, Metal chelation, radical scavenging and inhibition of Aβ 42-fibrillation by food constituents in relation to Alzheimer's disease, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem. 2015.11.118 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 These authors contributed equally to this work. Metal chelation, radical scavenging and inhibition of Aβ Abstract Various food constituents have been proposed as disease-modifying agents forAlzheimer's Disease (AD), due to epidemiological evidence of their beneficial effects, and for their ability to ameliorate factors linked to AD pathogenesis, namely by: chelating iron, copper and zinc; scavenging reactive oxygen species; and suppressing the fibrillation of amyloid-beta peptide (Aβ). In this study, nine different food constituents (L-ascorbic acid, caffeic acid, caffeine, curcumin, (−)-epigallocatechin gallate (EGCG), gallic acid, propyl gallate, resveratrol, and α-tocopherol) were investigated for their effects on the above factors, using metal chelation assays, antioxidant assays, and assays of Aβ 42 fibrillation. An assay method was developed using 5-Br-PAPS to examine the complexation of Zn(II) and Cu(II).EGCG, gallic acid, and curcumin were identified as a multifunctional compounds, however their poor brain uptake might limit their therapeutic effects. The antioxidants L-ascorbic acid and α-tocopherol, with better brain uptake, deserve further investigation for specifically addressing oxidative stress within the AD brain.

Pyrazinoic acid, the active form of the first-line antituberculosis drug pyrazinamide, decreased the proton motive force and respiratory ATP synthesis rates in subcellular mycobacterial membrane assays. Pyrazinoic acid also significantly lowered cellular ATP levels in Mycobacterium bovis BCG. These results indicate that the predominant mechanism of killing by this drug may operate by depletion of cellular ATP reserves.Shortening tuberculosis treatment duration is a key objective in order to reduce noncompliance and to combat recently emerging multidrug-resistant strains of Mycobacterium tuberculosis (6,19,36). Pyrazinamide (PZA), an important first-line drug employed in tuberculosis chemotherapy, played a key role in shortening the duration of tuberculosis treatment from 9 months to 6 months (22). PZA is a sterilizing drug that efficiently kills populations of Mycobacterium tuberculosis residing in acidic environments, as found during active inflammation (1,7,10,11,20,21,28,32). Despite the importance of PZA, no cellular target proteins have been clearly identified (4,23,30,41), and its mechanism of action is probably the least understood among all first-and second-line antituberculosis drugs. A better understanding of PZA action may help in development of new drugs to further shorten tuberculosis treatment.PZA constitutes a prodrug that is hydrolyzed in the mycobacterial cell by pyrazinamidase to yield the active entity pyrazinoic acid (POA) (15,16). According to the hypothesis put forward by Zhang and colleagues, POA, a weak acid (pK a , 2.9), acts as an uncoupling agent by breaking down the bacterial membrane potential (39,40). POA in its unprotonated form can leave the cell by means of an unknown efflux system (37, 40), take up a proton in the acidic environment, and enter the mycobacterial cell again in its protonated, less polar form (39). The resultant decrease in proton motive force then blocks, among other processes, uptake of metabolites required for growth (40). However, it is not known whether the decreased membrane potential observed for PZA in whole mycobacterial cells (40) is due to the postulated uncoupling effect or indirectly caused by interference of PZA or POA with other cellular targets. Moreover, the impact of POA on respiratory ATP synthesis and on cellular ATP levels has not been investigated. In the present study, we used subcellular and cellular assays to address these open issues. We used M. bovis BCG, which is resistant to PZA due to mutations in pyrazinamidase (16,29,30) but is fully susceptible to POA (15, 31, 37), as a model system.POA directly interferes with the proton motive force. We isolated membrane vesicles from M. bovis BCG as previously reported (9). In this subcellular system, the cytosolic fraction is removed, allowing a more specific investigation of drug action directed to membrane components (8, 9). First we determined whether pyrazinoic acid (POA) directly interferes with the proton motive force. The proton motive force was monitored with the ACMA (9-amino-6-chloro-2...

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ISCOMs have received much attention as vaccine adjuvants due to their immunostimulatory effects. They are colloidal particles typically comprised of phospholipids, cholesterol and Quil A, a crude mixture of saponins extracted from the bark of Quillaja saponaria Molina. We have previously shown that ISCOMs can be prepared by ether injection wherein an ether solution of phospholipids and cholesterol in a mass ratio of 5:2 is injected into a solution of Quil A at a mass ratio of 7 lipids: 3 Quil A. The aim of this study was firstly to isolate and characterise discrete fractions of Quil A and secondly to investigate which of these fractions were able to form ISCOMs by the method of ether injection. Six fractions of Quil A were isolated by semi-preparative reverse phase high performance liquid chromatography (RP-HPLC) and characterised by analytical HPLC, liquid chromatography tandem mass spectrometry (LC-MS) and the qualitative Liebermann-Burchard and Molisch tests for triterpenoids and carbohydrates respectively. ISCOMs were subsequently prepared from the isolated fractions by the method of ether injection and the resulting preparations characterized by photon correlation spectroscopy (PCS) and negative stain transmission electron microscopy (TEM). The molecular weights of the major compounds in the fractions ranged from approximately 1200 to approximately 2300 Da; all fractions tested positive for triterpenoids and saccharides and four of the fractions were identified as QS-7, QS-17, QS-18 and QS-21 by analysis (LC-MS and analytical HPLC). Injection of ether solutions of lipids into aqueous solutions of QS-17, QS-18 or QS-21 all resulted in homogeneous ISCOM dispersions. The combination of lipids and QS-7 by ether injection produced lamellae and liposomes as the prominent structures and a minor amount of ISCOMs. The remaining two hydrophilic, low molecular weight fractions of Quil A did not produce ISCOMs, instead liposomes and helical structures predominated in the samples.

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