Martin P. Bucknall

Practical quantitative biomedical applications of MALDI-TOF mass spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) is used to obtain fast and accurate determinations of molecular mass, but quantitative determinations are generally made by other techniques. In this study we illustrate the practical utility of automated MALDI-TOFMS as a tool for quantifying a diverse array of biomolecules covering an extensive molecular weight range, and present in biological extracts and fluids. Growth hormone was measured in rat pituitary tissue; insulin in human pancreatic tissue; homovanillic acid in human urine; and LVV-hemorphin-7, epinephrine and norepinephrine in human adrenal and pheochromocytoma tissues. Internal standards including compounds of similar molecular weight, structural analogs or isotopomers were incorporated into each analysis. We report on the current practical limitations of quantitative MALDI-TOFMS and highlight some of the potential benefits of this technique as a quantitative tool.

Induction of Settlement of Larvae of the Sea Urchin Holopneustes purpurascens by Histamine From a Host Alga

Larvae of the Australian sea urchin Holopneustes purpurascens are induced to settle and metamorphose (termed settlement herein) by a water-soluble compound produced by the red alga Delisea pulchra, the main host plant of new recruits. The settlement cue for H. purpurascens had previously been identified as a floridoside-isethionic acid complex, and this paper presents new evidence correcting that finding. The actual settlement cue produced by D. pulchra was isolated from the polar extract by cation-exchange chromatography and identified as histamine, using one- and two-dimensional nuclear magnetic resonance spectrometry. The chemical identity of the cue was confirmed by gas chromatography–mass spectrometry (GC-MS) and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry. Synthetic histamine and histamine at 4.5 μM isolated from D. pulchra both induced rapid settlement in 80%–100% of the larvae of H. purpurascens. Lower concentrations of histamine (0.9–2.3 μM) induced larval settlement, but this response varied from 0%–90%. The histamine content of two host plants of H. purpurascens—D. pulchra and Ecklonia radiata—and of four other common species was quantified using GC-MS. D. pulchra had the highest histamine content, which is consistent with H. purpurascens recruiting to this species. Histamine was also detected in the seawater surrounding these host algae. This is the first time that a settlement cue has been quantified in the habitat of a marine organism.

Ultrafiltration of protein mixtures: measurement of apparent critical flux, rejection performance, and identification of protein deposition

Abstract Crossflow ultrafiltration of binary protein solutions was carried out using flux-stepping and constant flux experiments to identify the apparent critical flux where fouling is rapid. The contributions of individual protein species to the apparent critical flux were evaluated as well as the separation performance. For mixtures of gunkeyable???-globulin/lysozyme and BSA/lysozyme the larger retained protein tended to control the critical flux behaviour while the observed rejection of the smaller transmitted protein went through a minimum close to the apparent critical flux. Identification of the respective protein species deposited onto membrane surfaces was carried out using Matrix-Assisted Laser Desorption Ionisation Mass Spectroscopy (MALDI-MS). Mass spectra showed that the transmitted proteins resulted in a higher incidence of peaks relative to the retained proteins. This was thought to be the result of desorption of proteins from the membrane surface, from inside pores and from the membrane substrate. It was shown that the MALDI-MS technique is a powerful tool for distinguishing between different proteins in fouling deposits and has potential for quantitative measurement of protein fouling on membrane surfaces.

Differential injurious effects of ambient and traffic-derived particulate matter on airway epithelial cells

Exposure to airborne particulate matter (PM) may promote development of childhood asthma and trigger acute exacerbations of existing asthma via injury to airway epithelial cells (AEC).

Signaling regulates activity of DHCR24, the final enzyme in cholesterol synthesis

The role of signaling in regulating cholesterol homeostasis is gradually becoming more widely recognized. Here, we explored how kinases and phosphorylation sites regulate the activity of the enzyme involved in the final step of cholesterol synthesis, 3β-hydroxysterol Δ24-reductase (DHCR24). Many factors are known to regulate DHCR24 transcriptionally, but little is known about its posttranslational regulation. We developed a system to specifically test human ectopic DHCR24 activity in a model cell-line (Chinese hamster ovary-7) using siRNA targeted only to hamster DHCR24, thus ensuring that all activity could be attributed to the human enzyme. We determined the effect of known phosphorylation sites and found that mutating certain residues (T110, Y299, and Y507) inhibited DHCR24 activity. In addition, inhibitors of protein kinase C ablated DHCR24 activity, although not through a known phosphorylation site. Our data indicate a novel mechanism whereby DHCR24 activity is regulated by signaling.

Interactions between phytochemicals from fruits and vegetables: Effects on bioactivities and bioavailability

ABSTRACT The combinations of two or more phytochemicals bring about changes in the ultimate biological effects and/or the bioavailability of each component. A number of mixtures of pure bioactive compounds or phytochemical-containing plant extracts provide synergy with regard to antioxidant status, anti-inflammation, anti-cancer and chemoprevention of several oxidative stress and metabolic disorders in vitro. The biological activities of food phytochemicals depend upon their bioaccessibility and bioavailability which can be affected by the presence of other food components including other bioactive constituents. The interactions between phytochemicals during intestinal absorption could result in changes in the bioavailability of the compounds, which in turn affects the intensity of their bioactivities. This paper provides an overview of combined biological effects of phytochemical mixtures derived from fruits and vegetables with a focus on anti-oxidative, anti-inflammatory and anti-carcinogenic activities. The bioavailability impairment or enhancement caused by the co-consumption of dietary phytochemicals is also discussed. Finally, research gaps for future studies on phytochemical interactions are identified.

Novel predators emit novel cues: a mechanism for prey naivety towards alien predators

Detecting enemies is crucial for survival and a trait that develops over an evolutionary timeframe. Introduced species disrupt coevolved systems of communication and detection in their new ranges, often leading to devastating impacts. The classic example is prey naivety towards alien predators, whereby prey fail to recognise a new predator. Yet exactly why native prey fail to recognise alien predators remains puzzling. Naivety theory predicts that it is because novel predators emit novel cues. Distantly related animals have distinct evolutionary histories, physiologies and ecologies, predicting they will emit different cues. Yet it also possible that all predators emit similar cues because they are carnivorous. We investigate whether odour cues differ between placental and marsupial carnivores in Australia, where native prey experienced only marsupial mammal predation until ~4000 years ago. We compared volatile chemical profiles of urine, scats and bedding from four placental and three marsupial predators. Chemical profiles showed little overlap between placental and marsupial carnivores across all odour types, suggesting that cue novelty is a plausible mechanism for prey naivety towards alien predators. Our results also suggest a role for olfactory cues to complement visual appearance and vocalisations as biologically meaningful ways to differentiate species.

Evaluating folate extraction from infant milk formulae and adult nutritionals: Enzymatic digestion versus enzyme-free heat treatment

This study compares enzymatic treatments to release folic acid (FA) and endogenous 5-methyltetrahydrofolate (5-MTHF) from infant milk formulae with enzyme-free heat extraction. The limits of detection and quantitation of FA were 1.4ng/mL and 3.1ng/mL, respectively; 7.5ng/mL and 16.2ng/mL for 5-MTHF. Absolute mean recoveries were 85% (FA) and 95% (5-MTHF). The RSD of the within-run variability was 6% and the inter-day variability was 8%. Averaged measurements of FA and 5-MTHF in SRM-1849a were within the certified value range. Analysed folate levels in three brands were greater than label values, because of inherently high 5-MTHF occurring in samples. The results indicate that enzyme-free heat treatment prior to UPLC-MS/MS analysis gives better sensitivity and reduces chromatographic interferences for the determination of FA and 5-MTHF in milk formulae than enzymatic treatments. Enzyme-free heat treatment is more compatible with UPLC-MS/MS than folate extraction techniques involving the addition of enzymes to milk.

Interferences of anthocyanins with the uptake of lycopene in Caco-2 cells, and their interactive effects on anti-oxidation and anti-inflammation in vitro and ex vivo

Lycopene was combined with the glucosides of each of the six common anthocyanidins at 3 different ratios to investigate their interactions on antioxidant and anti-inflammatory activities, and cellular uptake. The bioactivity interaction between lycopene and anthocyanins was studied in both chemical and cellular models. Anti-oxidative synergy was not seen in any of the tested lycopene-anthocyanin mixtures, nor in the models studied. When lycopene was paired with the methoxylated anthocyanins, the anti-inflammatory effect on the inhibition of the cytokine IL-8, which is a pro-inflammatory biomarker, was increased by 15-69% of the expected additive activity, indicating synergistic interaction between the compounds. The cellular uptake of lycopene was significantly impaired by the presence of the anthocyanins: reduced by 50-80% at the lycopene: anthocyanin combinatory ratios of 2.5:7.5 μM (1:3) or 5:5 μM (1:1). The reduced intracellular lycopene content might be partly responsible for the antagonistic cellular antioxidant property seen in some of the tested mixtures.

Effect of Different Anthocyanidin Glucosides on Lutein Uptake by Caco-2 Cells, and Their Combined Activities on Anti-Oxidation and Anti-Inflammation In Vitro and Ex Vivo

The interactive effects on anti-oxidation and anti-inflammation of lutein combined with each of the six common anthocyanidin glucosides were studied in both chemical and cellular systems. The combined phytochemicals showed an antagonism in the inhibition of lipid oxidation in a liposomal membrane, but showed an additive effect on cellular antioxidant activity in Caco-2 cells. Lutein was an active lipoxygenase inhibitor at 2–12 μM while anthocyanins were inactive. The concentration of lutein when it was used in combination with anthocyanins was 25–54% higher than when lutein was used alone (i.e., IC50 = 1.2 μM) to induce 50% of lipoxygenase inhibition. Only the combination of lutein with malvidin-3-glucoside showed anti-inflammatory synergy in the suppression of interleukin-8, and the synergy was seen at all three ratios tested. Some mixtures, however, showed anti-inflammatory antagonism. The presence of anthocyanins (5–7.5 μM) did not affect lutein uptake (2.5–5 μM) by Caco-2 cells.