Novel ingredients from brewers' spent grain - bioactivity in cell culture model systems and bioactivity retention in fortified food products

Functional food ingredients, with scientifically proven and validated bioactive effects, present an effective means of inferring physiological health benefits to consumers to reduce the risk of certain diseases. The search for novel bioactive compounds for incorporation into functional foods is particularly active, with brewers’ spent grain (BSG, a brewing industry co-product) representing a unique source of potentially bioactive compounds. The DNA protective, antioxidant and immunomodulatory effects of phenolic extracts from both pale (P1 - P4) and black (B1 – B4) BSG were examined. Black BSG extracts significantly (P < 0.05) protected against DNA damage induced by hydrogen peroxide (H2O2) and extracts with the highest total phenolic content (TPC) protected against 3-morpholinosydnonimine hydrochloride (SIN-1)-induced oxidative DNA damage, measured by the comet assay. Cellular antioxidant activity assays were used to measured antioxidant potential in the U937 cell line. Extracts P1 – P3 and B2 - B4 demonstrated significant (P < 0.05) antioxidant activity, measured by the superoxide dismutase (SOD) activity, catalase (CAT) activity and gluatathione (GSH) content assays. Phenolic extracts P2 and P3 from pale BSG possess anti-inflammatory activity measured in concanavalin-A (conA) stimulated Jurkat T cells by an enzyme-linked immunosorbent assay (ELISA); significantly (P < 0.05) reducing production of interleukin-2 (IL-2), interleukin-4 (IL-4, P2 only), interleukin-10 (IL-10) and interferon-γ (IFN-γ). Black BSG phenolic extracts did not exhibit anti-inflammatory effects in vitro. Hydroxycinnamic acids (HA) have previously been shown to be the phenolic acids present at highest concentration in BSG; therefore the HA profile of the phenolic extracts used in this research, the original barley (before brewing) and whole BSG was characterised and quantified using high performance liquid chromatography (HPLC). The concentration of HA present in the samples was in the order of ferulic acid (FA) > p-coumaric acid (p-CA) derivatives > FA derivatives > p-CA > caffeic acid (CA) > CA derivatives. Results suggested that brewing and roasting decreased the HA content. Protein hydrolysates from BSG were also screened for their antioxidant and anti-inflammatory potential. A total of 34 BSG protein samples were tested. Initial analyses of samples A – J found the protein samples did not exert DNA protective effects (except hydrolysate H) or antioxidant effects by the comet and SOD assays, respectively. Samples D, E, F and J selectively reduced IFN-γ production (P < 0.05) in Jurkat T cells, measured using enzyme linked immunosorbent assay (ELISA). Further testing of hydrolysates K – W, including fractionated hydrolysates with molecular weight < 3, < 5 and > 5 kDa, found that higher molecular weight (> 5 kDa) and unfractionated hydrolysates demonstrate greatest anti-inflammatory effects, while fractionated hydrolysates were also shown to have antioxidant activity, by the SOD activity assay. A commercially available yogurt drink (Actimel) and snack-bar and chocolate-drink formulations were fortified with the most bioactive phenolic and protein samples – P2, B2, W, W < 3 kDa, W < 5 kDa, W > 5 kDa. All fortified foods were subjected to a simulated gastrointestinal in vitro digestion procedure and bioactivity retention in the digestates was determined using the comet and ELISA assays. Yogurt fortified with B2 digestate significantly (P < 0.05) protected against H2O2-induced DNA damage in Caco-2 cells. Greatest immunomodulatory activity was demonstrated by the snack-bar formulation, significantly (P < 0.05) reducing IFN-γ production in con-A stimulated Jurkat T cells. Hydrolysate W significantly (P < 0.05) increased the IFN-γ reducing capacity of the snack-bar. Addition of fractionated hydrolysate W < 3 kDa and W < 5 kDa to yogurt also reduced IL-2 production to a greater extent than the unfortified yogurt (P < 0.05).

NOX-driven ROS formation in cell transformation of FLT3-ITD positive AML

In different types of myeloid leukemia, increased formation of reactive oxygen species (ROS) has been noted and associated with aspects of cell transformation including the promotion of leukemic cell proliferation and migration, as well as DNA-damage and accumulation of mutations. Work reviewed in this article has shown the involvement of NADPH oxidase (NOX)-derived ROS downstream of oncogenic protein-tyrosine kinases in both processes, and the related pathways have been partially identified. FLT3-ITD, an important oncoprotein in a subset of AML, causes activation of AKT and subsequently stabilization of p22phox, a regulatory subunit for NOX1-4. This process is linked to ROS formation and DNA damage. Moreover, FLT3-ITD signaling through STAT5 enhances expression of NOX4, ROS formation and inactivation of the protein-tyrosine phosphatase DEP-1/PTPRJ, a negative regulator of FLT3 signaling, by reversible oxidation of its catalytic cysteine residue. Genetic inactivation of NOX4 restored DEP-1 activity and attenuated cell transformation by FLT3-ITD in vitro and in vivo. Future work is required to further explore these mechanisms and their causal involvement in leukemic cell transformation, which may result in the identification of novel candidate targets for therapy.

Molecular genetic analysis of DNA alterations in Irish colorectal cancer

Colorectal cancer is the most common cause of death due to malignancy in nonsmokers in the western world. In 1995 there were 1,757 cases of colon cancer in Ireland. Most colon cancer is sporadic, however ten percent of cases occur where there is a previous family history of the disease. In an attempt to understand the tumorigenic pathway in Irish colon cancer patients, a number of genes associated with colorectal cancer development were analysed in Irish sporadic and HNPCC colon cancer patients. The hereditary forms of colon cancer include Familial adenomatous polyposis coli (FAP) and Hereditary Non-Polyposis Colon Cancer (HNPCC). Genetic analysis of the gene responsible for FAP, (the APC gene) has been previously performed on Irish families, however the genetic analysis of HNPCC families is limited. In an attempt to determine the mutation spectrum in Irish HNPCC pedigrees, the hMSH2 and hMLHl mismatch repair genes were screened in 18 Irish HNPCC families. Using SSCP analysis followed by DNA sequencing, five mutations were identified, four novel and a previously reported mutation. In families where a mutation was detected, younger asyptomatic members were screened for the presence of the predisposing mutation (where possible). Detection of mutations is particularly important for the identification of at risk individuals as the early diagnosis of cancer can vastly improve the prognosis. The sensitive and efficient detection of multiple different mutations and polymorphisms in DNA is of prime importance for genetic diagnosis and the identification of disease genes. A novel mutation detection technique has recently been developed in our laboratory. In order to assess the efficacy and application of the methodology in the analysis of cancer associated genes, a protocol for the analysis of the K-ras gene was developed and optimised. Matched normal and tumour DNA from twenty sporadic colon cancer patients was analysed for K-ras mutations using the Glycosylase Mediated Polymorphism Detection technique. Five mutations of the K-ras gene were detected using this technology. Sequencing analysis verified the presence of the mutations and SSCP analysis of the same samples did not identify any additional mutations. The GMPD technology proved to be highly sensitive, accurate and efficient in the identification of K-ras gene mutations. In order to investigate the role of the replication error phenomenon in Irish colon cancer, 3 polyA tract repeat loci were analysed. The repeat loci included a 10 bp intragenic repeat of the TGF-β-RII gene. TGF-β-RII is involved in the TGF-β epithelial cell growth pathway and mutation of the gene is thought to play a role in cell proliferation and tumorigenesis. Due to the presence of a repeat sequence within the gene, TGFB-RII defects are associated with tumours that display the replication error phenomenon. Analysis of the TGF-β-RII 10 bp repeat failed to identify mutations in any colon cancer patients. Analysis of the Bat26 and Bat 40 polyA repeat sequences in the sporadic and HNPCC families revealed that instability is associated with HNPCC tumours harbouring mismatch repair defects and with 20 % of sporadic colon cancer tumours. No correlation between K-ras gene mutations and the RER+ phenotype was detected in sporadic colon cancer tumours.

Osmotic stress tolerance mechanisms in Lactococcus lactis

The response of Lactococcus lactis subsp. cremoris NCDO 712 to low water activity (aw) was investigated, both in relation to growth following moderate reductions in the aw and in terms of survival following substantial reduction of the aw with NaCI. Lc.lactis NCDO 712 was capable of growth in the presence of ≤ 4% w/v NaCI and concentrations in excess of 4% w/v were lethal to the cells. The presence of magnesium ions significantly increased the resistance of NCDO 712 to challenge with NaCI and also to challenge with high temperature or low pH. Survival of Lc.lactis NCDO 712 exposed to high NaCI concentrations was growth phase dependent and cells were most sensitive in the early exponential phase of growth. Pre-exposure to 3% w/v NaCI induced limited protection against subsequent challenge with higher NaCI concentrations. The induction was inhibited by chloramphenicol and even when induced, the response did not protect against NaCI concentrations> 10% w/v. When growing at low aw, potassium was accumulated by Lc. lactis NCDO 712 growing at low aw, if the aw was reduced by glucose or fructose, but not by NaCI. Reducing the potassium concentration of chemically defined medium from 20 to 0.5 mM) produced a substantial reduction in the growth rate, if the aw was reduced with NaCI, but not with glucose or fructose. The reduction of the growth rate correlated strongly with a reduction in the cytoplasmic potassium concentration and in cell volume. Addition of the compatible solute glycine betaine, partially reversed the inhibition of growth rate and partially restored the cell volume. The potassium transport system was characterised in cells grown in medium at both high and low aw. It appeared that a single system was present, which was induced approximately two-fold by growth at low aw. Potassium transport was assayed in vitro using cells depleted of potassium; the assay was competitively inhibited by Na+ and by the other monovalent cations NH4+, Li+, and Cs+. There was a strong correlation between the ability of strains of Lc. lactis subsp. lactis and subsp. cremoris to grow at low aw and their ability to accumulate the compatible solute glycine betaine. The Lc. lactis subsp. cremoris strains incapable of growth at NaCI concentrations> 2% w/v did not accumulate glycine betaine when growing at low aw, whereas strains capable of growth at NaCI concentrations up to 4% w/v did. A mutant, extremely sensitive to low aw was isolated from the parent strain Lc. lactis subsp. cremoris MG 1363, a plasmid free derivative of NCDO 712. The parent strain tolerated up to 4% w/v NaCI and actively accumulated glycine betaine when challenged at low aw. The mutant had lost the ability to accumulate glycine betaine and was incapable of growth at NaCI concentrations >2% w/v or the equivalent concentration of glucose. As no other compatible solute seemed capable of substitution for glycine betaine, the data suggest that the traditional; phenotypic speciation of strains on the basis of tolerance to 4% w/v NaCI can be explained as possession or lack of a glycine betaine transport system.

Ecotoxicology of marine biotoxins in bivalve shellfish

A small proportion of harmful algae produce toxins which are harmful to human health. Strict monitoring programmes are in place within Ireland and the EU to effectively manage risk to human consumers of shellfish species that have accumulated marine biotoxins in their tissues. However, little is known about the impacts of HABs on shellfish health. This study used Solid Phase Adsorption and Toxin Tracking (SPATT) for the passive sampling of algal biotoxins at Lough Hyne Marine Nature Reserve in West Cork, Ireland. Spatial and temporal monitoring of the incidence of a wide range of lipophilic toxins was assessed over a 4-month period. Active sampling accumulated sufficient quantities of toxin for use in subsequent experimentation. In addition to commonly occurring Diarrhetic Shellfish Poisoning (DSP) toxins, Dinophysis toxin-1 and Pinnatoxin-G were both detected in the samples. This is the first identification of these latter two toxins in Irish waters. The effects of the DSP toxin okadaic acid (OA) were investigated on three shellfish species: Mytilus edulis, Ruditapes philippinarum and Crassostrea gigas. Histological examination of the gill, mantle and hepatopancreas tissues revealed varying intensity of damage depending both on the tissue type and the species involved. At the cellular level, flow cytometric analysis of the differential cell population distribution was assessed. No change in cell population distribution was observed in Mytilus edulis or Ruditapes philippinarum, however significant changes were observed in Crassostrea gigas granulocytes at the lower levels of toxin exposure. This indicated a chemically-induced response to OA. DNA fragmentation was measured in the haemolymph and hepatopancreas cells post OA-exposure in Mytilus edulis and Crassostrea gigas. A significant increase in DNA fragmentation was observed in both species over time, even at the lowest OA concentrations. DNA fragmentation could be due to genotoxicity of OA and/or to the induction of cell apoptosis.

The role of the IL-1 type 1 receptor in the life, death and differentiation of adult hippocampal neural precursor cells

Neurogenesis occurs in two distinct regions of the adult brain; the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus, and the subventricular zone (SVZ) lining the lateral ventricles. It is now well-known that adult hippocampal neurogenesis can be modulated by a number of intrinsic and extrinsic factors e.g. local signalling molecules, exercise, environmental enrichment and learning. Moreover, levels of adult hippocampal neurogenesis decrease with age, at least in rodents, and alterations in hippocampal neurogenesis have been reported in animal models and human studies of neuropsychiatric and neurodegenerative conditions. Neuroinflammation is a common pathological feature of these conditions and is also a potent modulator of adult hippocampal neurogenesis. Recently, the orphan nuclear receptor TLX has been identified as an important regulator of adult hippocampal neurogenesis as its expression is necessary to maintain the neural precursor cell (NPC) pool in the adult DG. Likewise, exposure of animals to voluntary exercise has been consistently demonstrated to promote adult hippocampal neurogenesis. Lentivirus (LV)- mediated gene transfer is a useful tool to elucidate gene function and to explore potential therapeutic candidates across an array of conditions as it facilitates sustained gene expression in both dividing and post-mitotic cell populations. Both intrinsic and extrinsic factors are important regulators of adult hippocampal neurogenesis. Examining how these factors are affected by an inflammatory stimulus, and the subsequent effects on adult hippocampal neurogenesis provides important information for the development of novel treatment strategies for neuropsychiatric and neurodegenerative conditions in which adult hippocampal neurogenesis is impaired. The aims of the series of experiments presented in this thesis were to examine the effect of the pro-inflammatory cytokine interleukin-1β (IL-1β) on adult hippocampal NPCs both in vitro and in vivo. In vitro, we have shown that IL-1β reduces proliferation of adult hippocampal NPCs in a dose and time-dependent manner. In addition, we have demonstrated that TLX expression is reduced by IL-1β. Blockade of IL-1β signalling prevented both the IL-1β-induced reduction in cell proliferation and TLX expression. In vivo, we examined the effect of short term and long term exposure to LV-IL-1β in sedentary mice and in mice exposed to voluntary running. We demonstrated that impaired hippocampal neurogenesis is only evident after long term exposure to IL-1β. In mice exposed to voluntary running, hippocampal neurogenesis is significantly increased following short-term but not long-term exposure to running. Moreover, short-term running effectively prevents any IL-1β-induced effects on hippocampal neurogenesis; however, no such effects are seen following long-term exposure to running.

Investigation of the genetic susceptibility to osteoporosis in the Irish population

Osteoporosis is a complex skeletal disorder characterized by compromised bone strength. Variation in bone mineral density (BMD) is a contributing factor. The aim of this research as to select informative single nucleotide polymorphisms (SNPs) in potential candidate genes from loci suggestively linked to BMD variation for fine mapping. The gene regulated by oestrogen in breast cancer 1 (GREB1), located at 2p25.1, was selected. GREB1 transcription is initiated early in the oestrogen receptor alpha regulated pathway. There was significant association between GREB1_03 and BMD variation at the lumbar spine and femoral neck (FN) in the discovery cohort. Significant association was observed between GREB1_04 and FN BMD in the replication cohort. The development and differentiation enhancing factor 2, the integrin cytoplasmic domain associated protein 1 and A-disintegrin and metalloprotease 17 were selected due to their respective roles in cell mobility and adhesion. There was no linkage or association observed between the Chr2 cluster SNPs and BMD. Two factors in bone remodelling are the attraction of bone cell precursors and endocrine regulation of the process, primarily through the action of parathyroid hormone (PTH). The C-C chemokine receptor type 3 (CCR3) encodes a CC chemokine receptor expressed in osteoclast precursors. The PTH receptor type 1 (PTHR1) encodes a G-protein coupled receptor for PTH. Association was observed between CCR3 haplotypes and BMD variation at the FN. There was no linkage or association observed between PTHR1 SNPs and BMD variation. Population genetic studies with complex phenotypes endeavour to elucidate the traits genetic architecture. This study presents evidence of association between GREB1 and BMD variation and as such, introduces GREB1 as a novel gene target for osteoporosis genetics studies. It affirms that common genomic variants in PTHR1 are not associated with BMD variation in Caucasians and supports the evidence that CCR3 may be contributing to BMD variation

The analysis of Bcr-Abl—Nox signalling in chronic myeloid leukaemia

Chronic Myeloid Leukaemia (CML) is a myeloproliferative disorder characterised by increased proliferation of haematopoietic stem cells. CML results following generation of the chimeric protein Bcr-Abl, a constitutively active tyrosine kinase which induces oncogenesis in part by promoting increased cell survival and proliferation. Since the development of Bcr-Abl-specific tyrosine kinase inhibitors (TKIs) there has been a substantial improvement in the clinical treatment of CML. Unfortunately, residual disease and the development of TKI resistance has become an ever growing concern, resulting in the need for a greater understanding of the disease in order to develop new treatment strategies. Interestingly, constitutive expression of the Bcr-Abl in CML is known to produce elevated levels of Reactive Oxygen Species (ROS) which are known to influence a variety of cellular processes. Previous studies have demonstrated that NADPH oxidase (Nox) activity contributes to intracellular-ROS levels in Bcr-Abl-positive cells, enhancing survival signalling. The objective of this study was to elucidate how Nox protein activity was influenced downstream of Bcr-Abl while examining how Nox-derived ROS influenced CML disease phenotype to identify the potential in targeting these proteins to improve CML treatment. These studies demonstrated that inhibition of Bcr-Abl signalling, led to a significant reduction in ROS levels which was concurrent with the GSK-3dependent, post-translational down-regulation of the small membrane-bound protein p22phox, an essential component of the Nox complex. siRNA knockdown of p22phox identified it to have a significant role in cellular proliferation and cell viability, demonstrating the importance of Nox protein activity in CML disease phenotype. Furthermore, removal of p22phox was demonstrated to make cells significantly more susceptible to Bcr-Abl-specific TKI treatment, while pharmacological silencing of Nox activity in combination with TKIs was demonstrated to produce substantial, synergistic increases in cell death through augmentation of apoptosis, demonstrating the therapeutic potential of targeting Nox proteins in combination with Bcr-Abl inhibition.

The use of different blood sample preparations in the development of biomarkers for the environmental monitoring of domestic farm animals

The application of biological effect monitoring for the detection of environmental chemical exposure in domestic animals is still in its infancy. This study investigated blood sample preparations in vitro for their use in biological effect monitoring. When peripheral blood mononuclear cells (PBMCs), isolated following the collection of multiple blood samples from sheep in the field, were cryopreserved and subsequently cultured for 24 hours a reduction in cell viability (<80%) was attributed to delays in the processing following collection. Alternative blood sample preparations using rat and sheep blood demonstrated that 3 to 5 hour incubations can be undertaken without significant alterations in the viability of the lymphocytes; however, a substantial reduction in viability was observed after 24 hours in frozen blood. Detectable levels of early and late apoptosis as well as increased levels of ROS were detectable in frozen sheep blood samples. The addition of ascorbic acid partly reversed this effect and reduced the loss in cell viability. The response of the rat and sheep blood sample preparations to genotoxic compounds ex vivo showed that EMS caused comparable dose-dependent genotoxic effects in all sample preparations (fresh and frozen) as detected by the Comet assay. In contrast, the effects of CdCl2 were dependent on the duration of exposure as well as the sample preparation. The analysis of leukocyte subsets in frozen sheep blood showed no alterations in the percentages of T and B lymphocytes but led to a major decrease in the percentage of granulocytes compared to those in the fresh samples. The percentages of IFN-γ and IL-4 but not IL-6 positive cells were comparable between fresh and frozen sheep blood after 4 hour stimulation with phorbol 12-myrisate 13-acetate and ionomycin (PMA+I). These results show that frozen blood gives comparable responses to fresh blood samples in the toxicological and immune assays used.