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The cell walls of forage chicory (Cichorium intybus) leaves are known to contain high proportions of pectic polysaccharides. However, little is known about the distribution of pectic polysaacharides among walls of different cell types/tissues and within walls. In this study, immunolabelling with four monoclonal antibodies was used to map the distribution of pectic polysaccharides in the cell walls of the laminae and midribs of these leaves. The antibodies JIM5 and JIM7 are specific for partially methyl-esterified homogalacturonans; LM5 and LM6 are specific for (1→4)-β-galactan and (1→5)-α-arabinan side chains, respectively, of rhamnogalacturonan I. All four antibodies labelled the walls of the epidermal cells with different intensities. JIM5 and JIM7, but not LM5 or LM6, labelled the middle lamella, tricellular junctions, and the corners of intercellular spaces of ground, xylem and phloem parenchyma. LM5, but not LM6, strongly labelled the walls of the few sclerenchyma fibres in the phloem of the midrib and lamina vascular bundles. The LM5 epitope was absent from some phloem parenchyma cells. LM6, but not LM5, strongly labelled the walls of the stomatal guard cells. The differential distribution of pectic epitopes among walls of different cell types and within walls may reflect the deposition and modification of these polysaccharides which are involved in cell wall properties and cell development.

Leishmaniasis is one of the most relevant neglected tropical diseases in the world, affecting 14 million people. Despite the high morbidity, mortality and socio-economic impact, few therapeutic options are available for this disease. To make matters worse, the available molecules have several limitations such as limited efficacy, high cost, side effects and increased resistance. In this context, our group previously synthesized new compounds with anti-leishmania potential being the bis(N-[4-(hydroxyphenyl)methyl]-2-pyridinemethamine)zinc perchlorate monohydrate 4 (complex 4) the most promising one. Therefore, this present work revealed some morphological and physiological changes promoted by complex 4 on Leishmania amazonensis promastigotes as well as it was evidenced its potential against intramacrophage amastigotes. Complex 4 promoted a progressive reduction on the promastigotes size and a remarkable increase on the granularity/complexity as judged by flow cytometry. Transmission electron microscopy (TEM) analysis revealed extensive mitochondrial and plasma membrane alterations, although plasma membrane integrity remained. The mitochondrial changes observed by TEM were accompanied by a decrease in the activity of mitochondrial dehydrogenases with increased production of reactive oxygen species. Interestingly, promastigotes also showed changes in lipid metabolism. Besides the very low toxicity to macrophages, complex 4 had a great effect on intramacrophage amastigotes, displaying an IC50 of 3.91 μM. Collectively, the data presented here reinforce the potential of aminopyridyl compounds complexed to zinc against L. amazonensis. Thus, our work serves as a basis for in vivo assays to be designed or even the synthesis of more selective/effective compounds with lower cost.

Environmental epigenetics is a relatively new area of discipline and represents a new approach for conducting risk assessments. To date, most focus studies have centered around the epigenetics of heavy metals such as arsenic, mercury, lead, and calcium. The induction of different epigenetic mechanisms in various tissues or different cell types may offer insight into complex nature of dioxin toxicity and aryl hydrocarbon receptor functions. Mounting evidence indicate that dioxins and dioxin-like compounds can induce neurological changes in the developing fetus and infant. The revelation that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can alter global methylation status engenders potential questions related to how dioxin promotes tumorigenesis. Dioxins are structurally diverse polyhalogenated aromatic hydrocarbons that include polychlorinated dibenzodioxins, polychlorinated dibenzofurans, and polychlorinated biphenyls. Toxicologists have long recognized that TCDD and dioxin-like polychlorinated biphenyls haves a suppressive effect on the immune system.

Simple Summary Ruminant production relies on nutrients obtained from feed fermentation in the rumen. In grazing ruminant production systems, fresh forage is the major or sole source of feed. Feeding frequency and feeding level are two important factors affecting rumen fermentation. This study investigated how these factors affect the fermentation of fresh forage in the rumen for in-depth evaluation of the nutritional value of two types of forages and for better understanding of their digestion by ruminants. To achieve the objectives of the study, fresh chicory or perennial ryegrass was fed to sheep hourly or twice-daily at low or high feeding levels. The results indicated that rumen fermentation was affected by forage species, feeding frequency, feeding level and their interactions and the differences in rumen fermentation were more apparent when feeding was performed hourly rather than twice-daily. This study highlighted the importance of feeding frequency on manipulating sheep ruminal metabolism when fed fresh forage. Abstract Feeding frequency and feeding level are two important factors affecting rumen fermentation characteristics, but few studies on these have been conducted on fresh forage. Eight rumen-fistulated sheep were fed either fresh chicory or perennial ryegrass hourly in the first period (d 14 to 21) of the experiment and twice-daily in the second period (d 22 to 27) at 1.3 or 2.2 times the requirement of metabolizable energy for maintenance. When fed hourly, but not twice-daily, rumen fluid pH value was affected by forage species and feeding level. The total concentrations of short-chain fatty acid (SCFA) were similar at both feeding levels when fed chicory hourly, but they were greater at the higher feeding level in comparison with the lower feeding level when fed perennial ryegrass. However, forage species and feeding level did not affect rumen fluid total SCFA concentration when sheep were fed twice-daily. Therefore, rumen fermentation characteristics were affected by forage species, feeding frequency, feeding level and their interactions and the differences in fermentation characteristics were more apparent when feeding was performed hourly rather than twice-daily. This study highlighted the importance of feeding frequency on manipulating sheep ruminal metabolism when fed fresh forage.

The aim of this paper was to give a basic understanding of studies on methane emissions of New Zealand, as we know the agriculture of New Zealand is pastoral farming, most livestock animals are grazed in pasture, and quantities of methane were released from the digestive tract and animals excreta. In New Zealand some 50% greenhouse gases (GHG) sources are attributed to agriculture and one third is methane from livestock enteric formation. For many years, many researchers have been exploiting the techniques and methods to measure the emission of methane of New Zealand, further more studing the available options to alleviate the methane emissions. Their pioneering work and successful experiences including the determined methods and mitigation strategies are worth learning for scholars around the world. Some of their approaches were not only suitable for New Zealand grazed livestock, but for many other countries, even the animals are intensively bred in pen. The calorimeter/respiration chamber is the most exactly method in present, but it needs expensive equipments and skilled manipulators, so there are still some difficulty in applying this approach extensively in practice. Sulfur hexafluoride (SF6) trace technique is much adopted for grazed livestock evaluating the methane emission, though its veracity was doubted by some researchers, it is still a good option in present for studying the GHG emissions for grazing animals. By measuring the rumen volatile fatty acid (VFA) concentration to estimate the methane emission is a relatively simple approach, it is just only a rough evaluation, and it is unsuitable for exact study, but this method may be used in China for extensively raised ruminant. In present China, the ruminants are fed in an extensively managed state, the diversities of roughage and animals varieties caused difficult to exactly estimate the methane emission. So exploiting the available options is much important for constituting the exhaustive emission inventory. This review just outline some practical techniques of New Zealand, those maybe a good reference for researchers to carry out their studies in this field, after all New Zealand have been persisting many years and acquired great achievements in methane mitigation area.

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous environment through self-assembly. The hydrophobic core of micelles functions as a drug reservoir and encapsulates hydrophobic drugs. The hydrophilic shell prevents the aggregation of micelles and also prolongs their systemic circulation in vivo. In this protocol, we describe a method to synthesize a doxorubicin lipophilic pro-drug, doxorubicin-palmitic acid (DOX-PA), which will enhance drug loading into micelles. A pH-sensitive hydrazone linker was used to conjugate doxorubicin with the lipid, which facilitates the release of free doxorubicin inside cancer cells. Synthesized DOX-PA was purified with a silica gel column using dichloromethane/methanol as the eluent. Purified DOX-PA was analyzed with thin layer chromatography (TLC) and (1)H-Nuclear Magnetic Resonance Spectroscopy ((1)H-NMR). A film dispersion method was used to prepare DOX-PA loaded DSPE-PEG micelles. In addition, several methods for characterizing micelle formulations are described, including determination of DOX-PA concentration and encapsulation efficiency, measurement of particle size and distribution, and assessment of in vitro anticancer activities. This protocol provides useful information regarding the preparation and characterization of drug-loaded micelles and thus will facilitate the research and development of novel micelle-based cancer nanomedicines.

Four forage brassicas, kale (Brassica oleracea L. cv. Kestrel), turnip (B. campestris L. cv. Appin), rape (B. napus L. cv. Titan) and swede (B. napus L. cv. Dominion) were compared with perennial ryegrass (Lolium perenne L. cv. mixture of Delish and Banquet) to examine their potential to mitigate CH4 emissions from sheep. Sixty healthy, one-year-old Romney cryptorchids (liveweight 36 ± 1.0 kg) were randomly allocated to 5 groups of 12. The sheep were initially adapted to their diets for 2 wks on paddocks and 1 wk in indoor pens, then fed at 1.5 times their metabolisable energy (ME) maintenance requirement during measurements. Apparent total tract digestibility (n = 5) and ME (n = 5) of brassicas and ryegrass, and rumen fermentation parameters (n = 10) were measured, and CH4 emissions (n = 9) were determined using open circuit respiration chambers. Correlations between nitrate, sulphur, sulphate, glucosinolates and S-methyl cysteine sulphoxide (SMCO) contents of forages and CH4 emissions were determined. Methane emissions/unit feed dry matter intake (g CH4/kg) from sheep were kale 19.8, rape 16.4, swedes 16.9, turnips 20.6 and ryegrass 22.0. Compared with ryegrass, rape and swedes reduced CH4 yield by 23% and 25%, respectively. Total tract digestibilities were higher for brassicas than for ryegrass: 25% higher for DM, organic matter (OM) and crude protein, and 12–16% higher for neutral detergent fibre (aNDF) and acid detergent fibre (ADF). The ME values (MJ/kg DM) were kale 12.7, rape 13.2, swedes 14.1, turnips 12.1 and ryegrass 9.4. Digestibility (P This paper is part of the special issue entitled: Plant Bioactive Compounds in Ruminant Agriculture – Impacts and Opportunities, Guest Edited by A.Z.M. Salem and S. Lopez, and Editor for Animal Feed Science and Technology, P.H. Robinson.

Forage chicory ( Cichorium intybus ) has the potential to mitigate methane emissions from ruminants. It was reported that the reduction can be up to 30% compared with perennial ryegrass ( Lolium perenne ). To accurately evaluate the reduction, fresh chicory and perennial ryegrass in the vegetative state were fed to 24 wethers, 8 of which rumen-fistulated, at 1.3 and 2.2 times maintenance metabolisable energy requirements. Dry matter (DM) intake, whole tract apparent digestibility, rumen fermentation parameters and rumen liquid passage rate were measured in metabolism crates, and methane emissions determined using a calorimetric technique. Chemical analyses showed that chicory contained less DM, organic matter (OM), crude protein, neutral detergent fibre (aNDF), acid detergent fibre (ADF), cellulose and hemicellulose, but more hot water-soluble carbohydrate and pectin, than perennial ryegrass. Methane yield (g/kg DM intake) of wethers fed chicory did not differ from that of those fed perennial ryegrass. Yield was lower at the high versus the low feeding level of ryegrass. Apparent digestibility of DM and OM was higher, and aNDF, ADF, hemicellulose and cellulose was lower, in wethers fed chicory versus perennial ryegrass. In situ DM degradation rate of chicory was higher than that of perennial ryegrass. Rumen liquid passage rate was the same for wethers fed the two forages and higher at the high feeding level. The reduction in methane emissions by feeding vegetative chicory to wethers was limited, but increased feeding level reduces methane yields per unit of DM intake.

Published reports of CH4 yields as g CH4/kg dry matter (DM) intake suggest that emissions from sheep fed fresh forage chicory (Cichorium intybus) are about 30% lower than from those fed fresh ryegrass. In this study, 2 year old wethers (16; 54 ± 3.8 kg liveweight) were fed either mature chicory or perennial ryegrass at 1.3 times maintenance metabolisable energy requirements in the late spring/early summer of 2009. Methane emissions were determined using individual animal respiration chambers. Feeds differed in their chemical composition with chicory containing 856 g/kg organic matter (OM), 117 g/kg crude protein (CP) and 281 g/kg neutral detergent fibre (aNDF), whereas ryegrass contained 916 g/kg OM, 85 g/kg CP and 499 g/kg aNDF. The DM intake was similar for both forages at 0.76 kg/d, and CH4 yields did not differ between forages being 22.8 and 23.8 g CH4/kg DM intake for chicory and ryegrass, respectively. In vitro incubations of chicory and perennial ryegrass in the vegetative or mature states had similar CH4 yields. Despite large differences in chemical composition, especially aNDF, chicory and ryegrass had similar CH4 yields in vitro and in vivo. Chicory is not a viable alternative to perennial ryegrass for mitigating CH4 in pastoral based sheep production systems. This paper is part of the special issue entitled: Greenhouse Gases in Animal Agriculture – Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors: K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson.

The sulphur hexafluoride (SF6) tracer technique is a means to estimate CH4 emissions from individual ruminants, particularly whilst grazing. The success of the SF6 technique is reliant upon accurate determination of the release rate of SF6 gas from the permeation tubes (PT). Our study aimed to determine effects of SF6 PT rumen residence time on the release rate of SF6 gas and calculated CH4 production (as g CH4/d) in grazing red deer. The CH4 emissions of red deer stags grazing permanent perennial ryegrass based pasture were estimated daily for 5 d at 4.5, 6.5, 9.0 and 11.5 months of age, corresponding to a PT release period of 55, 111, 195 and 258 d. Permeation tube charge time refers to the number of days since PT were charged with SF6. The PT were recovered from 3 deer after the second CH4 measurement and from the remaining 10 deer at the end of the experiment. Release rates of SF6 gas from PT were determined pre-insertion to the rumen (i.e., uncorrected SF6 release rates), or by pre-insertion SF6 release rate with adjustment for SF6 release rates from PT recovered at the termination of the experiment (i.e., corrected SF6 release rates), after the PT had resided in the rumen of red deer for 212 d. A difference of 12.8% between corrected and uncorrected SF6 release rates (P=0.07) occurred after 9 d of PT rumen residence. In contrast, after 65, 150 and 212 d of PT residence, corrected release rates were lower (P This paper is part of the special issue entitled: Greenhouse Gases in Animal Agriculture – Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors: K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson.

There are increasing concerns regarding the relative safety of chlorpyrifos (CPF) to various facets of the environment. Although published works suggest that CPF is relatively safe in adult animals, recent evidence indicates that juveniles, both animals and humans, may be more sensitive to CPF toxicity than adults. In young animals, CPF is neurotoxic and mechanistically interferes with cellular replication and cellular differentiation, which culminates in the alteration of synaptic neurotransmission in neurons. However, the effects of CPF on glial cells are not fully elucidated. Here we report that chlorpyrifos is toxic to oligodendrocyte progenitors. In addition, CPF produced dose-dependent increases in 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCF-DA) and dihydroethidium (DHE) fluorescence intensities relative to the vehicle control. Moreover, CPF toxicity is associated with nuclear condensation and elevation of caspase 3/7 activity and Heme oxygenase-1 mRNA expression. Pan-caspase inhibitor QVDOPh and cholinergic receptor antagonists' atropine and mecamylamine failed to protect oligodendrocyte progenitors from CPF-induced injury. Finally, glutathione (GSH) depletion enhanced CPF-induced toxicity whereas nitric oxide synthetase inhibitor L-NAME partially protected progenitors and the non-specific antioxidant vitamin E (alpha-tocopherol) completely spared cells from injury. Collectively, this data suggests that CPF induced toxicity is independent of cholinergic stimulation and is most likely caused by the induction of oxidative stress.

The polysaccharide compositions of cell walls isolated from the laminae and midribs of chicory (cv. Grasslands Puna II) leaves were investigated. The walls, which, except for the walls of xylem tracheary elements in vascular bundles, were non-lignified, were fractionated progressively with 50 mM CDTA, 50 mM Na 2 CO 3 , 1 M KOH, 4 M KOH, 4 M KOH + 3.5% H 3 BO 3 , and hot water. Monosaccharide and linkage analyses of the polysaccharides in the walls and wall fractions indicated that the polysaccharides were similar to those in the non-lignified walls of other dicotyledons. However, the proportions of pectic polysaccharides were particularly high (67% of the total wall polysaccharides in the laminae). These included homogalacturonans (50% of the total wall polysaccharides in the laminae) and rhamnogalacturonan-I (RG-I). In contrast, the proportions of cellulose, xyloglucans, heteroxylans and glucomannans were particularly low. The xyloglucans were probably fucogalactoxyloglucans. The wall polysaccharides from the leaf laminae differed from those from the midribs in having higher proportions of homogalacturonans, lower proportions of non-pectic polysaccharides, and RG-I with a higher ratio of galactan to arabinan.

The present study investigated the relative importance of glutamine as a transamination source in the ovine liver by examination of the labelling of amino acids (AA) in the hepatic free pool, mixed liver and plasma proteins of fed and fasted sheep, following infusion of isotopically-labelled glutamine. In a cross-over design four sheep were either fasted for 3 d or fed to 1·2×energy maintenance and finally euthanased. At each intake, the sheep were infused for 6 h with [2-15N]glutamine (150 μmol/h) and samples of total plasma protein isolated. Following the terminal infusion, liver tissue total proteins were prepared and hydrolysed and15N-enrichments in seventeen AA were determined by GC–combustion–isotope-ratio mass spectrometry. All AA were enriched (relative to natural abundance) except lysine and threonine, with the lowest enrichments in phenylalanine and histidine. There was no effect of the fedv.fasted state, except for leucine and isoleucine in liver protein (P

The mechanisms by which prolonged exposure to morphine leads to tolerance are not fully understood. We investigated the effects of etorphine (ET) on [(35)S]guanosine 5'-(-thio)-triphosphate ([(35)S]GTP-gamma-S) binding in brains of rats made tolerant to morphine via the implantation of morphine (or placebo) pellets. Binding surface analysis was used to characterize the interactions of ET, Gpp(Np)H and GTP-gamma-S with sites labeled by [(35)S]GTP-gamma-S. Data sets were fitted to one- and two-site binding models using the nonlinear least squares curve fitting program MLAB-PC (Civilized Software, Bethesda, MD, USA). Two binding sites were readily resolved. Chronic morphine significantly increased the B(max) and K(d) of the high affinity binding site. ET stimulated [(35)S]GTP-gamma-S binding in placebo membranes via an increase in the B(max) of the high affinity binding site. In contrast, ET stimulated [(35)S]GTP-gamma-S in chronic morphine membranes via a large decrease in the K(d) of the high affinity site. These results suggest that chronic morphine treatment alters the mechanism by which ET stimulates [(35)S]GTP-gamma-S binding to G-proteins. Since proper G-protein/receptor coupling increases [(35)S]GTP-gamma-S binding via an increase in B(max) values, these results suggest that opioid receptors in chronic morphine membranes are not normally coupled to G-proteins. These findings corroborate earlier studies that reported changes in G-protein function in morphine tolerant animals.

There is strong evidence supporting the existence of multiple kappa receptors. Previous studies proposed that U69,593 and (+)-tifluadom act on different kappa receptor subtypes, kappa(1) (kappa(1)) and kappa(2) (kappa(2)), respectively. In this study, we investigated the effects of the kappa selective antagonist nor-binaltorphimine (Nor-BNI) on U69,593- and (+)-tifluadom-induced receptor-mediated stimulation of [(35)S]-GTP-gamma-S binding in the guinea pig caudate. The IC(50) value of Nor-BNI in the presence of a stimulating concentration of U69,593 (1 microM) was 0.19+/-0.02; while the IC(50) for Nor-BNI in the presence of (+)-tifluadom (1 microM) was 13.9+/- 1.62 nM. The mu-opioid receptor antagonist CTAP (10,000 nM) significantly reduced (+)-tifluadom-stimulated [(35)S]-GTP-gamma-S binding in rat brain sections and guinea pig brain membranes, indicating that (+)-tifluadom has mu agonist activity. Under conditions in which the mu agonist activity of (+)-tifluadom was blocked by 1000 nM CTAP the Ki value for Nor-BNI for inhibition of U69,593-stimulated [(35)S]-GTP-gamma-S binding was 0.036+/-.004 nM, whereas, its Ki value for the (+)-tifluadom-stimulated [(35)S]-GTP-gamma-S binding was 0.27+/-.015 nM. These results suggest that (+)-tifluadom and U69,593 activate pharmacologically different receptors. This study provides functional evidence in support of kappa receptor heterogeneity.

The development of addictive states in response to chronic opioid use may be regulated partially by the release of endogenous peptides. These anti-opiate peptides (AOP) are secreted or released into the CNS and produce diverse actions that counterbalance the effects of prolonged opiate exposure. Though the mechanism(s) by which these peptides exert their physiological properties remain largely unknown, there is some indication that AOP's modulate opioid receptor levels. In this study, we investigated the effects of chronically infused alpha-melanocyte stimulating hormone (alpha-MSH), dynorphin(1-8) (DYN(1-8)), dynorphin A (DYNA), and NPFF antibodies on delta-opioid receptor expression in rat brains. Quantitative autoradiographic experiments revealed that antibodies directed against alpha-MSH and DYNA produced significant increases in delta receptor levels in the caudate, claustrum, and cingulate cortex of the rat brain. Conversely, NPFF monoclonal antibodies caused significant decreases in the caudate, nucleus accumbens, olfactory tubercle, and cingulate cortex. These results suggest that the density of delta-opioid receptors is affected by changes in the levels of the anti-opioid peptides in the extracelluar fluid in the rat brain.

We utilized the [(35)S]-GTP-gamma-S functional binding assay to determine the selectivity of opioid receptor agonists in guinea pig caudate membranes. The study focused on two opioid agonists used for treating opioid-dependent patients: methadone and buprenorphine. Selective antagonists were used to generate agonist-selective conditions: TIPP + nor-BNI to measure mu receptors, CTAP + nor-BNI to measure gamma receptors and TIPP + CTAP to measure kappa receptors. The assay was first validated with opioid agonists of known subtype specificity (DAMGO for mu, SNC80 for delta, and U69, 593 for kappa receptors). Methadone-stimulated [(35)S]-GTP-gamma-S binding was mu-specific and less potent and efficacious than etorphine (K(d) = 1,537 nM vs. K(d) = 7.8 nM). Buprenorphine failed to stimulate [(35)S]-GTP-gamma-S binding but inhibited agonist-stimulated [(35)S]-GTP-gamma-S binding. The antagonist-K(i) values (nM) of buprenorphine at mu, delta, and kappa receptors were 0.088 nM, 1.15 nM, and 0.072 nM, respectively. The antagonist-K(i) values (nM) of naloxone at mu, delta, and kappa receptors were 1.39 nM, 25.0 nM, and 11.4 nM, respectively. Autoradiographic studies showed that buprenorphine failed to stimulate [(35)S]-GTP-gamma-S binding in caudate-level rat brain sections but blocked DAMGO-stimulated [(35)S]-GTP-gamma-S binding. In cells expressing the cloned rat mu receptor, buprenorphine was a partial agonist and potent mu antagonist. Administration of buprenorphine to rats produced a long-lasting (>24 h) decrease in mu and kappa2 receptor binding and attenuated mu-stimulated [(35)S]-GTP-gamma-S binding. Viewed collectively, these data indicate that, in this assay system, buprenorphine is a potent mu and gamma receptor antagonist. The clinical implications remain to be elucidated. Synapse 34:83-94, 1999. Published 1999 Wiley-Liss, Inc.

Pharmacological study of Phe-Met-Leu-Phe-amide (FMRFa) receptors is hindered by the lack of selective ligands. The classification of these selective ligands is further hampered by the limited availability of functional assays. In this study, we evaluated several synthetic FMRFa analogs for agonist and antagonist activity by measuring their abilities to produce [35S]-GTP-γ-S stimulation or to inhibit FMRFa-induced [35S]-GTP-γ-S binding in squid optic lobes. Analogs included acetyl-Phe-norLeu-Arg-Phe-amide (acFnLRFa), desamino-Tyr-Phe-Leu-Arg-amide (daYFLRa), desamino Tyr-Phe-norLeu-Arg-Phe-amide (daYFnLRFa), desamino Tyr-Phe-norLeu-Arg-[TIC]-amide (daYFnLR[TIC]a), desamino Tyr-Trp-norLeu-Arg-amide (daYWnLRa), ( d )-Tyr-Phe-norLeu-Arg-Phe-amide ( d -YFnLRFa), Phe-Leu-Arg-Phe-amide (FLRFa), and the d -amino acid analogs of FMRFa ( d -FMRFa, F-( d )-MRFa and FM-( d )-RFa). For agonist studies, full dose–response curves were generated and analyzed for potency and efficacy (maximal percent effect). FMRFamide as well as analogs ac-FnLRFa, daYFnLRFa, daYFnLR[TIC]a, D-YFnLRFa, FLRFa, and ( d )-FMRFa stimulated [35S]-GTP-γ-S binding. Analogs daYWnLRa, daYFLRa, F-( d )-MRFa, and FM-( d )-RFa failed to stimulate either [35S]-GTP-γ-S binding or to inhibit FMRFa-induced [35S]-GTP-γ-S binding. The rank order of potency was daYFnLRFa ≥ daYFnLRF[TIC]a > acFnLRFa > ( d )YFnLRFa > FLRFa ≥ FMRFa ≫ ( d )-FMRFa. The order of efficacy was daYFnLRFa = acFnLRFa = ( d )-YFnLRFa>FLRFa = FMRFa≥( d )-FMRFa≥daYFnLRF[TIC]a. Peptide analog daYFnLR[TIC]a was less efficacious (59% maximal stimulation) than analogs daYFnLRFa, acFnLRFa, and ( d )-YFnLRFa (113-146% maximal stimulation). A maximal concentration of daYFnLR[TIC]a (10 μM) reduced daYFnLRFa, acFnLRFa, and ( d )-YFnLRFa induced [35S]-GTP-γ-S stimulation, indicating that daYFnLR[TIC]a is a partial agonist at the receptor stimulated by the FMRFamide analogs. Analysis of the structural requirements needed for promoting [35S]-GTP-γ-S binding show that elongation (i.e., daYFnLRFa, d -YFnLRFa) or modification of Phe1 (ac-FnLRFa) leads to increased efficacy and potency. Moreover, elimination of the C-terminal Phe (daYWnLRa, daYFLRa,) leads to a loss of biological activity. However, substitution with l -1,2,3,4 tetrahydroisoquinoline-3-carboxylic acid, a rigid analog of the C-terminal Phe (daYFnLR[TIC]a), leads to decreased efficacy but not loss of potency. The data suggest that immobilization or modification of the C-terminal Phe may produce highly selective and potent FMRFamide antagonists. These results agree with published receptor radioligand studies and indicate that the [35S]GTP-γ-S assay may be useful in classifying novel FMRFamide-selective ligands.

There is some indication that anti-opiate peptides (AOP) modulate opioid receptor systems by altering mu-receptor density. To further characterize this phenomenon, we investigated the effects of continuous infusion of anti-AOP IgG on mu binding sites in the brains of rats. Specifically, male Sprague-Dawley rats received intracerebroventricular (i.c.v.) infusions for 13 days of either control (rabbit) IgG or test IgGs: anti-dynorphin A IgG, anti-dynorphin A1-8 IgG, anti-alpha-MSH IgG, or the monoclonal anti-NPFF IgG. Administration of anti-NPFF IgG or the anti-dynorphin1-8 IgG significantly increased mu labeling by 40-70% in several brain regions at the caudate level. Contrary to these findings, anti-alpha-MSH IgG decreased (19-32%) [125I]-DAMGO labeling in several thalamic nuclei. The results suggest that the density of mu-opioid receptors is regulated in part by anti-opiate peptides in the extracellular fluid of the brain.

Objective To assess the magnitude of sodium reduction that can be made without significantly changing the perception of saltiness and acceptability of a broad range of common food items. Design The investigation was carried out in two phases. Military and civilian volunteers (N=190 in study 1; N=380 in study 2) from the US Army Natick Research, Development and Engineering Center rated the saltiness and acceptability of foods containing differing sodium concentrations. Setting Consumers rated food items in the sensory testing laboratory. Intervention "Regular" and "low-sodium" entrees were rated for saltiness and acceptability in study 1. Prepared food, commercially prepared food, and beverages containing various concentrations of sodium were rated for saltiness and acceptability in study 2. Statistical analyses performed Results were analyzed using Pearson correlation coefficients, Student's t test, and analysis of variance. Results The perception of saltiness increased as the concentration of sodium increased. Acceptance ratings varied considerably over a broad range of sodium concentrations, which indicates that the relationship was product specific. Results suggest that a reduction of one third or more in added sodium can be made to some foods without significantly affecting consumer acceptance. Applications The sodium content of food can be reduced by consumer-guided food engineering and food preparation. Alterations in food preparation and product formulation, in conjunction with alterations in diet, can be effective methods for reducing sodium consumption. J Am Diet Assoc. 1995; 95:447–453.

Aims: Determine the susceptibility of forage chicory (Cichorium intybus L.) to degradation by ruminal fibrolytic bacteria and measure the effects on cell-wall pectic polysaccharides. Methods and Results: Large segments of fresh forage chicory were degraded in vitro by Lachnospira multiparus and Fibrobacter succinogenes, but not by Ruminococcus flavefaciens or Butyrivibrio hungatei. Cell-wall pectins were degraded extensively (95%) and rapidly by L. multiparus with a simultaneous release of uronic acids and the pectin-derived neutral monosaccharides arabinose, galactose and rhamnose. Fibrobacter succinogenes also degraded cell-wall pectins extensively, but at a slower rate than L. multiparus. Immunofluorescence microscopy using monoclonal antibodies revealed that, after incubation, homogalacturonans with both low and high degrees of methyl esterification were almost completely lost from walls of all cell types and from the middle lamella between cells. Conclusions: Only two of the four ruminal bacteria with pectinolytic activity degraded fresh chicory leaves, and each showed a different pattern of pectin breakdown. Degradation was greatest for F. succinogenes which also had cellulolytic activity. Significance and Impact of the Study: The finding of extensive removal of pectic polysaccharides from the middle lamella and the consequent decrease in particle size may explain the decreased rumination and the increased intake observed in ruminants grazing forage chicory.

The mechanism by which chlorpyrifos exerts its toxicity in fetal and perinatal animals has yet to be elucidated. Since the placenta is responsible for transport of nutrients and is a major supplier hormone to the fetus, exposure to xenobiotics that alter the function or viability of placenta cells could ostensibly alter the development of the fetus. In this study, JAR cells were used to determine if CPF and the metabolites 3,5,6-trichloro-2-pyridinol (TCP) and chlorpyrifos-oxon (CPO) are toxic to the placenta. Our results indicate that chlorpyrifos (CPF), and its metabolite chlorpyrifos-oxon (CPO) caused a dose-dependent reduction in cellular viability with CPF being more toxic than its metabolites. Chlorpyrifos-induced toxicity was characterized by the loss of mitochondrial potential, the appearance of nuclear condensation and fragmentation, down-regulation of Bcl-2 as well as up-regulation of TNFalpha and FAS mRNA. Pharmacological inhibition of FAS, nicotinic and TNF-alpha receptors did not attenuate CPF-induced toxicity. Atropine exhibited minimal ability to reverse toxicity. Furthermore, signal transduction inhibitors PD98059, SP600125, LY294002 and U0126 failed to attenuate toxicity; however, SB202190 (inhibitor of p38alpha and p38beta MAPK) sensitized cells to CPF-induced toxicity. Pan-caspase inhibitor Q-VD-OPh produced a slight but significant reversal of CPF-induced toxicity indicating that the major caspase pathways are not integral to CPF-induced toxicity. Taken collectively, these results suggest that chlorpyrifos induces apoptosis in placental cells through pathways not dependent on FAS/TNF signaling, activation of caspases or inhibition of cholinesterase. In addition, our data further indicates that activation of p38 MAPK is integral to the protection cells against CPF-induced injury.

Doxorubicin (DOX) is a chemotherapeutic agent widely used in the treatment of various cancers. However, its clinical use is restricted due to serious cardiotoxicity and the development of drug resistance. Many delivery strategies, including the use of liposomes, micelles, and polymer drug conjugates, have been used to improve the therapeutic efficacy and reduce the toxicity of DOX. Recently, lipid-drug conjugates have also been investigated for the delivery of anticancer drugs. The conjugation of lipids with DOX can increase its lipophilicity, thus enhance its loading in lipid-based nanoparticles, facilitate interaction with cells, and improve pharmacokinetic properties. In this study, we developed a micelle formulation with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino polyethylene glycol-2000 (DSPE-PEG) to deliver a DOX-lipid conjugate to treat breast cancers. DOX was conjugated with palmitic acid (PA) through a pH sensitive hydrazone bond to yield a lipophilic pro-drug of DOX (PA-DOX). PA-DOX (Log P = 2) showed significantly increased lipophilicity than DOX (Log P = - 0.5). The micelles were prepared using a thin-film dispersion method. Due to the lipophilic nature of PA-DOX, it can be efficiently loaded into the hydrophobic core of DSPE-PEG micelles. In addition, the micelle formulation demonstrated superior stability, showing no leakage or precipitation of PA-DOX for at least two weeks during storage at 4°C. In vitro drug release was tested in sodium phosphate buffer (pH 7.5) and sodium acetate buffer (pH 5.0), respectively. DOX-PA was stable at pH 7.4 and showed minimal DOX release (less than 10% at 24 hours). The release of DOX was significantly increased at pH 5 (approximately 75% at 24 hours). Anticancer activities were evaluated with MCF-7 breast cancer cells and MCF-7/MDR drug-resistant breast cancer cells. Cell viability was determined by a MTT assay. PA-DOX showed similar anticancer activity on MCF-7 cells as DOX itself. Anticancer activity of PA-DOX was significantly reduced in MCF-7/MDR cells but restored when used in combination with lapatinib. Citation Format: Feng Li, Marilyn Saulsbury, Simone Heyliger, Mikhail Bondarev, Chengan Du, Corinne C. Ramaley, KiTani P. Lemieux. A pH-responsive doxorubicin-lipid conjugate loaded nanomedicine for breast cancer treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5538. doi:10.1158/1538-7445.AM2015-5538

Apart from the obvious role of plants in herbivore nutrition, they are also a rich source of bioactive products that can operate either to the benefit or the detriment of grazing animals. Here, we review the available evidence for the potential beneficial effects that plant-derived bioactive substances can have on gastrointestinal parasites. Tannin-rich plants have attracted most attention for their effect on internal nematodes in ruminants. These plants could act through direct antiparasitic activity but might also act indirectly by increasing host resistance. The effects vary with the species of plant, parasite and host. More research is required to understand better the mechanisms of action, and therefore make more pertinent use of these bioactive plants in livestock systems.

New N-heteroarylcarbonylalanines of the D-series were stereoselectively prepared from enoates derived from D-mannitol. These compounds were active in binding and functional assays of the NMDA sub-type of glutamate receptors. A pyridine derivative inhibited MK801 binding, protected neurons from excitotoxic damage and blocked NMDA-induced currents in neurons. A thiophene derivative positively modulated the NMDA receptor, possibly through the allosteric glycine site.

International audience; While both the quantity and quality of food ingested are potent regulators of whole body protein metabolism in ruminants, little data are available on responses across a wide range of intakes. The current study examined the responses in whole body protein flux (PrF) to such intake changes and compared these with the responses across the hind-quarters (in a companion study). Six growing sheep (6-8 months, 30-35 kg) received each of four intakes of dried grass pellets (0.5, 1.0, 1.5 and 2.5 times maintenance energy; M) for a minimum of 7 days. At each intake, a mixture of U-$^{13}$C amino-acids (AA) was infused intravenously for 10 h. Arterial plasma and blood were obtained over the last 4 h of infusion and the concentrations and the enrichments of thirteen $^{13}$C labelled AA were determined. The absolute values for plasma Irreversible Loss Rate (ILR) but also converted PrF varied between the AA. PrF values were lower for histidine, methionine, aspartate, glycine and proline (range 68 to 174 g$\cdot$d$^{-1}$ at 1.5 M) than for isoleucine, leucine, valine and glutamate (range 275 to 400 g$\cdot$d$^{-1}$ at 1.5 M). These discrepancies may be explained by (1) the differential AA removal by the splanchnic tissues, (2) the de novo synthesis of the non-essential AA, (3) the transfer of AA from the erythrocytes or plasma to the tissues. The first two assumptions require further investigation whereas recent work has shown a minor role for AA transfers between erythrocytes and tissues. For most AA, ILR and PrF responded linearly to intake but curvilinear responses were observed for phenylalanine, lysine, leucine, isoleucine and tyrosine. These differences were not due to hind-quarter metabolism and may involve the digestive tract and liver.

Responses in variables of amino acid (AA) metabolism across peripheral tissues to feed intake were studied in six sheep (mean live weight 32 kg) prepared with arterio–venous catheters across the hindquarters. Four intakes (0·5, 1·0, 1·5 and 2·5 × maintenance energy) were offered over 2-week periods to each sheep in a Latin square design with two animals replicated. Animals were infused intravenously with a mixture of U-13C-labelled AA for 10 h and integrated blood samples withdrawn from the aorta and vena cava hourly between 5 and 9 h of infusion. Biopsy samples were also taken from skin andm. vastus lateralis. Data from both essential (histidine, isoleucine, leucine, lysine, phenylalanine, threonine) and nonessential (glycine, proline, serine, tyrosine) AA were modelled to give rates of inward and outward transport, protein synthesis and degradation, plus the fraction of total vascular inflow that exchanged with the hindquarter tissues. Rates of inward transport varied more than 10-fold between AA. For all essential AA (plus serine), inward transport increased with food intake (PPP

It has been suggested that protein synthesis in peripheral tissues: (1) responds in a curvilinear manner to increasing feed intake over a wide range of feeding levels; and (2) has a greater sensitivity to intake than protein breakdown. The aim of the present experiment was to test these hypotheses across the ovine hindlimb. Six growing sheep (6 ‐8 months, 30 ‐35 kg), with catheters in the aorta (two), posterior vena cava and jugular vein, received each of four intakes of dried grass pellets (0:5, 1:0, 1:5 and 2:5 maintenance energy; M) for a minimum of 7 d. A U- 13 C-labelled algal hydrolysate was infused intravenously for 10 h and from 3 ‐ 9 h para-aminohippuric acid was infused to measure plasma flow. Arterial and venous plasma were obtained over the last 4 h and the concentrations and enrichments of thirteen 13 C-labelled amino acids (AA) were determined by GC ‐ MS. As intake increased, a positive linear response was found for plasma flow, arterial concentrations of the aromatic and branched-chain AA, total flow of all AA into the hindquarters and net mass balance across the hindquarters (except glycine and alanine). Based on two separate statistical analyses, the data for protein synthesis showed a significant linear effect with intake (except for phenylalanine, glycine and alanine). No significant curvilinear effect was found, which tends not to support hypothesis 1. Nonetheless, protein synthesis was not significantly different between 0:5, 1:0 and 1:5 M and thus the 2:5 M intake level was largely responsible for the linear relationship found. There was no significant response in protein breakdown to intake, which supports hypothesis 2. Amino acids: Protein metabolism: Feed intake: Hindlimb: Arterio ‐ venous: Sheep

With its many proposed metabolic roles, glutamine would seem to have major potential in normal animal production systems as well as during situations involving adverse challenges. In practice, however, responses to glutamine supplementation have been inconsistent. Thus, during lactation and growth studies in ruminants, both positive and null effects on production responses have been reported. Similarly, therapeutic responses to glutamine supplementation during various digestive tract disorders have been inconsistent in both pigs and ruminants. This is despite a proven involvement in the nucleic acid biosynthesis necessary to support cell proliferation. In sheep, at least, glutamine may exert a protective effect against hepatic amino acid (AA) oxidation, particularly for methionine. This may offer anabolic potential because methionine is the first limiting AA in a number of animal feedstuffs. Glutamine is also important in control of metabolic acidosis, but, in contrast to rodents, the main site of production seems to be extra-hepatic. In the immune system, while lymphocyte proliferation is glutamine-dependent, intracellular concentrations are low (in contrast to other tissues, such as muscle and liver). Instead, glutamate is accumulated, but the majority of this (approximately 65%) is derived in vivo from plasma glutamine. In sheep, endotoxin challenge elevates the plasma flux of glutamine, with a corresponding decrease in plasma concentration. At the same time, both the glutamate accumulation and fractional rate of protein synthesis within lymphocytes are enhanced. These lymphocyte responses, however, are not altered by an AA supplement that contains glutamine. Overall, although glutamine obviously plays important metabolic roles within the body, supplementation does not appear to provide consistent beneficial or therapeutic effects, except during certain catabolic situations. Glutamine availability, therefore, does not seem to be a limitation in many challenge situations. Rather, glutamine may signal alterations in nutrient demands among organs and a better understanding of this role may increase understanding of where modulation of glutamine status would be beneficial.

Both plasma and red blood cells contain amino acids (AA), but the relative amount of AA transferred from each vascular compartment to the tissues remains unclear. For splanchnic tissues, the relative transfers between the plasma, the red blood cells and the tissues may vary with nutritional state, but whether the same situation pertains for other tissues is not known. The current study focused on the transfer of lysine from plasma and red blood cells across the hindquarters of sheep offered four levels of intakes (0.5, 1.0, 1.5 and 2.5×maintenance energy). This design, coupled with use of [U-13C]lysine as tracer, also allowed the effect of intake on protein kinetics to be examined. At all intakes, the concentration of lysine in the sheep’ red blood cells exceeded that in plasma by 50 % (PPPP

Male Sprague–Dawley rats weighing 150–200 g were given doses of tryptophan methyl ester or its metabolites; kynurenine sulphate, kynurenic acid, xanthurenic acid, quinolinic acid, anthranilic acid methyl ester or picolinic acid methyl ester. Doses administered intraperitoneally were 50, 100, 200, 300, 400 and 600 mg kg−1. Pain sensitivity was assessed using the hotplate and tailflick methods at 30 min before and at 30-min interval after the injection of test compounds. The administrations of tryptophan, kynurenic acid, quinolinic acid, anthranilic acid, xanthurenic acid, picolinic acid, and kynurenine were associated with analgesia. Animals given 300 or 600 mg kg−1of tryptophan exhibited a significant decrease (P

Organophosphates are chemicals that inhibit cholinesterases and are employed widely as pesticides. Concerns are increasing regarding the relative safety of these chemicals to the environment. Recent studies suggest organophosphate exposure is associated with increased expression of fragile sites and DNA damage at concentrations that are not associated with cholinesterase inhibition. Chronic exposure to the organophosphate has been associated with high incidences of prostate cancer in farm workers as well as leukemia and non-Hodgkin lymphoma in adults and children. Moreover, a higher degree of DNA damage has been reported in pesticide applicators that have polymorphisms in the base excision repair genes XRCC1 and OGG1. Our laboratory reports that cells deficient in BER enzymes APE1 and OGG1 exhibit a higher cytotoxicity when exposed, in a dose-dependent manner, to organophosphates Chlorpyrifos, Chlorpyrifos-oxon and Isofenphos. However, it should be noted that other organophosphates such as Diazinon and Dichlorvos had very little effects on cellular viability within the APE1 and OGG1 deficient cells. It should be noted that Chlorpyrifos, Chlorpyrifos-oxon and Isofenphos also produce significant oxidative stress in cells. Our data is consistent with previous studies which report a higher degree of DNA damage has been reported in pesticide applicators that have polymorphisms in the base excision repair genes XRCC1 and OGG1. Moreover, our data further suggests that only certain organophosphates, particularly those that induce oxidative stress, may be responsible for toxicity in APE1 and OGG1 deficient cells. Citation Format: Marilyn D. Saulsbury, Simone Heyliger, Feng Li, KiTani A. Parker-Johnson. Knock-down of base excision repair genes increases susceptibility to organophospate pesticide toxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 615. doi:10.1158/1538-7445.AM2013-615