Brucellosis: 'One Health' challenges and opportunities.

Brucellosis is an ancient disease with host-specific evolutionary mechanisms that allow itto hide from or manipulate cellular immunity and achieve intracellular persistence. The disease yields low fatality rates but can cause substantial disabilities. Zoonotic brucellosis remains widespread and neglected in many areas despite notable advances in science, technology, and management in the 19th and 20th Centuries. The burden appears to remain greatest, and yet most under-prioritised globally, amongst pastoral peoples and small-scale livestock farmers. Capacity building for zoonotic brucellosis diagnosis, surveillance, management, and treatment in developing countries faces numerous challenges. Adaptive risk management can provide a framework to build stakeholder support for addressing the complexities and uncertainties, and learning from management actions. The challenges and opportunities for brucellosis management must be recognised as fundamentally multivariate, multifaceted, and integrative; it is thus crucial for veterinary, public health, and wildlife/conservation professions to collaboratively develop, adopt and promulgate a brucellosis One Health paradigm.

Vaccination in conservation medicine.

Unprecedented human population growth and anthropogenic environmental changes have resulted in increased numbers of people living in closer contact with more animals (wild, domestic, and peridomestic) than at any other time in history. Intimate linkage of human and animal health is not a new phenomenon. However, the global scope of contemporary zoonoses has no historical precedent. Indeed, most human infectious diseases classed as emerging are zoonotic, and many of these have spilled over from natural wildlife reservoirs into humans either directly or via domestic or peridomestic animals. Conservation medicine has recently emerged as a meaningful discipline to address the intersection of animal, human, and ecosystem health. Interest in the development of novel vaccines for wildlife encounters important challenges that may prevent progress beyond the conceptual phase. Although notable examples of successful wildlife immunisation programmes exist, depending upon key considerations, vaccination may or may not prove to be effective in the field. When implemented, wildlife vaccination requires a combination of novel zoonosis pathogen management strategies and public education to balance conservation, economic, and public health issues.

Purification of cytosolic beta-glucosidase from pig liver and its reactivity towards flavonoid glycosides.

Flavonoid glycosides are common dietary components which may have health-promoting activities. The metabolism of these compounds is thought to influence their bioactivity and uptake from the small intestine. It has been suggested that the enzyme cytosolic beta-glucosidase could deglycosylate certain flavonoid glycosides. To test this hypothesis, the enzyme was purified to homogeneity from pig liver for the first time. It was found to have a molecular weight (55 kDa) and specific activity (with p-nitrophenol glucoside) consistent with other mammalian cytosolic beta-glucosidases. The pure enzyme was indeed found to deglycosylate various flavonoid glycosides. Genistein 7-glucoside, daidzein 7-glucoside, apigenin 7-glucoside and naringenin 7-glucoside all acted as substrates, but we were unable to detect activity with naringenin 7-rhamnoglucoside. Quercetin 4'-glucoside was a substrate, but neither quercetin 3, 4'-diglucoside, quercetin 3-glucoside nor quercetin 3-rhamnoglucoside were deglycosylated. Estimates of K(m) ranged from 25 to 90 microM while those for V(max) were about 10% of that found with the standard artificial substrate p-nitrophenol glucoside. The non-substrate quercetin 3-glucoside was found to partially inhibit deglycosylation of quercetin 4'-glucoside, but it had no effect upon activity with p-nitrophenol glucoside. This study confirms that mammalian cytosolic beta-glucosidase can deglycosylate some, but not all, common dietary flavonoid glycosides. This enzyme may, therefore, be important in the metabolism of these compounds.

Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase.

Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane-bound, family 1 beta-glycosidase found on the brush border of the mammalian small intestine. LPH, purified from sheep small intestine, was capable of hydrolysing a range of flavonol and isoflavone glycosides. The catalytic efficiency (k(cat)/K(m)) for the hydrolysis of quercetin-4'-glucoside, quercetin-3-glucoside, genistein-7-glucoside and daidzein-7-glucoside was 170, 137, 77 and 14 (mM(-1) s(-1)) respectively. The majority of the activity occurred at the lactase and not phlorizin hydrolase site. The ability of LPH to deglycosylate dietary (iso)flavonoid glycosides suggests a possible role for this enzyme in the metabolism of these biologically active compounds.

Glucosinolates and phenolics as antioxidants from plant foods.

Dietary glucosinolates and flavonoids exhibit anticarcinogenicity by blocking formation of endogenous or exogenous carcinogens and so preventing initiation of carcinogenesis. Glucosinolates act by induction of antioxidant and detoxifying enzymes such as glutathione-S-transferases and UDP-glucuronosyl transferase, and by inhibition of carcinogen-activating enzymes such as cytochrome P450 1A1. Flavonoids and other phenolic antioxidants act by direct free-radical scavenging. The distribution in foods and structure-function relationships for these classes of compounds are reviewed.

Antioxidant properties of the major polyphenolic compounds in broccoli.

We have examined the antioxidant activity of the major phenolic compounds in Broccoli: two flavonol glycosides (quercetin 3-O-sophoroside and kaempferol 3-O-sophoroside) and four hydroxycinnamic acid esters (1,2'-disinapoyl-2-feruloyl gentiobiose, 1-sinapoyl-2-feruloyl gentiobiose, 1,2,2'-trisinapoyl gentiobiose and 1,2-disinapoyl gentiobiose). The Trolox C equivalent antioxidant capacity (TEAC) and inhibition of iron/ascorbate-induced lipid peroxidation of phosphatidyl choline vesicles were measured. In the aqueous phase TEAC assay, the two flavonol glycosides were less active than their respective aglycones. TEAC values for the hydroxycinnamic acid esters were less than the sum of their constituent hydroxycinnamic acids on a molar basis. Quercetin 3-O-sophoroside was a potent inhibitor of lipid peroxidation, in contrast to kaempferol 3-O-sophoroside. The hydroxycinnamic acid esters were highly effective at preventing lipid damage with the exception of 1,2,2'-trisinapoyl gentiobiose. The six compounds analysed herein demonstrate the antioxidant activity of the major phenolics in broccoli and indicate the effect on antioxidant activity of sugar substitutions in the phenolic B ring.

Human cytochrome P450's are pro-oxidants in iron/ascorbate-initiated microsomal lipid peroxidation.

We have examined the effect of human cytochrome P450's (1A1,1A2,3A4,2A6,2B6,2D6,2E1) on ascorbate/iron-induced lipid peroxidation. Using microsomes prepared from human lymphoblastic cells enriched in recombinant cytochrome P450 isoenzymes, we have shown that the degree of peroxidation is a function of the amount of P450 present rather than the presence of any specific isoenzyme. Incorporated P450 increased the amount of peroxidation products by up to 2.1-fold compared to the control microsomes with no P450. It is therefore concluded that cytochrome P450's play a significant role in ascorbate/iron peroxidation.

Antioxidant properties of catechins and proanthocyanidins: effect of polymerisation, galloylation and glycosylation.

A range of catechins and oligomeric procyanidins was purified by high performance liquid chromatography (HPLC) from grape seed, apple skin, lentil and almond flesh. Catechins, galloylated epicatechin, glycosylated catechin, procyanidin dimers, galloylated dimers, trimer, and tetramer species were all identified, purified and quantified by HPLC, LC-MS and NMR. The antioxidant properties of these compounds were assessed using two methods: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes; (b) scavenging of the radical cation of 2,2'-azinobis(3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue Trolox C (expressed as Trolox C equivalent antioxidant capacity, TEAC). Antioxidant activity in the lipid phase decreased with polymerisation in contrast with antioxidant action in the aqueous phase which increased from monomer to trimer and then decreased from trimer to tetramer. Galloylation of catechin and dimeric procyanidins decreased lipid phase and increased aqueous phase antioxidant activity. Glycosylation of catechin demonstrated decreased activity in both phases.

Are whole extracts and purified glucosinolates from cruciferous vegetables antioxidants?

Fruits and vegetables contain several classes of compounds that can potentially contribute to antioxidant activity, including vitamins, simple and complex phenolics, sulphur-containing compounds and glucosinolates. The glucosinolates are found in high concentration in many cruciferous vegetables, and it is well established that their breakdown products induce endogenous antioxidant defences such as quinone reductase and glutathione S-transferase in cells and in vivo. Despite the anticarcinogenic effect of these compounds in animal models, the direct antioxidant properties of this class of compounds have not been systematically studied. We therefore examined the free radical-scavenging properties of representative extracts and of purified glucosinolates from cruciferous vegetables, by measuring their effect on ascorbate- or NADPH/iron-induced peroxidation of human liver microsomes, ascorbate/iron-induced peroxidation on phospholipid liposomes, iron chelation and hydroxyl radical scavenging using the deoxyribose assay, total antioxidant potential using ABTS (2,2'-azinobis(3-ethyl-benzothiazoline-6-sulphonate)) and the bleomycin assay. Most of the extracts from cruciferous vegetables exhibited some antioxidant properties, although extracts from cooked Brussels sprouts increased the rate of microsomal lipid peroxidation. The effects in these assays were dependent upon processing and species of crucifer, and the glucosinolate content appeared to play a minor role in these effects, since purified glucosinolates exhibited only weak antioxidant properties. The total antioxidant activities of extracts from cooked and autolysed Brussels sprouts were identical within experimental error. This is probably due to the content of phenolics which is unaltered by autolysis, despite the differences between these samples in other assays especially NADPH-iron-induced lipid peroxidation of human liver microsomes. The results demonstrate that glucosinolates are unlikely to account for the direct antioxidant effects of extracts from cruciferous vegetables.

Antioxidant properties of flavonol glycosides from green beans.

We have examined the antioxidant activity of one class of polyphenolic compounds in green beans: two novel flavonol glycosides (quercetin 3-O-[xylosyl(1-->2)]-rhamnosyl (1-->6)-glucoside and the corresponding kaempferol analogue), quercetin 3-O-glucuronide, kaempferol 3-O-glucuronide, quercetin 3-O-rutinoside and kaempferol 3-O-rutinoside. The Trolox equivalent antioxidant capacity (TEAC) and inhibition of iron/ascorbate-induced lipid peroxidation of phosphatidyl choline vesicles were measured. In the aqueous phase TEAC assay, the glucuronide and rutinoside of quercetin were good antioxidants, but not as effective as the quercetin aglycone. TEAC values for the glucuronide and other glycosides of kaempferol were much lower than the corresponding quercetin species but similar to that of the kaempferol aglycone. Quercetin 3-O-glucuronide and quercetin 3-O-rutinoside were both potent inhibitors of lipid peroxidation, in contrast to the those of kaempferol. The compounds described herein demonstrate the antioxidant activity of the flavonols present in green beans and indicate the effect on antioxidant activity of sugar substitutions in the phenolic C ring.

Antioxidant properties of flavonol glycosides from tea.

We have determined the antioxidant activity of the major flavonols found in tea: a monoglycoside, a diglycoside and two triglycosides of kaempferol and three monoglycosides, a diglycoside and two triglycosides of quercetin. The Trolox equivalent antioxidant capacity (TEAC) and inhibition of iron/ascorbate-induced lipid peroxidation of phosphatidyl choline vesicles were measured. In the aqueous phase TEAC assay, the quercetin monoglycosides and diglycoside were approximately half as effective as quercetin aglycone. The quercetin triglycosides were much less effective than the monoglycosides and the diglycoside. The kaempferol glycosides were 32-39% less effective in the aqueous phase antioxidant assay compared to the kaempferol aglycone. Quercetin monoglycosides and diglycoside were potent inhibitors of lipid peroxidation, in contrast to the triglycoside which was much less effective. All the kaempferol glycosides were significantly less potent inhibitors of lipid peroxidation compared to the kaempferol aglycone. The compounds described herein demonstrate the antioxidant activity of the major flavonols in tea and indicate the effect of substituting a range of sugar moieties in the phenolic C ring.

Antioxidant properties of gallocatechin and prodelphinidins from pomegranate peel.

Gallocatechins and a range of prodelphinidins were purified by high performance liquid chromatography from pomegranate peel. Gallocatechin, gallocatechin-(4-8)-catechin, gallocatechin-(4-8)-gallocatechin and catechin-(4-8)-gallocatechin were all identified, purified and quantified by LC-DAD-MS and MS-MS. The antioxidant properties of these compounds were assessed using two methods: (i) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes; and (ii) scavenging of the radical cation of 2,2-azinobis (3-ethyl-benzothiazoline-6-sulphonate, ABTS) relative to the water-soluble vitamin E analogue Trolox C (expressed as Trolox C equivalent antioxidant capacity, TEAC). The prodelphinidin dimers were potent antioxidants in the aqueous phase, being much more effective than the gallocatechin monomer. However, in the lipid phase, only one of the dimers (gallocatechin-(4-8)-catechin) was significantly more effective than the monomer in the inhibition of lipid peroxidation of phosphatidylcholine vesicles. This study represents the first report on the antioxidant properties of prodelphinidins.

Ferulic acid dehydrodimers from wheat bran: isolation, purification and antioxidant properties of 8-O-4-diferulic acid.

Wheat bran contains several ester-linked dehydrodimers of ferulic acid, which were detected and quantified after sequential alkaline hydrolysis. The major dimers released were: trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl)-7-methoxy-2, 3- dihydrobenzofuran-3-carboxylic acid (5-8-BendiFA), (Z)-beta-[4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy]-4-hydroxy-3-methox ycinnamic acid (8-O-4-diFA) and (E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid (5-5-diFA). trans-7-hydroxy-1-(4-hydroxy-3methoxyphenyl)-6-methoxy-1,2-dihydro - naphthalene-2,3-dicarboxylic acid (8-8-diFA cyclic form) and 4,4'-dihydroxy-3,3'-dimethoxy-beta,beta'-bicinnamic acid (8-8-diFA non cyclic form) were not detected. One of the most abundant dimers, 8-O-4-diFA, was purified from de-starched wheat bran after alkaline hydrolysis and preparative HPLC. The resultant product was identical to the chemically synthesised 8-O-4-dimer by TLC and HPLC as confirmed by 1H-NMR and mass spectrometry. The absorption maxima and absorption coefficients for the synthetic compound in ethanol were: lambda max: 323 nm, lambda min: 258 nm, epsilon lambda max (M-1 cm-1): 24,800 +/- 2100 and epsilon 280 (M-1 cm-1): 19,700 +/- 1100. The antioxidant properties of 8-O-4-diFA were assessed using: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes and; (b) scavenging of the radical cation of 2,2'-azinobis (3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue, Trolox C. The 8-O-4-diFA was a better antioxidant than ferulic acid in both lipid and aqueous phases. This is the first report of the antioxidant activity of a natural diferulate obtained from a plant.

Antioxidant properties of ferulic acid dimers.

Dimers of ferulic acid were chemically synthesized and the antioxidant properties assessed using (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes and (b) scavenging of the radical cation of 2, 2'-azinobis (3-ethyl-benzothiazoline-6-sulphonate (ABTS) relative to the water-soluble vitamin E analogue, Trolox C (expressed as Trolox C equivalent antioxidant capacity, TEAC). The dimers examined were (E, E)-4, 4'- dihydroxy-5, 5'-dimethoxy-3, 3'-bicinnamic acid (5-5' diFA), trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl)-7-methoxy-2, 3-dihydrobenzofuran-3-carboxylic acid (5, 8'-BenDiFA) and the methyl ester of 5, 8'-BenDiFA, dimethyl-5, 8'-BenDiFA. In both assays, the order of antioxidant efficacy was: 5, 5'-diFA > 5, 8'-BenDiFA > dimethyl-5, 8'-BenDiFA. From these results, methyl esterification decreases the antioxidant action. Comparison of the TEAC values shows that 1 mol of each of the ferulic acid dimers tested is less effective than 2 mol of free ferulic acid, and so dimerization decreases antioxidant action of these hydroxycinnamates.

A Critical Review of Assumptions About the Prairie Dog as a Keystone Species.

/ Prairie dogs (Cynomys spp.) have been labeled as keystone species because of their influence on biological diversity and ecosystem function. However, the validity of several assumptions used to support keystone status is questionable. We review the strength of the evidence and the magnitude of the prairie dog's effects on ecosystem structure and function. We use this review to reevaluate the keystone role for prairie dogs. Our goal is to encourage sound management of the prairie dog ecosystem by improving the ecological foundation of their keystone status. Our review confirms that prairie dogs affect a number of ecosystem-level functions but that their influence on prairie vertebrates may be less than previously suggested. Species richness and abundance patterns were variable among plants, mammals, and birds and were not consistently higher on prairie dog colonies compared to uncolonized areas. In addition, only nine of the 208 species listed in the literature as observed on or near prairie dogs colonies had quantitative evidence of dependence on prairie dogs. Abundance data indicated opportunistic use of colonies for an additional 20 species. A total of 117 species may have some relationship with prairie dogs, but we lacked sufficient data to evaluate the strength of this relationship. The remaining 62 species may be accidental or alien to the system. Despite our conclusion that some prairie dog functions may be smaller than previously assumed, collectively these functions are quite large compared to other herbivores in the system. We suggest that prairie dogs also provide some unique functions not duplicated by any other species and that continued decline of prairie dogs may lead to a substantial erosion of biological diversity and landscape heterogeneity across prairie and shrub-steppe landscapes. Thus, we concur that keystone status for prairie dogs is appropriate and may aid conservation efforts that help protect species dependent on prairie dogs and support other important ecosystem functions.KEY WORDS: Prairie dogs; Cynomys spp.; Keystone species; Ecosystem functions; Biological diversityhttp://link.springer-ny.com/link/service/journals/00267/bibs/24n2p177.html

Application of high-performance liquid chromatography to the assessment of subunit heterogeneity in plant 11S storage globulins.

Plant 11S storage proteins from a wide range of species have been resolved into their component subunits by ion-exchange fast protein liquid chromatography under denaturing conditions. The complex profiles obtained were reproducible and characteristic for each plant species analysed. The technique is shown to have distinct advantages over the more conventional electrophoretic approaches used to assess 11S subunit heterogeneity. The potential for the fast protein liquid chromatographic method as a simple screening system is discussed.

Human metabolic pathways of dietary flavonoids and cinnamates.

Flavonoids and cinnamates are widespread phenolic secondary metabolites synthesized by plants for defensive purposes. Many foods and beverages contain high levels of phenolic compounds. Certain phenolics in the diet are particularly bioactive and have pronounced effects on mammalian cells. These effects, together with epidemiological studies and animal models, have led to the hypothesis that dietary phenolics contribute to the health benefits of a diet rich in fruit and vegetables. This paper examines the biochemistry of the uptake and metabolic route of two groups of plant phenolics, the flavonols and hydroxycinnamates.

Dietary quercetin glycosides: antioxidant activity and induction of the anticarcinogenic phase II marker enzyme quinone reductase in Hepalclc7 cells.

It has recently been shown by Hollman et al. (Am. J. Clin. Nutr., 62, 1276-1282) that flavonoid glycosides are preferentially absorbed from dietary onions compared to the flavonoid aglycone. In the light of this, we have compared the bioactivities of the two most abundant flavonoid glycosides that we have purified from onions (quercetin-3,4'-diglucoside and quercetin-4'-glucoside) to the quercetin aglycone, and also to the more commonly studied commercially-available flavonoid glycosides, rutin (quercetin-3-rutinoside) and isoquercitrin (quercetin-3-glucoside). Quercetin aglycone was the most effective inducer of the anticarcinogenic phase II marker enzyme, quinone reductase (QR), in mouse Hepalclc7 cells. Of the glycosides, only quercetin-4'-glucoside was able to induce QR activity in this assay. Inhibition of NADPH/iron- and ascorbate/iron-induced lipid peroxidation of human liver microsomes, and the Trolox C-equivalent antioxidant capacity (TEAC), were also measured. The 4'-glycosylation dramatically decreased activity in the 'antioxidant' assays, whereas 3-substitutions produced much smaller changes. These results show that the preferentially-absorbed quercetin glycosides in onions have markedly different biological properties compared with the aglycone.

Resolution of pea legumin subunits by high-performance liquid chromatography.

Pea legumin was dissociated into its component subunits by 6 M urea: these were subsequently fractionated by FPLC using a combination of Mono P, Mono Q, and Mono S columns. The resolution and speed of separation were greatly improved in comparison with previous fractionations. Twelve discrete fractions were obtained and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six "normal" legumin subunits (Mr 60,000) were identified as well as some "large" (Mr 66,000) and "small" (Mr 44,000) subunits. A few polypeptides of unknown origin were also observed. Four subunits were purified to homogeneity as adjudged by electrophoresis and HPLC and in sufficient yields to permit further studies. Anomalous electrophoretic behavior of the legumin subunits was also observed.