Ken Ng

Chemical characterisation and speciation of organic selenium in cultivated selenium-enriched Agaricus bisporus

The selenium concentration in Agaricus bisporus cultivated in growth compost irrigated with sodium selenite solution increased by 28- and 43-fold compared to the control mushroom irrigated solely with water. Selenium contents of mushroom proteins increased from 13.8 to 60.1 and 14.1 to 137 μgSe/g in caps and stalks from control and selenised mushrooms, respectively. Selenocystine (SeCys; detected as [SeCys]2 dimer), selenomethionine (SeMet), and methyl-selenocysteine (MeSeCys) were separated, identified and quantified by liquid chromatography-electrospray ionisation-mass spectrometry from water solubilised and acetone precipitated proteins, and significant increases were observed for the selenised mushrooms. The maximum selenoamino acids concentration in caps and stalks of control/selenised mushrooms was 4.16/9.65 μg/g dried weight (DW) for SeCys, 0.08/0.58 μg/g DW for SeMet, and 0.031/0.10 μg/g DW for MeSeCys, respectively. The most notable result was the much higher levels of SeCys accumulated by A. bisporus compared to SeMet and MeSeCys, for both control and selenised A. bisporus.

Sulfate activation and transport in mammals: system components and mechanisms

Extensive studies on the mammalian sulfate-activating enzymes and PAPS translocase have enhanced our understanding of the overall pathway of sulfate activation and utilization. Isolation of the PAPS-synthesizing activities from rat chondrosarcoma and preparation of stable non-hydrolyzable analogs of APS and PAPS have facilitated the kinetic characterization of mammalian ATP sulfurylase and APS kinase. These studies provided the basis for further experimental work showing that APS, the labile intermediate product, is channeled directly between the sulfurylase and kinase active sites. The defect in the brachymorphic mutant mouse lies in this channeling mechanism, thus interfering with efficient PAPS production. The rat chondrosarcoma ATP sulfurylase and APS kinase activities, in fact, reside in a single bifunctional cytoplasmic protein, which has now been cloned and expressed. The mechanism by which PAPS reaches its sites of utilization in the Golgi lumen has also been elucidated: The PAPS translocase is a 230-kDa integral Golgi membrane protein which functions as an antiport.

The antimalarial drug, chloroquine, interacts with lactate dehydrogenase from Plasmodium falciparum

We have previously shown that a radioiodinated photoreactive analogue of chloroquine, [125I]N-(4-(4-diethylamino-1-methylbutylamino)quinolin-6-yl) -4-azido-2-hydroxybenzamide ([125I]ASA-Q), specifically labels two proteins in Plasmodium falciparum with apparent molecular weights (Mr) of 42 and 33 kDa (Foley M, Deady LW, Ng K, Cowman AF, Tilley L. J Biol Chem 1994:269:6955-6961). We now report the identification of the 33 kDa protein. The 33 kDa protein was purified from Plasmodium falciparum using photoaffinity labeling with [125I]ASA-Q to monitor the enrichment process. N-terminal sequence analysis of the purified protein revealed exact identity of the first 35 amino acids with P. falciparum lactate dehydrogenase (PfLDH). The plasmodial enzyme was cloned and expressed in E. coli and the recombinant protein used to produce a rabbit antiserum. Immunoprecipitation using affinity-purified anti-PfLDH antibodies confirmed the identity of the 33 kDa CQ-binding protein. The enzyme activity of purified PfLDH was not significantly affected by chloroquine indicating that PfLDH is not a direct target of CQ. PfLDH was, however, shown to be exquisitely sensitive to inhibition by free heme and chloroquine protected against this inhibitory effect.

Interactions of buttermilk with curcuminoids.

The ability of buttermilk to carry and stabilise a preparation of curcuminoids was examined. The quenching of intrinsic protein fluorescence confirmed that the curcuminoids interacted with proteins in buttermilk. The Stern-Volmer quenching constant was ≥ 9.4 × 10³ M⁻¹. The apparent binding constant of curcuminoids to whole buttermilk was ≥ 2.2 × 10⁴ M⁻¹. Centrifugation of buttermilk (5% total solids, TS)--curcuminoid mixtures demonstrated that curcuminoids were partitioned into the cream (18.0%w/w, 0.64% TS), milk serum (73.3%w/w, 2.86% TS) and the casein-rich precipitate (6.76% w/w, 1.87% TS) fractions in the ratio of 1:3.7:3.5. The interaction of curcuminoids with components in the buttermilk improved its stability, as evidenced by the faster degradation of curcuminoids in phosphate buffer (pH=6.8) than in buttermilk. The ability of buttermilk to carry and stabilise curcuminoids has the potential to enable the delivery of these components into functional foods.

Specificity of Binding of [beta]-Glucoside Activators of Ryegrass (1->3)-[beta]-Glucan Synthase and the Synthesis of Some Potential Photoaffinity Activators

Structure-activity relationships among glycoside activators of ryegrass (Lolium multiflorum) (1–<3)-[beta]-glucan synthase were investigated using a number of natural and synthetic glycosides, including some carrying photoaffinity functions. There is an absolute requirement for a [beta]-D-glucosyl moiety in the activator, both S- and N-glucosides are active, and the position of the glucosidic linkage in [beta]-glucose disaccharides has a significant effect on the affinity of binding. However, the binding requirement does not extend beyond a single [beta]-D-glucosyl residue, and [beta]-D-oligoglucosides are less effective than disaccharides. The nature of the aglycon has a major influence on the binding affinity. Hydrophobic aglycons lower the concentration required for half-maximal stimulation of the enzyme obtained from an Eadie-Hofstee plot of kinetic data (Ka) for activation, but charged aglycons increase Ka. Relative to methyl-[beta]-D-glucoside and cellobiose (Ka 1.1 mM), the most potent compounds tested were N-[4-(benzoyl)benzoyl]-[beta]-D-glucosylamine and 2[prime]-[4-azidosalicylamino]ethyl-1-thio-[beta]-D-glucoside with Kas of approximately 30 [mu]M. The latter also was tested for its potential to specifically label the [beta]-glucoside-binding site on the synthase, but under the conditions used the binding was found to be nonspecific.

Selenium-enriched Agaricus bisporus increases expression and activity of glutathione peroxidase-1 and expression of glutathione peroxidase-2 in rat colon

The effect of dietary supplementation with Se-enriched Agaricus bisporus on cytosolic gluthathione peroxidase-1 (GPx-1), gastrointestinal specific glutathione peroxidase-2 (GPx-2), thioredoxin reductase-1 (TrxR-1) and selenoprotein P (SeP) mRNA expression and GPx-1 enzyme activity in rat colon was examined. Rats were fed for 5weeks with control diet (0.15μg Se/g feed) or Se-enriched diet fortified with selenised mushroom (1μg Se/g feed). The mRNA expression levels were found to be significantly (P<0.01) up-regulated by 1.65-fold and 2.3-fold for GPx-1 and GPx-2, respectively, but were not significantly different for TrxR-1 and SeP between the 2 diet treatments. The up-regulation of GPx-1 mRNA expression was consistent with GPX-1 activity level, which was significantly (P<0.05) increased by 1.77-fold in rats fed with the Se-enriched diet compared to the control diet. The results showed that selenised A. bisporus can positively increase GPx-1 and GPx-2 gene expression and GPx-1 enzyme activity in rat colon.

Antioxidant capacity and mineral contents of edible wild Australian mushrooms

Five selected edible wild Australian mushrooms, Morchella elata, Suillus luteus, Pleurotus eryngii, Cyttaria gunnii, and Flammulina velutipes, were evaluated for their antioxidant capacity and mineral contents. The antioxidant capacities of the methanolic extracts of the dried caps of the mushrooms were determined using a number of different chemical reactions in evaluating multi-mechanistic antioxidant activities. These included the Trolox equivalent antioxidant capacity, ferric ion reducing antioxidant power, and ferrous ion chelating activity. Mineral contents of the dried caps of the mushrooms were also determined by inductively coupled plasma–optical emission spectroscopy. The results indicated that these edible wild mushrooms have a high antioxidant capacity and all, except C. gunnii, have a high level of several essential micro-nutrients such as copper, magnesium, and zinc. It can be concluded that these edible wild mushrooms are good sources of nutritional antioxidants and a number of mineral elements.

Selenium-Enriched Agaricus bisporus Mushroom Protects against Increase in Gut Permeability ex vivo and Up-Regulates Glutathione Peroxidase 1 and 2 in Hyperthermally-Induced Oxidative Stress in Rats

Dietary effects of organic Se supplementation in the form of Se-enriched Agaricus bisporus mushroom on ileal mucosal permeability and antioxidant selenoenzymes status in heat induced oxidative stress in rats were evaluated. Acute heat stress (40 °C, 21% relative humidity, 90 min exposure) increased ileum baseline short circuit current (Isc; 2.40-fold) and epithelial conductance (Ge; 2.74-fold). Dietary supplementation with Se-enriched A. bisporus (1 µg Se/g feed) reduced (p < 0.05) ileum Isc and Ge during heat stress to 1.74 and 1.91 fold, respectively, indicating protection from heat stress-induced mucosal permeability increase. The expression of ileum glutathione peroxidase (GPx-) 1 and 2 mRNAs were up-regulated (p < 0.05) by 1.90 and 1.87-fold, respectively, for non-heat stress rats on the Se-enriched diet relative to the control. The interplay between heat stress and dietary Se is complex. For rats on the control diet, heat stress alone increased ileum expression of GPx-1 (2.33-fold) and GPx-2 (2.23-fold) relative to thermoneutral conditions. For rats on the Se-enriched diet, heat stress increased (p < 0.05) GPx-1 expression only. Rats on Se-enriched + α-tocopherol diet exhibited increased expression of both genes (p < 0.05). Thus, dietary Se-enriched A. bisporus protected against increase in ileum permeability and up-regulated GPx-1 and GPx-2 expression, selenoenzymes relevant to mitigating oxidative stress.

Bioaccessibility of curcuminoids in buttermilk in simulated gastrointestinal digestion models.

In vitro gastrointestinal digestion models were used to investigate bioaccessibility of curcuminoids delivered with buttermilk. The percentage of solubilised curcuminoids that partitioned into the micelle in aqueous phase was determined. In fasted states (0-2.5 mg bile extract/mL sample), the bioaccessibility of curcuminoids (2% v/v ethanol) ranged from 16.3% to 26.7% in buttermilk, and from 11.4% to 18.7% with neat curcuminoids. In fed states (10-40 mg bile extract/mL sample), the bioaccessibility of curcuminoids in buttermilk was 21.3% (no ethanol) and ranged from 37.1% to 69.2% (2% v/v ethanol), while for neat curcuminoids bioaccessibility was 14.1% (no ethanol), ranging from 45.6% to 79.6% (2% v/v ethanol). The in vitro bioaccessibility of curcuminoids was influenced by the presence of the carrier (buttermilk) and ethanol, and increased significantly with increasing amount of bile extract. Curcuminoids did not markedly influence the digestibility of protein or lipids. These findings demonstrated that buttermilk could be used as a carrier for curcuminoids especially if delivered with food.

Genistein- and daidzein 7-O-β-D-glucuronic acid retain the ability to inhibit copper-mediated lipid oxidation of low density lipoprotein

Two isoflavones in vivo metabolites, genistein-7-O-beta-D-glucuronic acid (G7G) and daidzein-7-O-beta-D-glucuronic acid (D7G) were synthesised chemically. The ability of these metabolites to scavenge an organic radical was measured by the trolox equivalent antioxidant capacity (TEAC) assay, while their reducing ability was measured by the ferric reducing antioxidant power (FRAP) assay. The TEAC and FRAP values of G7G were 45 and 51% of that of genistein, while those of D7G were 52 and 77% of that of daidzein, respectively. A direct assessment of G7G and D7G antioxidant activity by their ability to delay copper(II)-mediated lipid oxidation of human LDL showed that these metabolites retained the ability to prevent oxidation in the lipid phase, but activity was diminished compared to their corresponding aglycones. However, G7G and D7G also decreased the rate of lipid oxidation to 53 and 86% of control without isoflavones, respectively, indicating a continuous exchange of antioxidants between the aqueous environment and the LDL lipid phase during the whole oxidation period.