in arable fields (Reading University and Syngenta) will be produced with advice from The Arable Group and Patchwork Technology. Natural infestations will be mapped in the fields but we will also introduce specimen plants in pots to facilitate more rigorous system evaluation and testing. Manual weed maps of the same fields will be generated by Reading University, Syngenta and Peter Lutman so that the accuracy of automated mapping can be assessed. The principal hypothesis and concept to be tested is that by combining maps from several surveys, a weed map with acceptable accuracy for endusers can be produced. If the concept is proved and can be commercialised, systems could be retrofitted at low cost onto existing farm machinery. The outputs of the weed mapping software would then link with the precision farming options already built into many commercial sprayers, allowing their use for targeted, site-specific herbicide applications. Immediate economic benefits would, therefore, arise directly from reducing herbicide costs. SSWM will also reduce the overall pesticide load on the crop and so may reduce pesticide residues in food and drinking water, and reduce adverse impacts of pesticides on non-target species and beneficials. Farmers may even choose to leave unsprayed some non-injurious, environmentally-beneficial, low density weed infestations. These benefits fit very well with the anticipated legislation emerging in the new EU Thematic Strategy for Pesticides which will encourage more targeted use of pesticides and greater uptake of Integrated Crop (Pest) Management approaches, and also with the requirements of the Water Framework Directive to reduce levels of pesticides in water bodies. The greater precision of weed management offered by SSWM is therefore a key element in preparing arable farming systems for the future, where policy makers and consumers want to minimise pesticide use and the carbon footprint of farming while maintaining food production and security. The mapping technology could also be used on organic farms to identify areas of fields needing mechanical weed control thereby reducing both carbon footprints and also damage to crops by, for example, spring tines. Objective i. To develop a prototype machine vision system for automated image capture during agricultural field operations; ii. To prove the concept that images captured by the machine vision system over a series of field operations can be processed to identify and geo-reference specific weeds in the field; iii. To generate weed maps from the geo-referenced, weed plants/patches identified in objective (ii).

An isotope dilution model for partitioning phenylalanine uptake by the liver of lactating dairy cows

An isotope dilution model for partitioning phenylalanine uptake by the liver of the lactating dairy cow was constructed and solved in the steady state. If assumptions are made, model solution permits calculation of the rate of phenylalanine uptake from portal vein and hepatic arterial blood supply, phenylalanine release into the hepatic vein, phenylalanine oxidation and synthesis, and degradation of hepatic constitutive and export proteins. The model requires the measurement of plasma fow rate through the liver in combination with phenylalanine concentrations and plateau isotopic enrichments in arterial, portal and hepatic plasma during a constant infusion of [1-13C]phenylalanine tracer. The model can be applied to other amino acids with similar metabolic fates and will provide a means for assessing the impact of hepatic metabolism on amino acid availability to peripheral tissues. This is of particular importance for the dairy cow when considering the requirements for milk protein synthesis and the negative environmental impact of excessive nitrogen excretion.

Hypoxic modulation of ca(2+) signaling in human venous and arterial endothelial cells

Our understanding of vascular endothelial cell physiology is based on studies of endothelial cells cultured from various vascular beds of different species for varying periods of time. Systematic analysis of the properties of endothelial cells from different parts of the vasculature is lacking. Here, we compare Ca(2+) homeostasis in primary cultures of endothelial cells from human internal mammary artery and saphenous vein and how this is modified by hypoxia, an inevitable consequence of bypass grafting (2.5% O(2), 24 h). Basal [Ca(2+)]( i ) and store depletion-mediated Ca(2+) entry were significantly different between the two cell types, yet agonist (ATP)-mediated mobilization from endoplasmic reticulum stores was similar. Hypoxia potentiated agonist-evoked responses in arterial, but not venous, cells but augmented store depletion-mediated Ca(2+) entry only in venous cells. Clearly, Ca(2+) signaling and its remodeling by hypoxia are strikingly different in arterial vs. venous endothelial cells. Our data have important implications for the interpretation of data obtained from endothelial cells of varying sources.

Janus PEG-based dendrimers for use in combination therapy: controlled multi-drug loading and sequential release

The increasing use of drug combinations to treat disease states, such as cancer, calls for improved delivery systems that are able to deliver multiple agents. Herein, we report a series of novel Janus dendrimers with potential for use in combination therapy. Different generations (first and second) of PEG-based dendrons containing two different “model drugs”, benzyl alcohol (BA) and 3-phenylpropionic acid (PPA), were synthesized. BA and PPA were attached via two different linkers (carbonate and ester, respectively) to promote differential drug release. The four dendrons were coupled together via (3 + 2) cycloaddition chemistries to afford four Janus dendrimers, which contained varying amounts and different ratios of BA and PPA, namely, (BA)2-G1-G1-(PPA)2, (BA)4-G2-G1-(PPA)2, (BA)2-G1-G2-(PPA)4, and (BA)4-G2-G2-(PPA)4. Release studies in plasma showed that the dendrimers provided sequential release of the two model drugs, with BA being released faster than PPA from all of the dendrons. The different dendrimers allowed delivery of increasing amounts (0.15–0.30 mM) and in exact molecular ratios (1:2; 2:1; 1:2; 2:2) of the two model drug compounds. The dendrimers were noncytotoxic (100% viability at 1 mg/mL) toward human umbilical vein endothelial cells (HUVEC) and nontoxic toward red blood cells, as confirmed by hemolysis studies. These studies demonstrate that these Janus PEG-based dendrimers offer great potential for the delivery of drugs via combination therapy.

Short communication: plasma concentration of glucose-dependent insulinotropic polypeptide may regulate milk energy production in lactating dairy cows

In dairy cows, an increase in plasma concentration of glucose-dependent insulinotropic polypeptide (GIP) is associated with an increase in metabolizable energy intake, but the role of GIP in energy partitioning of dairy cattle is not certain. The objective of this study was to examine the relationship between plasma GIP concentrations and energy partitioning toward milk production. Four mid-lactation, primiparous, rumenfistulated Holstein-Friesian cows were fed a control diet of 55% forage and 45% concentrate [dry matter (DM) basis] in a 4 × 4 Latin square design with 4-wk periods. The 4 treatments were (1) control diet fed at 1000 and 1600 h, and (2) once-daily (1000 h) feeding, (3) twice daily (1000 and 1600 h) feeding, and (4) 4 times/d (1000, 1600, 2200 and 0400 h) feeding of the control diet plus 1 dose (1.75 kg on a DM basis at 0955 h) into the rumen of supplemental vegetable proteins (Amino Green; SCA NuTec Ltd., Thirsk, UK). Measurements of respiratory exchange and energy balance were obtained over 4 d during the last week of each period while cows were housed in open-circuit respiration chambers. Blood was collected from the jugular vein every 30 min for 12 h, using indwelling catheters, starting at 0800 h on d 20 of each period. Plasma GIP concentration was measured in samples pooled over each 5 consecutive blood samplings. The relationships between plasma GIP, DM intake, heat production, respiratory quotient, milk yield, and milk energy output were analyzed using linear correlation procedures, with metabolizable intake as a partial variant. Plasma GIP concentration was not correlated with heat production, or milk yield, but was positively correlated with milk energy yield (correlation coefficient = 0.67) and negatively correlated with RQ (correlation coefficient = −0.72). The correlations between GIP and RQ and milk energy output do not imply causality, but suggest that a role for GIP may exist in the regulation of energy metabolism in dairy cows.