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Presenilin-1 (PS-1) is the catalytic core of the ã-secretase complex, which is best known for its role in the generation of the Alzheimer disease (AD)-related â-amyloid peptide. Mutated forms of PS-1 are known to be associated with particularly aggressive forms of AD. Changes in monoaminergic neurotransmitter systems, including the serotonin and norepinephrine systems, have long been associated with some of the earliest events in AD, whereas changes in the availability of these same monoamines have historically been associated with clinical depression. Therefore, it is not surprising that depression has now been proposed as a risk factor for developing AD and that pre-demented carriers of mutated forms of PS-1 are more prone to developing depression. MAO-A is historically associated with depression and is also a known risk factor for AD. Given this, I hypothesized that MAO-A represents a neurochemical link between depression and AD, and I chose to examine the influence of PS-1 mutations on MAO-A function in vivo/ex vivo and in vitro.<p> I first focused on the PS-1(M146V) knock-in mouse model of AD-related PS-1/ã-secretase function. I used a radioenzymatic assay to estimate MAO-A catalytic activity and western blot analysis to determine MAO-A protein expression, and found that MAO-A activity does not correlate with MAO-A expression in the cortex and cerebellum of the PS-1(M146V) mice. Furthermore, the potency of the MAO-A inhibitor clorgyline (CLG) is greater in both the cortex and cerebellum of the PS-1(M146V) mice compared to the potency of CLG in wildtype littermates. CLG dose-response curves suggest that there might be a change in cooperativity in the MAO-A protein from PS-1(M146V) cortex (which would suggest a change in conformation and/or access of the substrate to the catalytic pocket in MAO-A). High-pressure liquid chromatography was used to analyze monoamine levels in these same regions. The levels of monoamines (i.e. serotonin, dopamine and norepinephrine) suggest that PS-1 (M146V) inhibits MAO-A function in the cortex, but not in the cerebellum. Furthermore, CLG has no significant effect on amine levels in cortex, but tends to increase their accumulation in cerebellum.<p> The overexpression of PS-1 (M146V) in neuronal cultures reveals that this protein affects MAO-A activity and, more importantly, the PS-1(M146V) protein co-precipitates with MAO-A, thus suggesting a possibility for a direct protein-protein interaction. This is supported by the observation that MAO-A activity is increased in cell extracts incubated with the PS-1 substrate-competitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Preliminary studies have been undertaken to determine the motif in MAO-A that could be acting as a binding site/target site for PS-1.<p> These combined results support the hypothesis that PS-1 proteins can influence MAO-A function and, furthermore, that MAO-A is a novel interactor for PS-1/ã-secretase. This could well explain some of the ambiguous literature associated with both of these proteins in disorders as diverse as depression and AD.

The enzyme monoamine oxidase (MAO) is linked to mental disorders such as depression and neurodegenerative diseases. Our laboratory has recently demonstrated that increases in calcium (Ca2+) can enhance MAO activity and that this might contribute to Alzheimer disease (AD). AD has been linked to gain-of-function mutations in the presenilin-1 (PS-1) protein that not only promote the generation of the toxic amyloid-â peptide, but that also alter intracellular Ca2+ availability. <p>Radioenzymatic MAO assays were used to demonstrate that over-expression of different AD-related PS-1 mutant proteins, i.e. Y115H, ÄEx9 and M146V, in hippocampal-derived HT-22 cells alter either basal and/or Ca2+-sensitive MAO-A activity. The effects of PS-1 mutant proteins on the availability of intracellular Ca2+ are not consistent suggesting that this may not be the primary means of regulating MAO activity. The sensitivity of MAO to Ca2+ was also demonstrated in cortical (both MAO-A and MAO-B responded to Ca2+) and cerebellar (only MAO-A responded to Ca2+) samples from transgenic mice overexpressing the PS-1 (M146V) mutation. These changes in MAO coincided with changes in the availability of the neurotransmitters dopamine, noradrenaline and serotonin in the cerebellum, but not in the cortex, and reflect the known regional differences in neurotransmitter regulation. Immunoprecipitation studies and the observed increase in MAO-A activity following in vitro chemical inhibition of the ã-secretase complex (comprising several proteins including PS-1) support the notion that PS-1 constitutively associates with MAO-A. These effects on Ca2+-sensitive MAO function could contribute to AD-related pathology and could also contribute to the depression often associated with AD.

Choline is indispensable for a number of fundamental processes in the body. Besides being the precursor of the acetylcholine, an important neurotransmitter, choline is found in the cell membrane structure combining with fatty acids, phosphate and glycerol. Its deficiency may result in nervous system disorders, fatty acid build up in the liver, along with increased cholesterol levels, high blood pressure and memory loss. Thus, rapid detection methods are required for the determination of choline in biological fluids. In this study a choline oxidase biosensor was constructed for the determination of choline. During construction of the biosensor, glucose oxidase was used as a model enzyme, before choline oxidase used. The Teflon (PTFE) membrane of the oxygen electrode was grafted with 2-hydroxyethyl methacrylate (HEMA, 15%, v/v) in the presence of ferrous ammonium sulphate (FAS, 0.1%, w/v) by gamma irradiation and ethyleneglycol dimethacrylate (EGDMA, 0.15 %, v/v) was used as a crosslinker in a series of membranes. HEMA-grafted membranes were activated with epichlorohydrin or glutaraldehyde to maintain covalent immobilization of enzyme. The enzyme activity was measured with an oxygen electrode unit based on oxygen consumption upon substrate addition. Membranes were characterized in terms of grafting conditions and mechanical properties. Membranes, gamma irradiated in a solution of HEMA (15%) and FAS (0.1%) for 24 h, were found to be suitable for use in the further studies. Mechanical test results revealed that HEMA grafting made Teflon membrane more flexible and the presence of EGDMA made the grafted membrane stiffer. During optimization stage, it was found that the immobilized enzyme amount was not sufficient to obtain enzyme activity. Thus, the membrane preparation stage was modified to obtain thinner membranes. The immobilized glucose oxidase and choline oxidase contents on thin HEMA grafted membranes were determined by Bradford and Lowry methods. The influence of EGDMA presence and the epichlorohydrin activation duration on enzyme activity studies revealed that the membrane should be prepared in the absence of EGDMA and 30 min activation duration is appropriate for epichlorohydrin coupling. The study on the influence of membrane activation procedures revealed that the membranes activated with glutaraldehyde had a higher specific activity than the membranes activated with epichlorohydrin. Upon stretching membrane on the electrode directly rather than placing in the sample unit, the response of the enzyme immobilized sensor improved with high specific activity. The optimum choline oxidase concentration was found to be 2 mg/mL considering the effect of immobilization concentration on enzyme activity. With the choline oxidase biosensor, the linear working range was determined as 0.052-0.348 mM, with a 40 &plusmn / 5 &micro / M minimum detection limit. The response of the sensor decreased linearly upon successive measurements.

Monoamine oxidase-A (MAO-A) is an enzyme that has historically been linked to major depressive disorder (MDD). The prevalence of MDD among breast cancer patients is almost 25%, but realistically it is underdiagnosed within this patient population. Most breast cancer is deemed estrogen receptor positive [ER(+)] and is commonly treated with the anti-estrogenic chemotherapeutic compound tamoxifen. Resistance to tamoxifen has been associated with a paradoxical activation of the stress-associated kinase, p38(MAPK) (normally associated with cell death). Our research group has recently demonstrated that p38(MAPK) can regulate the function of MAO-A in glial cells. Taken together, MAO-A, depression and p38(MAPK) are all associated with a poor prognosis in breast cancer patients, particularly those with an ER(+) status. Several mechanisms have been proposed in each respect and we hope to further elucidate this relationship by focussing on the interaction between MAO-A and p38(MAPK) in the context of breast cancer. The hypothesis states that a functional interaction between the p38(MAPK) and MAO-A systems alters breast cancer cells in an ER-dependent manner. The proposed objectives of this project are to determine what might be influencing MAO-A function in breast cancer cells, and how changes in MAO-A function affect cell phenotype. Using pharmacological approaches (i.e. antidepressant drugs), we investigated the role of MAO-A and p38(MAPK) on selected characteristics of ER(+) (e.g. MCF-7) and ER(-) (e.g. MDA-MB-231) breast cancer cells under four treatment conditions, which include clorgyline (CLG), an antidepressant MAO-A inhibitor, and SB203580, an inhibitor of p38(MAPK). Our results indicate that the very high MAO-A activity in MDA-MB-231 (MB-231) cells was partly dependent on p38(MAPK) activity. The tumourigenic properties (e.g. anchorage-independent growth, migration) of MB-231 cells depended on both MAO-A and p38(MAPK) functions, although the effects were not additive suggesting that both inhibitors were exerting their respective effects via common signalling targets. The role of MAO-A and p38(MAPK) on MB-231 mitochondrial function and cell growth was negligible. In contrast, MAO-A and p38(MAPK) only influenced mitochondrial function in MCF-7 cells and did not affect any of the other tumourigenic properties measured. Immunocytochemical methods, supported by Western blotting, revealed an increase in E-cadherin expression in both cell lines. This suggested that MAO-A and p38(MAPK) could be influencing transitions between epithelial and mesenchymal phenotypes. Our in vitro findings suggest that MAO-A and p38(MAPK) might contribute to a common mechanism in breast cancer cell lines, but that their influence on cell phenotype is less dependent on the respective cell's ER status and perhaps more so dependent on the cell's metastatic potential. If this is the case, then the contribution of MAO-A and p38(MAPK) to [clinical] metastatic breast cancer should be duly considered. Our ongoing investigations are focussing on the influence of clinically relevant antidepressants on breast cancer cell phenotype in vitro.

Presenilin-1 (PS-1) is the catalytic core of the ã-secretase complex, which is best known for its role in the generation of the Alzheimer disease (AD)-related â-amyloid peptide. Mutated forms of PS-1 are known to be associated with particularly aggressive forms of AD. Changes in monoaminergic neurotransmitter systems, including the serotonin and norepinephrine systems, have long been associated with some of the earliest events in AD, whereas changes in the availability of these same monoamines have historically been associated with clinical depression. Therefore, it is not surprising that depression has now been proposed as a risk factor for developing AD and that pre-demented carriers of mutated forms of PS-1 are more prone to developing depression. MAO-A is historically associated with depression and is also a known risk factor for AD. Given this, I hypothesized that MAO-A represents a neurochemical link between depression and AD, and I chose to examine the influence of PS-1 mutations on MAO-A function in vivo/ex vivo and in vitro.<p> I first focused on the PS-1(M146V) knock-in mouse model of AD-related PS-1/ã-secretase function. I used a radioenzymatic assay to estimate MAO-A catalytic activity and western blot analysis to determine MAO-A protein expression, and found that MAO-A activity does not correlate with MAO-A expression in the cortex and cerebellum of the PS-1(M146V) mice. Furthermore, the potency of the MAO-A inhibitor clorgyline (CLG) is greater in both the cortex and cerebellum of the PS-1(M146V) mice compared to the potency of CLG in wildtype littermates. CLG dose-response curves suggest that there might be a change in cooperativity in the MAO-A protein from PS-1(M146V) cortex (which would suggest a change in conformation and/or access of the substrate to the catalytic pocket in MAO-A). High-pressure liquid chromatography was used to analyze monoamine levels in these same regions. The levels of monoamines (i.e. serotonin, dopamine and norepinephrine) suggest that PS-1 (M146V) inhibits MAO-A function in the cortex, but not in the cerebellum. Furthermore, CLG has no significant effect on amine levels in cortex, but tends to increase their accumulation in cerebellum.<p> The overexpression of PS-1 (M146V) in neuronal cultures reveals that this protein affects MAO-A activity and, more importantly, the PS-1(M146V) protein co-precipitates with MAO-A, thus suggesting a possibility for a direct protein-protein interaction. This is supported by the observation that MAO-A activity is increased in cell extracts incubated with the PS-1 substrate-competitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Preliminary studies have been undertaken to determine the motif in MAO-A that could be acting as a binding site/target site for PS-1.<p> These combined results support the hypothesis that PS-1 proteins can influence MAO-A function and, furthermore, that MAO-A is a novel interactor for PS-1/ã-secretase. This could well explain some of the ambiguous literature associated with both of these proteins in disorders as diverse as depression and AD.

The enzyme monoamine oxidase (MAO) is linked to mental disorders such as depression and neurodegenerative diseases. Our laboratory has recently demonstrated that increases in calcium (Ca2+) can enhance MAO activity and that this might contribute to Alzheimer disease (AD). AD has been linked to gain-of-function mutations in the presenilin-1 (PS-1) protein that not only promote the generation of the toxic amyloid-â peptide, but that also alter intracellular Ca2+ availability. <p>Radioenzymatic MAO assays were used to demonstrate that over-expression of different AD-related PS-1 mutant proteins, i.e. Y115H, ÄEx9 and M146V, in hippocampal-derived HT-22 cells alter either basal and/or Ca2+-sensitive MAO-A activity. The effects of PS-1 mutant proteins on the availability of intracellular Ca2+ are not consistent suggesting that this may not be the primary means of regulating MAO activity. The sensitivity of MAO to Ca2+ was also demonstrated in cortical (both MAO-A and MAO-B responded to Ca2+) and cerebellar (only MAO-A responded to Ca2+) samples from transgenic mice overexpressing the PS-1 (M146V) mutation. These changes in MAO coincided with changes in the availability of the neurotransmitters dopamine, noradrenaline and serotonin in the cerebellum, but not in the cortex, and reflect the known regional differences in neurotransmitter regulation. Immunoprecipitation studies and the observed increase in MAO-A activity following in vitro chemical inhibition of the ã-secretase complex (comprising several proteins including PS-1) support the notion that PS-1 constitutively associates with MAO-A. These effects on Ca2+-sensitive MAO function could contribute to AD-related pathology and could also contribute to the depression often associated with AD.

Many oxidoreductases need nicotinamide cofactors for their reactions. The big obstacle of using these syntheses in industry is the high cost of these nicotinamide cofactors. The work here is about finding novel NADH oxidases from Lactococcus lactis and applying in a cofactor regeneration system with carbonyl reductase or alcohol dehydrogenase. NADH oxidases are useful biocatalysts for regenerating nicotinamide cofactors of biological redox reactions. The annotated alkyl hydroperoxide reductase (AhpR) and the H2O-forming enzyme (nox-2) genes from Lactococcus lactis (L. lactis, L.lac-Nox2) were cloned and proteins were expressed and characterized. They were compared with the H2O-former from Lactobacillus sanfranciscensis (L. sanfranciscensis, L.san-Nox2). AhpR is composed of H2O2-forming NADH oxidase (nox-1) and peroxidase and the net reaction of AhpR is the same as nox-2 when oxygen is the substrate. Both nox-1 and nox-2 are flavoproteins and turnover-limited. In the absence of exogenously added thiols, both nox-1 and nox-1/peroxidase are considerably more stable against overoxidation than nox-2. L.san-Nox2 was crystallized and was found to have ADP ligand, but according to the HPLC results, no ADP ligand was found in the L. lac-Nox-2. Enzyme membrane reactor was used for the application of oxidative reaction of cyclohexanol to cyclohexanone, with isolated enzymes horse liver alcohol dehydrogenase and L.lac-Nox2.

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Induction imposex of gastropod snails by organotins has been reported worldwide. However, the relationship between imposex and detoxification capability is not well studied in molluscs. In the present study, the relationship between the mixed function oxidase (MFO) system and the degree of imposex in drills Thais spp. was investigated. Thais clavigera, T. rufotincta and T. sp. were collected from the west coast of Taiwan. The degree of imposex was determined by percent imposex, relative penis size (RPS) and vas defenes sequence (VDS). Enzymes of cytochrome P450, NADPH-cytochrome c reductase, EROD¡]7-ethoxyresorufin-O-deethylation¡^ and cytochrome P420 (an inactive form of cytochrome P450) in the MFO system were examined. In the three Thais species, the degrees of imposex expressed as percent imposex, RPS and VDS were 71-100%, 8.8-62.5% and 0.7-2.5, respectively. Contents of cytochrome P450 and cytochrome P420 were 0.043-0.097 nmol/mg protein and 0.129-0.296 nmol/mg protein, respectively. The activity of NADPH-cytochrome c reductase was in the range of 1.52-6.73 nmol/min/mg protein. The activity of EROD was not detected. Negative trends between the degree of imposex and the contents of cytochrome P450 and cytochrome P420 were found in all the Thais species. And, a positive trend betweed the degree of imposex and the activity of NADPH-cytochrome c reductase was also observed. Based on the results, it is suggested that the induction of imposex was possibly due to the imbalance of steroid hormone metabolism caused by the inhibition of cytochrome P450 activity.