Dissertations / Theses on the topic 'Fatty Acid Metabolites'

In the presence of arachidonic acid (AA), Saccharomyces cerevisiae produces prostaglandin E₂ (PGE₂). S. cerevisiae and its metabolites may be consumed in products manufactured using the yeast (e.g. beer). Neutrophils are immune cells present in the gastrointestinal (GI) tract during inflammation. As a lipid-signaling molecule, PGE₂ can potentially modify neutrophil functions and exacerbate pre-existing inflammation. As neutrophil migration is a hallmark of inflammation, we investigated the impact of PGE₂ on neutrophil chemotaxis. Chemotaxis assays were performed on neutrophils isolated from human whole blood using the chemotactic agents f-Met-Leu-Phe (fMLP) or interleukin-8 (IL-8). Neutrophil chemotaxis was concentration dependent as it was enhanced 3.5-fold at low concentrations of PGE₂ (0.1 nM-10 nM) and reduced 3.0-fold at higher concentrations of PGE₂ (100 nM).

The biochemical pathway utilized by S. cerevisiae to produce PGE₂ is unknown. Identifying enzymes that metabolize AA may direct approaches to reduce the impact that yeast PGE₂ may have on neutrophils. S. cerevisiae does not have genes homologous to those involved in mammalian AA metabolism. We employed RNAseq transcriptome sequencing to study the lipid biosynthetic pathway in S. cerevisiae and observed 1248 genes upregulated in yeast that were cultured in the presence of AA relative to yeast that were cultured without AA. Notably, genes that mediate beta-oxidation of fatty acids (Pot1, Pox1, Faa1 and Faa2) were upregulated up to 2.3-fold.

The results demonstrate that low concentrations of PGE₂ enhance neutrophil chemotaxis that is mediated by fMLP or IL-8, suggesting that PGE ₂ may aid in recruiting neutrophils from regions that are distant to a site of inflammation. Once a higher concentration of PGE₂ is encountered by neutrophils, neutrophils may halt their migration and engage effector functions such as phagocytosis and superoxide production. Increased expression of genes involved with fatty acid metabolism points to enzymes that may utilize AA to produce PGE₂ in S. cerevisiae. Experiments testing PGE₂ levels in knock-out strains of yeast will identify genes involved in PGE₂ production. Results of this study have implications to reduce potential off-target effects caused by yeast PGE ₂ in consumables.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第19766号
農博第2162号
新制||農||1040(附属図書館)
学位論文||H28||N4982(農学部図書室)
32802
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 菅原 達也, 教授 澤山 茂樹, 教授 佐藤 健司
学位規則第4条第1項該当

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第22664号
農博第2419号
新制||農||1080(附属図書館)
学位論文||R2||N5295(農学部図書室)
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 菅原 達也, 教授 佐藤 健司, 教授 澤山 茂樹
学位規則第4条第1項該当

Streptomyces coelicolor produces fatty acids for both primary metabolism and for production of the components of natural products such as undecylprodiginine. Primary metabolism makes the longer and predominantly branched-chain fatty acids, while undecylprodiginine utilizes shorter and almost exclusively straight chain fatty acids. The first step in fatty acid biosynthetic process is catalyzed by FabH (β-ketoacyl synthase III), which catalyzes a decarboxylative condensation of an acyl-CoA primer with malonyl-acyl carrier protein (ACP). The resulting 3-ketoacyl-ACP product is reduced by NADPH-dependent FabG into 3-hydroxyacyl-ACP, which is dehydrated by FabA to form enoyl-ACP. The NADH-dependent FabI (InhA) completes the cycle. Subsequent rounds of elongations in the pathways are catalyzed by the condensing enzyme FabF. For undecylprodiginine biosynthesis in S. coelicolor, homologues of the condensing enzymes (FabH and FabF) and the ACP (FabC) are encoded by redP, redR and redQ respectively in the red gene cluster. The genes encoding 3-ketoacyl-ACP reductase (FabG), 3-hydroxyacyl-ACP dehydratase (FabA), and enoyl-ACP reductase (FabI), are putatively shared between fatty acid and undecylprodigine biosynthesis, since the corresponding genes are not present within the red gene cluster of S. coelicolor. RedP is proposed to initiate biosynthesis of undecylprodiginine alkane chain by condensing an acetyl-CoA with a malonyl-RedQ, in contrast to FabH which process a broad range of acyl-CoA with malonyl-FabC. The 3-keto group of the resulting 3-ketoacyl-RedQ is then reduced to provide butyryl-RedQ, presumably by the type II FAS enzymes FabG, FabA and FabI. These enzymes would not differentiate between straight and branched-chain substrates, and have equal preference for FabC and RedQ ACPs. RedR would then catalyze four subsequent elongation steps with malonyl-RedQ, with appropriate 3-keto group processing after each step. The proposed role and substrate specificities of condensing enzymes RedP and FabH have not been investigated in S. coelicolor. The genes encoding FabG, FabA, and FabI have not been characterized in Streptomyces. Analysis of the S. coelicolor genome sequence has revealed the presence of one fabI gene (SCO1814, encoding an enoyl-ACP reductase), and three likely fabG genes (SCO1815, SCO1345, and SCO1346, encoding β-ketoacyl-ACP reductase). In the current study the substrates specificities of both RedP and FabH were determined from assays using pairings of two acyl-CoA substrates (acetyl-CoA and isobutyryl-CoA) and two malonyl-ACP substrates (malonyl-RedQ and malonyl-FabC) (FabC is a dedicated ACP for fatty acid biosynthesis and RedQ for undecylprodiginine biosynthesis in S. coelicolor). For RedP, activity was only observed with a pairing of acetyl-CoA and malonyl-RedQ. No activity was observed with isobutyryl-CoA consistent with the proposed role for RedP and the observation that acetyl CoA-derived prodiginines predominate in S. coelicolor. Malonyl-FabC is not a substrate for RedP, indicating that ACP specificity is one of the factors that permit a separation between prodiginine and fatty acid biosynthetic processes. In contrast to RedP, FabH was active with all pairings but demonstrated the greatest catalytic efficiency with isobutyryl-CoA using malonyl-FabC. Lower catalytic efficiency was observed using an acetyl-CoA and malonyl-FabC pairing consistent with the observation that in streptomycetes, a broad mixture of fatty acids are biosynthesized, with those derived from branched chain acyl-CoA starter units predominating. Diminished but demonstrable FabH activity was also observed using malonyl-RedQ, with the same preference for isobutyryl-CoA over acetyl-CoA, completing biochemical and genetic evidence that in the absence of RedP this enzyme can also play a role in prodiginine biosynthesis, producing branched alkyl chain prodiginines. The identification and characterization of both enzymes FabG and FabI was also carried out. A series of straight and branched-chain β-ketoacyl and enoyl substrates tethered to either NAC or ACP were synthesized and used to elucidate the functional role and substrate specificity of these enzymes. Kinetic analysis demonstrates that of the three S. coelicolor enzymes, SCO1815 and SCO1345 have NADPH-dependent β-ketoacyl-reductase activity, in contrast to SCO1346, which has NADH-dependent β-ketoacyl-reductase activity. Spectrophotometric assays revealed that all three FabGs are capable of utilizing both straight and branched-chain β-ketoacyl-NAC substrates. These results are consistent with FabGs role in fatty acid and undecylprodiginine biosynthesis, wherein it processes branched-chain for primary metabolism as well as straight-chain products for undecylprodiginine biosynthesis. LC/MS assays demonstrate that these FabG enzymes do not discriminate between primary metabolism ACP (FabC) and secondary metabolism ACP (RedQ) (except for SCO1345, which does not have any activity with RedQ). This relaxed substrate specificity allows these enzymes to process 3-ketoacyl-FabC substrates for fatty acid biosynthesis as well as 3-ketoacyl-RedQ substrates for undecylprodiginine biosynthesis. Similar to FabG, spectrophotometric and LC/MS assays were also carried out to elucidate the functional role and substrate specificity of S. coelicolor FabI. The kinetic analyses demonstrate that SCO1814 has NADH-dependent enoyl-ACP reductase activity. Spectrophotometric and LC/MS assays demonstrated that FabI does not differentiate between straight and branched-chain substrates, and has equal preference for FabC and RedQ ACPs. These observations provide experimental support for the hypothesis that these enzymes are shared and process the intermediates in the elongation cycle of both fatty acid and undecylprodiginine biosynthesis. In summary, these studies have demonstrated the activity of enzymes RedP, FabH, FabG and FabI (InhA) previously uncharacterized in S. coelicolor and clarified their role in fatty acid and undecylprodiginine biosynthesis.

Vitamin E exhibits anti-tumor activity by regulating pathways in cancer cells, potentially the lipoxygenase (LOX) pathway. We studied the effects of alpha tocopherol (AT), gamma tocopherol (GT), gamma tocotrienol (GT3), and an alpha-gamma tocopherol mixture (ATGT) on the production of the LOX metabolites 13-hydroxyoctadecaenoic acid (HODE), 15-hydroxyeicosatetraenoic acid (HETE), 12-HETE, and 5-HETE in colorectal cancer. These metabolites were examined in the HCT-116 cell line after 24 h treatment with select vitamin E isoforms and quantified by LC/MS/MS. Under physiological conditions, we find that treatment with varying vitamin E isoforms have different effects on the production of 13-HODE, 15-HETE, 12-HETE, and 5-HETE. GT increases 13-HODE and decreases 12-HETE. AT reverses the effects of GT regulation on the LOX pathway, while GT3 has no significant effect on the metabolites tested. GT shows superiority in regulating the LOX pathway as it increases 13-HODE and decreases 12-HETE for possible prevention of colorectal cancer.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第18327号
農博第2052号
新制||農||1022(附属図書館)
学位論文||H26||N4834(農学部図書室)
31185
京都大学大学院農学研究科食品生物科学専攻
(主査)教授 河田 照雄, 教授 金本 龍平, 教授 入江 一浩
学位規則第4条第1項該当

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第19046号
農博第2124号
新制||農||1032(附属図書館)
学位論文||H27||N4928(農学部図書室)
31997
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 小川 順, 教授 加納 健司, 教授 植田 充美
学位規則第4条第1項該当

Epidemiological and experimental data implicate branched chain amino acids (BCAAs) in the development of insulin resistance, but the mechanisms underlying this link remain unclear. Insulin resistance in skeletal muscle stems from excess accumulation of lipid species, a process that requires blood-borne lipids to first traverse the blood vessel wall. Little is known, however, of how this trans-endothelial transport occurs or is regulated. Here, we identify 3-hydroxy-isobutyrate (3-HIB), a catabolic intermediate of the BCAA valine, as a novel paracrine regulator of trans-endothelial transport of fatty acids. PGC-1α, a transcriptional co-activator that regulates broad programs of fatty acid consumption, induces the secretion from muscle of 3-HIB, which then triggers fatty acid uptake and transport in endothelial cells. Conversely, inhibiting the synthesis of 3-HIB in muscle cells blocks the promotion of endothelial fatty acid uptake. Providing animals with 3-HIB in drinking water, or inducing 3-HIB levels in skeletal muscle by over-expressing PGC-1α, stimulates muscle to take up fatty acids in vivo, leading to muscle lipid accumulation, and systemic glucose intolerance. 3-HIB levels are elevated in muscle from patients with diabetes. These data thus unveil a novel mechanism that regulates trans-endothelial flux of fatty acids, revealing 3-HIB as a new bioactive signaling metabolite that links the regulation of fatty acid flux to BCAA catabolism and provides a mechanistic explanation for how increased BCAA catabolic flux can cause diabetes.
Medical Sciences

Short-chain fatty acids (SCFAs) and the tricarboxylic acid (TCA) cycle metabolites aresmall hydrophilic compounds that play crucial roles in biological species ranging fromenergy metabolism, immune homeostasis to cellular signalling. There is a need for reliableand precise quantification of these metabolites in biological matrices as they can providecrucial information of metabolic status and potentially be used as diagnostic biomarkersfor different pathological and physiological conditions. However, their retention andseparation in traditional reversed-phase system, without chemical derivatization, is oftenproblematic due to their volatile and hydrophilic characteristics. The aim of this studywas to implement a facile and effective derivatization method for the simultaneousquantitation of SCFAs and TCA cycle metabolites by LC-qToF-MS in negative ion mode. Inthis work, 3-nitrophenylhydrazine (3-NPH) was employed for preanalyticalderivatization to convert the compounds to their respective 3-nitrophenylhydrazones.Analytical standards and faecal samples were used to assess the linearity, matrix effect,accuracy, extraction efficiency, precision, retention-time shift and short-term stability.The compounds were successfully separated within 6 minutes on a reverse-phase C18column. All the compounds showed good linearity (R2≥ 0.97) in both solvent-only andfaecal samples. The matrix effect was minimal and did not affect the compoundsquantitation. The extraction efficiency ranged from 80% to 110% (CV≤9.7%, n = 6). Theaccuracy of quantitation was determined to be between 82.8% to 113.8% (CV≤9.0%, n =6). The intra-day (CV%) demonstrated good precision for all analytes, the inter-day (%)were more variable due to the derivatives’ chemical instability. However, most of thederivatives were chemical stable up to 5 days in the autosampler (10°C). The method wasalso applied to explore the levels of these metabolites in human faecal samples and mousebrain samples.

Prostaglandins Ej and Fja play major roles in the initiation and maintenance of parturition. There is evidence that they also prime the uterus prior to parturition. Diets high in n3 fatty acids have been reported to be associated with impaired parturition, whereas uterine infection by the intracellular pathogen Chlamydia psiltaci is associated with abortion and premature labour. In each case the course of disruption of normal parturition is unknown, however, impaired or excessive 2-series prostaglandin production has been postulated to play an important part. The effect of high dietary n3 and n6 fatty acid intake on uterine fatty acid composition and metabolism by desaturase, elongase, phospholipase and cydooxygenase enzymes was investigated. The effect of C. psittaci infection on 2-series prostaglandin production and its control was also studied. The uterine fatty acid content of rats maintained on diets high in n6 and n3 essential fatty acids (EFA) for various periods was analysed and compared with a control group fed a normal pelleted diet. Rapid changes in uterine n6 and n3 fatty acid content were observed after three weeks feeding. However, in all three diet groups conservation of arachidonic acid was observed, which was highest in rats fed the n6 fatty acid diet and lowest in rats fed the n3 fatty acid diet. The 20C and 22C EFA were incorporated into phospholipids to a greater extent than into neutral lipids. The distribution of EFA in the individual lipid classes in the three diet groups indicated selective release of arachidonic acid and eicosapentaenoic acid into the free fatty acid pool. Phosphatidylethanolamine arachidonic acid levels were more susceptible to changes in dietary fatty acid content than those of phosphatidycholine and phosphatidylinositol. Analysis of prostaglandins produced by uteri of rats on the three diets, by mass spec-trometry, demonstrated an inhibitory effect of the n3 fatty acids on total prostaglandin production, and the synthesis of the 3-series prostaglandins E and F was detected. Pregnant sheep experimentally infected with an ovine abortion strain of C. psittaci were found to prematurely release prostaglandin Ej (PGEj) into the amniotic and allantoic fluids. Impaired release of PGE3 into the utero-ovarian vein was also detected in infected sheep. The plasma oestradiol-17/3 also increased earlier than that of control sheep. The study detected competitive inhibition of uterine n6 EFA metabolism at the level of esterification, chain elongation, desaturation and cyclooxy-genase metabolism by the dietary n3 EFA. In infectious abortion, abnormalities in PGEj and oestradiol-170 were detected. The first evidence of 3-series prostaglandin production by uterine tissue is presented.

Doctor of Philosophy
Department of Human Nutrition
Mark D. Haub
Background: Dietary fiber has been shown to help improve several metabolic disorders including obesity and type II diabetes. However, the mechanism by which this occurs is poorly understood. Purpose: This study was designed to compare the effects of energy restriction and dietary fiber and subsequent production of short chain fatty acids on body composition, biomarkers of health, and hepatic and myocellular expression of key regulators of lipid metabolism Methods: Crossbred gilts (n=17) were randomly assigned to either a control (CON), high fiber (HF) or energy restricted (ER) diet for 42 days. Gilts on the CON and HF diets were fed ad libitum. The ER Gilts were pair fed HF gilts and matched for body weight gain. Blood samples were collected and glucose, insulin, triglycerides, non-esterified fatty acids and short chain fatty acids (SCFA) concentrations were measured. Liver and muscle tissue were biopsied and expression of peroxisome proliferator-activated receptor gama (PGC-1α) and carnitine palmitoyltransferase 1 (CPT1) were determined via RT-PCR. Results: HF gilts had significantly higher plasma TG and lower NEFA concentrations when compared to the CON and ER. The HF diet elicited a significant increase in all plasma SCFA concentrations. No differences in fold change of myocyte CPT1 and PGC-1α mRNA expression were found while they tended to be lower in hepatic samples of the HF gilts. HF gilts also had a lower (P < 0.05) back fat thickness when compared to the ER even though energy intakes were similar. Minimal changes were observed in fasting glucose and insulin as a result of diet. Conclusions: Gilts consuming a diet high in dietary fiber (DF) significantly altered their plasma lipid profiles independently to that of energy restriction and body weight and appears to be a result of plasma SCFA concentration. DF and/or SCFA appear to have minimal affects on CPT1 and PGC-1α in the liver and muscle of gilts.

Introduction: Microbial fermentation of carbohydrates in forestomach of ruminants produces large amounts of short-chain fatty acids (SCFA, mainly acetic acid, propionic acid, and n-butyric acid). The majority of these substrates is taken up directly across the ruminal wall. After luminal uptake into the epithelial cells, SCFA mainly occur in the dissociated form due to the intracellular pH of ~7.4. Moreover, a big portion of SCFA is metabolised within the cytosol. Main end products of epithelial SCFA metabolism are ketone bodies (D-3-hydroxybutyric acid and acetoacetic acid) and lactic acid. Both intact SCFA and ketone bodies and lactate need to be efficiently extruded from the ruminal epithelial cells to prevent a lethal drop of intracellular pH and counteract osmotic load of the cytosol. All these substances are less lipophilic in comparison to the undissociated form of SCFA. Thus, dissociated SCFA (SCFA-) and their metabolites need Protein mediated mechanisms for the extrusion across the basolateral side of ruminal epithelium. One mechanism suggested to be involved in the extrusion of SCFA- across basolateral membrane of the ruminal epithelium is the monocarboxylate transporter 1 (MCT1). Functionally, MCT1 was first assumed to operate as proton-coupled transporter for monocarboxylates including SCFA. Nonetheless, a recent study found a bicarbonate dependent anion exchange mechanism which turned out to be sensitive to MCT1 Inhibitors at the basolateral side of the ruminal epithelium pointing to the ability of MCT1 to act as an anion exchanger. However, in these experiments the inhibition of MCT1 abolished bicarbonate dependent transport only by half. This suggests the involvement of further anion exchanger(s) in the transport of SCFA across the basolateral membrane of ruminal epithelium. Promising candidates to underlie this exchange are the putative Anion exchanger 1 (PAT1) and a transport protein designated „down-regulated in adenoma“ (DRA). Materials and Methods: Sheep rumen epithelium was mounted in Ussing Chambers under short-circuit conditions. Radioactively labelled acetate (ac) was added to the serosal side. Serosal to mucosal flux of ac (Jsm ac) was measured with or without anion Exchange inhibitors (50 mM NO3- or 1 mM DIDS) or the MCT1 inhibitor p-hydroxy mercuribenzoic acid (pHMB; 1.5 mM) in the serosal buffer solution. The inhibitors were added alone or in combination with each other. Furthermore, mucosal to serosal flux of radioactivelly labelled ac or butyrate (bu) (Jms ac, bu) was measured in the presence or absence of SO42-, Cl- or NO3- (50 mM respectively) as exchange substrate in the serosal buffer solution. Immunohistochemical staining was conducted to locate PAT1 and DRA by use of commercially available antibodies. Results: NO3- and pHMB significantly reduced Jsm ac by 57 % and 51 %, respectively. When pHMB was applied after pre-incubation with NO3- an additional inhibition of Jsm ac was observed. Vice versa, NO3- further inhibited Jsm ac when epithelia were pre-incubated with pHMB before. DIDS had no inhibitory effect on SCFA flux. Serosal presence of SO42- or Cl- enhanced Jms ac significantly. Regarding bu, Cl- or SO4 2- also enhanced Jms bu significantly. The different anions available in the serosal buffer solution numerically enhanced Jms in the order of SO4 2- > Cl- for both ac and bu, which corresponds to the known affinity sequence of PAT1 and DRA. Immunohistochemistry revealed localization of PAT 1 in the stratum basale, whereas DRA was not detectable using this method. Conclusions: Basically, this study supports the suggestion that MCT1 works as an Anion exchanger in ruminal epithelium. In addition, it clearly shows that there is at least one further anion exchanger involved in the basolateral extrusion of SCFA and their metabolites. The functional and immunohistochemical findings suggest that PAT1 holds a significant role in this respect.

Kyoto University (京都大学)
0048
新制・論文博士
博士(農学)
乙第12822号
論農博第2795号
新制||農||1025(附属図書館)
学位論文||H26||N4817(農学部図書室)
31309
京都大学農学研究科食品生物科学専攻
(主査)教授 喜多 恵子, 教授 三上 文三, 教授 栗原 達夫
学位規則第4条第2項該当

La production de microalgues en hétérotrophie présente plusieurs avantages pour la production de biocarburants par rapport à la production autotrophe, comme une productivité plus importante en termes de biomasse et de lipides. Cependant, le développement industriel de ce procédé est limité par les coûts de productions associés au substrat organique (i.e. glucose) et à ceux liés à la stérilisation des fermenteurs. Les effluents de fermentation sombre, composés principalement d’acétate et de butyrate, pourraient être utilisés comme milieux de culture peu onéreux pour la culture hétérotrophe ou mixotrophe de microalgues. Les objectifs de cette thèse étaient i) de mieux appréhender la croissance algale sur des mélanges variés d’acétate et de butyrate en fonction de la présence ou l’absence de lumière et de la température de croissance et ii) d’évaluer la faisabilité d’utiliser des effluents de fermentation non stérilisés pour soutenir la croissance de microalgues oléagineuses. Tout d’abord, un modèle basé sur des bilans de masse a été construit afin de caractériser la croissance hétérotrophe de Chlorella sorokiniana et Auxenochlorella protothecoides (taux de croissance et rendements) sur des mélanges d’acétate et de butyrate. Les résultats ont montré que le rapport acétate:butyrate et la concentration en butyrate étaient deux paramètres clés pour soutenir la croissance hétérotrophe. Puis, il a été démontré que la présence de lumière et l’utilisation d’une température suboptimale (30 °C) pour la croissance algale permettaient de réduire l’inhibition du butyrate en permettant une production de biomasse autotrophe ou en améliorant la croissance sur acétate. Enfin, il a été montré que les microalgues peuvent être compétitives sur l’acétate lors de la croissance sur des effluents bruts de fermentation sombre en présence de bactéries fermentaires, grâce à la croissance rapide des microalgues sur acétate (1.75 j-1) et à un changement drastique des conditions de culture peu favorables à la croissance des bactéries d’origine fermentaire
Growing microalgae in heterotrophic mode present several advantages over autotrophic mode such as a higher productivity in terms of biomass and lipids for biofuels production. Nevertheless, this process is limited by the production cost associated with the organic substrate (i.e. glucose) and fermenters sterilization costs. Dark fermentation effluents, mainly composed of acetate and butyrate, could be used as a low-cost medium to grow microalgae heterotrophically or mixotrophically. The aims of this PhD were i) to optimize microalgae growth on various mixtures of fermentations metabolites using the presence or absence light and different cultivation temperatures and ii) to assess the feasibility of using unsterilized fermentation effluents. First, a model based on mass balance was built to characterize heterotrophic growth rates and yields when Chlorella sorokiniana and Auxenochlorella protothecoides were supplemented with different mixtures of acetate and butyrate. Results showed that the acetate:butyrate ratio and the butyrate concentration per se were two key parameters for promoting heterotrophic growth. Then, further studies showed that the presence of light and the use of suboptimal temperature (30 °C) could reduce the butyrate inhibition on growth by either triggering autotrophic production of biomass or enhancing growth on acetate. Finally, it was shown that microalgae could outcompete fermentation bacteria for acetate when growing on raw dark fermentation effluents, thanks to a fast algal growth on acetate (1.75 d-1) and a drastic change of culture conditions to the detrimental of bacterial growth

La preparation des composes du titre se fait a partir du d-mannitol via la decyclisation par des nucleophiles de diepoxydes; les alpha -hydroxy aldehydes obtenus ont ete utilises pour preparer le leucotriene b**(4) et un de ses homologues en c21

La synthese d'inhibiteurs de la biosynthese des leucotrienes a ete developpee selon deux voies: -synthese de metabolites naturels de l'acide arachidonique (monoepoxides) -synthese de produits analogues de l'acide arachidoniques susceptibles de stabiliser l'intermediaire radicalaire forme au cours de la premiere etape de la 5-lipoxygenase

Abstract Coronary heart disease (CHD) is a clinical manifestation of atherosclerosis. It is a major cause of mortality and morbidity both in Finland and globally. Even after the best known treatments a significant residual risk of CHD remains. A low plasma HDL cholesterol level (HDL, high-density lipoprotein) is a common lipid abnormality in patients affected by premature CHD and also a component of the metabolic syndrome, a cluster of risk factors for atherosclerosis associated with central obesity. In this study, a phenotype of low HDL cholesterol level and premature CHD was investigated in two Northern Finnish family populations. The aim was to find new biological factors accounting for the elevated CHD risk in the phenotype. In the subjects of family population I, plasma levels of antibodies (IgG, IgM, IgA) against experimental epitopes (malondialdehyde-acetaldehyde-modified, copper-oxidized) of oxidized LDL (low-density lipoprotein) particles were measured. In the subjects of family population II, capacity of HDL fractions (total HDL, HDL2 and HDL3) to accept cholesterol from a THP-1 experimental foam cell model was assayed (cholesterol efflux). In addition, a phospholipid composition of their HDL fractions (HDL2 and HDL3) was measured using liquid chromatography-mass spectrometry. The antibody levels were not related to CHD or to HDL cholesterol level. Instead, the cholesterol efflux to HDL2 fraction was clearly impaired in CHD, which was associated with the low HDL cholesterol level of the patients. The impaired cholesterol efflux to HDL2 fraction was primarily in conjunction with the metabolic syndrome. The phospholipid composition of HDL fractions was different between the affected and the non-affected subjects. As an example, characteristic of the metabolic syndrome were elevated contents of palmitic, palmitoleic or oleic acids relative to linoleic acid in lysophosphatidylcholines and phosphatidylcholines. In conclusion, the HDL fraction is both functionally and compositionally modified in the phenotype of low HDL cholesterol level and premature CHD. Especially the cholesterol efflux capacity of the HDL2 fraction and thus its many functional properties may be impaired. There are many characteristic features in the phospholipid composition of the HDL in the phenotype which were detected in HDL2 and HDL3 fractions
Tiivistelmä Sepelvaltimotauti on ateroskleroosin kliininen ilmenemismuoto. Se on merkittävimpiä kuolleisuuden ja sairastavuuden aiheuttajia niin Suomessa kuin maailmalla. Parhaillakin tunnetuilla hoidoilla sepelvaltimotaudille jää huomattava jäännösriski. Plasman matala HDL-kolesterolitaso (HDL, high-density lipoprotein) on yleinen lipidipoikkeavuus varhaista sepelvaltimotautia sairastavilla ja myös eräs metabolisen oireyhtymän, eli keskivartalolihavuuteen liittyvän ateroskleroosin riskitekijäkasauman, komponentti. Tässä väitöskirjassa tutkittiin matalan HDL-kolesterolitason ja varhaisen sepelvaltimotaudin fenotyyppiä kahdessa pohjoissuomalaisessa sukuaineistossa. Tavoitteena oli löytää uusia biologisia tekijöitä fenotyypin kohonneen sepelvatimotautiriskin taustalta. Ensimmäisen aineiston henkilöiden plasmasta mitattiin vasta-ainetasoja (IgG, IgM, IgA) LDL-hiukkasten (LDL, low-density lipoprotein) kokeellisia hapettuneita epitooppeja (malonidialdehydi-asetaldehydi-modioitu ja kuparilla hapetettu LDL) vastaan. Toisessa aineistossa mitattiin henkilöiden HDL-fraktioiden (kokonais-HDL, HDL2 ja HDL3) kykyä saada aikaan kolesterolin ulosvirtausta kokeellisesta THP-1 vaahtosolumallista. Lisäksi heidän HDL-fraktioidensa (HDL2, HDL3) fosfolipidikoostumus mitattiin nestekromatografi-massaspektrometri-laitteistolla. Vasta-ainetasot eivät liittyneet sepelvaltimotautiin tai HDL-kolesterolitasoon. Sen sijaan kolesterolin ulosvirtaus HDL2-fraktioon oli selkeästi alentunut sepelvaltimotaudissa, mikä liittyi potilaiden pieneen HDL-kolesterolipitoisuuteen. Alentunut ulosvirtaus HDL2-fraktioon liittyikin ensisijaisesti metaboliseen oireyhtymään. HDL-fraktioiden fosfolipidikoostumus erosi terveiden ja sairaiden välillä. Esimerkiksi metabolisessa oireryhtymässä tunnusomaista oli lysofosfatidyylikoliinien ja fosfatidyylikoliinien sisältämän palmitiinihapon, palmitoleiinihapon tai oleiinihapon suurentunut määrä suhteessa niiden sisältämän linoleenihapon määrään. Loppupäätelmä on, että matalan HDL-kolesterolitason ja varhaisen sepelvaltimotaudin fenotyypin HDL-fraktio on sekä toiminnaltaan että koostumukseltaan muuntunut. Erityisesti HDL2-fraktion kyky saada aikaan kolesterolin ulosvirtausta ja näin ollen sen monet toiminnalliset ominaisuudet voivat olla alentuneet. Fenotyypin HDL:n fosfolipidikoostumuksessa on monia tunnusomaisia piirteitä, joita havaittiin sekä HDL2- että HDL3-fraktiossa

У оквиру ове докторске дисертације испитана је биолошка активност екстраката плодних тела и потопљених култура (мицелије и филтрата) аутохтоних врста гљива Coprinus comatus и Coprinellus truncorum. Такође, испитан је  метаболизам фосфата мицелија обе врсте употребом нуклеарно магнетне резонантне спректроскопије (³¹Р NMR), утицај ванадијума на метаболизам фосфата као и идентификација облика ванадата присутних у ћелији мицелије (⁵¹V NMR). Утврђена је антирадикалска и антиоксидативна активност  етанолних,метанолних и водених екстраката гљива при чему су се екстракти потопљених култура издвојили по антирадикалској, а екстракти плодних тела по антиоксидативној активности. Екстракти потопљених култура истакли су се и у погледу антибактеријске активности, где се као најпотентнији показао  хлороформски екстракт филтрата потопљене културе C. comatus. Такође, етанолни екстракт филтрата потопљене културе C. comatus показао се као најпотентнији у анти-ацетилхолинестеразној активности у односу на  конвенционални лек донепезил. Испитан је и утицај екстраката на вијабилност ћелијских линија HepG2 (хумане хепатома ћелије) и Rin-5F (ß ћелије панкреаса пацова).Спектрофотометријским методама одређен је укупан садржај фенола и флавоноида у већини анализираних екстраката.LC/MS идентификацијом и квантификацијом фенолних киселина уочена је разлика између фенолних једињења присутних у плодном телу, мицелији и филтрату потопљене културе. Екстракти потопљених култура бележе већи број и већи садржај једињења. Укупан садржај протеина одређен само у воденим екстрактима, а укупан садржај угљених хидрата у полисахаридним екстрактима.Употребом Фуријеве инфрацрвене спектроскопске методе (FTIR) детектоване су везе између угљених хидрата  присутних у полисахаридним екстрактима, а планарном  хроматографијом показано је да екстракти плодног тела и филтрата врсте С. truncorum, као и екстракт плодног тела врсте C. comatus, садрже велику  количину D-глукозе, док екстракт мицелије C. truncorum, баш као и екстракти филтрата и мицелије C. comatus, садрже највише галактозе. Квалитативном и квантитативном елементарном анализом (ААS) утврђен је виши садржај  калијума и гвожђа у анализираним узорцима. GC-МS идентификацијом и квантификацијом масних киселина указано је на значајно присуство линолне киселине код обе врсте. Како за аутохтону врсту  C.truncorum постоји мало података у литератури, подаци о њеном хемијском саставу могу се сматрати иновативним.Компаративним прегледом биолошке активности и хемијског састава екстраката плодног тела и мицелије и филтрата (потопљених култура) указано је да су анализирани екстракти извори биоактивних супстанци са медицинским потенцијалом, а потопљене културе датих гљива представљају атрактивне кандидате за даља биотехнолошка истраживања.
U okviru ove doktorske disertacije ispitana je biološka aktivnost ekstrakata plodnih tela i potopljenih kultura (micelije i filtrata) autohtonih vrsta gljiva Coprinus comatus i Coprinellus truncorum. Takođe, ispitan je  metabolizam fosfata micelija obe vrste upotrebom nuklearno magnetne rezonantne sprektroskopije (³¹R NMR), uticaj vanadijuma na metabolizam fosfata kao i identifikacija oblika vanadata prisutnih u ćeliji micelije (⁵¹V NMR). Utvrđena je antiradikalska i antioksidativna aktivnost  etanolnih,metanolnih i vodenih ekstrakata gljiva pri čemu su se ekstrakti potopljenih kultura izdvojili po antiradikalskoj, a ekstrakti plodnih tela po antioksidativnoj aktivnosti. Ekstrakti potopljenih kultura istakli su se i u pogledu antibakterijske aktivnosti, gde se kao najpotentniji pokazao  hloroformski ekstrakt filtrata potopljene kulture C. comatus. Takođe, etanolni ekstrakt filtrata potopljene kulture C. comatus pokazao se kao najpotentniji u anti-acetilholinesteraznoj aktivnosti u odnosu na  konvencionalni lek donepezil. Ispitan je i uticaj ekstrakata na vijabilnost ćelijskih linija HepG2 (humane hepatoma ćelije) i Rin-5F (ß ćelije pankreasa pacova).Spektrofotometrijskim metodama određen je ukupan sadržaj fenola i flavonoida u većini analiziranih ekstrakata.LC/MS identifikacijom i kvantifikacijom fenolnih kiselina uočena je razlika između fenolnih jedinjenja prisutnih u plodnom telu, miceliji i filtratu potopljene kulture. Ekstrakti potopljenih kultura beleže veći broj i veći sadržaj jedinjenja. Ukupan sadržaj proteina određen samo u vodenim ekstraktima, a ukupan sadržaj ugljenih hidrata u polisaharidnim ekstraktima.Upotrebom Furijeve infracrvene spektroskopske metode (FTIR) detektovane su veze između ugljenih hidrata  prisutnih u polisaharidnim ekstraktima, a planarnom  hromatografijom pokazano je da ekstrakti plodnog tela i filtrata vrste S. truncorum, kao i ekstrakt plodnog tela vrste C. comatus, sadrže veliku  količinu D-glukoze, dok ekstrakt micelije C. truncorum, baš kao i ekstrakti filtrata i micelije C. comatus, sadrže najviše galaktoze. Kvalitativnom i kvantitativnom elementarnom analizom (AAS) utvrđen je viši sadržaj  kalijuma i gvožđa u analiziranim uzorcima. GC-MS identifikacijom i kvantifikacijom masnih kiselina ukazano je na značajno prisustvo linolne kiseline kod obe vrste. Kako za autohtonu vrstu  C.truncorum postoji malo podataka u literaturi, podaci o njenom hemijskom sastavu mogu se smatrati inovativnim.Komparativnim pregledom biološke aktivnosti i hemijskog sastava ekstrakata plodnog tela i micelije i filtrata (potopljenih kultura) ukazano je da su analizirani ekstrakti izvori bioaktivnih supstanci sa medicinskim potencijalom, a potopljene kulture datih gljiva predstavljaju atraktivne kandidate za dalja biotehnološka istraživanja.
The biological activity of extracts of basidiocarps (fruiting bodies)  and submerged cultures (mycelium and filtrate) of autochthonous mushroom species  Coprinus comatus and  Coprinellus truncorum  was examined. Furthermore, the metabolism of phosphate  of mycelia  of both types was studied using nuclear magnetic  resonance spectros-copy ( ³¹ R NMR), the influence of vanadium on phosphate metabolism and the identification of vanadate oxidation states present in the mycelia cell ( ⁵¹ V NMR). The antiradical and antioxidant activity of methanolic, ethanolic and water fungal extracts was determined. Extracts of submerged cultures achieved the best anti- radical activity while fruit body extracts showed the best antioxidant activity. Extracts of submerged cultures also highlighted in terms of antibacterial activity, where the chloroform extract of the submerged culture  C. comatus  showed as the most potent. Also, the ethanolic extract of the submerged culture of  C. comatus  was found to be most relevant in anti-acetylcholinesterase activity  compared with  the conventional donepezil drug. The influence of extracts on the viability of cell lines HepG2 (human hepatocytes cells) and Rin-5F (ß pancreatic cells of the rat) was also examined.Spectrophotometric methods determined the total con-tent of phenol and flavonoids in most of the analyzed extracts.The LC/MS identification and quantification of phenolic acids revealed the difference between the phenolic compounds present in the fruiting body, mycelium, and the submerged culture filtrate. Extracts of submerged cultures record a greater number and higher content of compounds.The total content of proteins determined only in water extracts  and the total content of  carbohydrates in poly-saccharide extracts. Using the Fourier infrared spectro-scopic method (FTIR), the links between the sugar pre-sent in the  polysaccharide extracts were detected, and planar chromatography showed that the extracts  of the fruiting body and the filtrate of type  C. truncorum, as well as the extract of the fruiting body of the species  C. comatus, contain a large amount of D-glucose, while the extract of the  C. truncorum  mycelia  and  mycelia  of  C. comatus, contain the most galactose. GC-MS identification and quantification of fatty acids indicated a significant presence of linoleic acid in both species, while qualitative and quantitative elemental analysis (AAS) has determined a higher content of potas-sium and iron in the analyzed samples. Since there is no data in the literature for the autochtho-nous species  C. truncorum, the studies on its chemical composition can be considered advanced аs innovative. A comparative review of the biological activity and the chemical composition of the extracts of the fruiting body and  mycelia  and filtrates  of  medium of  submerged cultures  indicated that the extracts were analyzed by sources of bioactive substances with medical potential, and the submerged cultures of these mushrooms are attractive candidates for biotechnological research.
В рамках данной работы была исследованна биологическая активность экстракта плодородных тел и погружонных видов култур (мицелии и филтрата) автотоных видов грибов Coprinus comatus и Coprinellus truncorum. Также, исследованн метаболизм фосфата обеих видов  мицелий с помощью ядерного магнитного резонанса спектроскопии (³¹Р ЯМР), влияние на содержание ванадия в метаболизме фосфата, а также идентификация формы ванадата присущего в клеток мицеллий (⁵¹V ЯМР). Установленная антирадикальная и антиоксидантная активность метанольных, этанольных и водных экстрактов гриб, причём выделяются экстракты погружённых культур по антирадикальной активности и  экстракты плодородных тел по антиоксидантной активности.Экстракты погружённых культур выделялись и в плане антибактериальной активности, причем,  наиболее мощным из филтратов показался экстракт хлороформа погруженной культуры C. comatus. А также этанольный экстракт филтрата погружённой культуры C. comatus оказался найболее мощным в анти-ацетихолинестеразной активностипо сравнению с традиционным лекарством донепезилом. Было исследовано и влияние экстрактов на виябильность клеток линий   HepG2 (гуманые хепатома клетки) и Rin-5F (ß клетки поджелудочной железы крыс).Методом спектрофотометрии определена совокупность фенола и флавоноида в большинстве проанализированных экстрактах.С помощью ЛС ̸МС идентификации и квантификации фенолных кислот была замечена разница между соединениями фенола, присущих в плодородном теле, и мицелии, и филтрата погружённой культуры. Экстракты погружённых культур отражают больше количество и более высокое содержание соединений.Общее содержание белков выделен только в водяных экстрактах, и общее содержание углеводов в полисахаридных экстрактах. Используя инфракрасный метод спектроскопии Фурия (ИКМСФ) были обнаружены связи между сахарами, присущими в полисахаридных экстрактах, а планарной хромотографиой было показано, что экстракты плодородного тела и филтратов вида С. truncorum,  а  также и экстракты плодородного тела вида C. comatus содержат большое количество D-глюкозы, в то время как экстракт мицелии C. truncorum, именно как и экстракт фильтрата и мицелии C. comatus, содержат больше всего галактозы.GC-МS идентификацией и квантификацией жирных кислот показано значительное наличие линолевой кислоты у обоих видах. А качественным и квантитативным элементарным анализом установленно большее содержание калиума и железа в анализированых шаблонах.Из-за того, что для автохтонного вида C. truncorum практически не было данных в литературе, данные о её химическом составе можно считать прогрессивным и инновационным.Сравнительный анализ биологической активности и химического состава экстрактов плодородного тела и мицелии и фильтрат (погружённых культур) показаывает, что проанализированные экстракты — источники биологически активных веществ с медицинским потенциалом, и погружённые культуры данных гриб являются привлекательными кандидатами для биотехнологических исследований.

No
Nutritionally important PUFAs (polyunsaturated fatty acids) mediate some of their bioactivities through formation of oxygenated metabolites. These bioactive lipids are formed by COX (cyclo-oxygenase), LOX (lipoxygenase) and cytochrome-P450-catalysed reactions, as well as non-enzymatic lipid peroxidation. These reactions produce numerous species, some of which can be formed through more than one pathway. MS-based lipidomics offers the selectivity and sensitivity required for qualitative and quantitative analysis of multiple lipid species, in a variety of biological systems, and can facilitate the study of these mediators.

(hCysLT2R) based on a β-galactosidase complementation system. The EC50 values for LTC4 and LTD4 are consistent with previous literature values determined based on radio-labelling affinity assays for hCysLT2R. Activity of a synthetic analogue of LTC4, N-Methyl LTC4 (NMLTC4), gave an EC50 value of 8.5 nM. Also described in this thesis, is the synthesis of a cysLT2R selective antagonist, 3-{[(3- carboxycyclohexyl)amino]carbonyl}-4-{3-{4-(4-phenoxybutoxy)phenyl}- propoxy}benzoic acid. Through the in vitro assay system, this selective antagonist showed a dose-dependent inhibition (IC50 value of 86 nM) when CysLT2R was stimulated with 30 nM of LTD4. Both the selective agonist (NMLTC4) as well as the selective cysLT2R antagonist were also tested in vivo in a localized vascular ear inflammation assay. Results show NMLTC4 is able to promote vascular leakage through stimulation of cysLT2R, and this extravasation can be significantly attenuated by the cysLT2R antagonist.
Thesis (Master, Biochemistry) -- Queen's University, 2011-06-29 22:31:03.11

no
Lipid mediators are produced from the oxidation of polyunsaturated fatty acids through enzymatic and free radical-mediated reactions. When subject to oxygenation via cyclooxygenases, lipoxygenases, and cytochrome P450 monooxygenases, polyunsaturated fatty acids give rise to an array of metabolites including eicosanoids, docosanoids, and octadecanoids. These potent bioactive lipids are involved in many biochemical and signaling pathways, with inflammation being of particular importance. Moreover, because they are produced by more than one pathway and substrate, and are present in a variety of biological milieus, their analysis is not always possible with conventional assays. Liquid chromatography coupled to electrospray mass spectrometry offers a versatile and sensitive approach for the analysis of bioactive lipids, allowing specific and accurate quantitation of multiple species present in the same sample. Here we explain the principles of this approach to mediator lipidomics and present detailed protocols for the assay of enzymatically produced oxygenated metabolites of polyunsaturated fatty acids that can be tailored to answer biological questions or facilitate assessment of nutritional and pharmacological interventions.

In the human gut, bacterial fermentation of dietary fibers and proteins produces metabolites, primarily as short-chain fatty acids (SCFA), that are highly beneficial for host health. However, unlike dietary fiber, bacterial fermentation of protein additionally generates potentially toxic substances such as ammonia, hydrogen sulfide, amines, and indoles. It is believed that most gut bacteria favor utilization of dietary fiber over that of protein for energy. Therefore, when fermentable dietary fiber is readily available to colonic bacteria, protein fermentation, and its subsequent potentially toxic metabolites, remains relatively low. Dietary intake primarily determines the quantity of dietary fiber and protein substrate available to the gut microbiota and the resulting profile of metabolites produced. Increased protein consumption is associated with deleterious health outcomes such as higher risk of colorectal cancer and type II diabetes. Conversely, diets following US dietary recommendations are high in fiber, which promote a healthy microbiome and are protective against disease. Diets following the recommendation are also moderate in protein intake so that, ultimately, far more fiber than protein is available for colonic bacterial fermentation. On the contrary, dietary fiber intake is chronically low in a standard Western diet, while protein consumption is above dietary recommendations, which results in nearly equal amounts of dietary fiber and protein available for gut microbial fermentation. Furthermore, the popularity of high-protein diets for athletes, as well as that of high-protein low-carbohydrate diets for weight loss, may flip fiber and protein substrate proportions upside down, resulting in more protein than fiber available in the gut for fermentation. The objective of this study was to elucidate how substrate ratios in protein-fiber mixtures affect protein fermentation and metabolites, as well as examine the degree to which fiber source may influence these outcomes. Each dietary fiber source [fructooligosaccharides (FOS), apple pectin (Pectin), a wheat bran and raw potato starch mixture (WB+PS), and an even mixture of the three aforementioned fibers (Even Mix)] and protein were combined in three ratios and provided as substrate for in vitro fecal fermentation to understand how low, medium, and high fiber inclusion levels influence fermentation outcomes. They were compared to 100% protein and fiber (each different fiber) controls. Branched-chain fatty acids (BCFAs), metabolites produced exclusively from protein fermentation, were used as a measure of protein fermentation; the data were normalized based on the initial quantity of protein within the substrate. In protein-fiber substrate mixtures, only FOS and Even Mix inhibited BCFAs (mM/g protein basis) and only when they made up at least half of the substrate. Unexpectedly, the rate of protein fermentation was increased when the protein-fiber substrate contained 25% WB+PS fiber, possibly due to the starch component of the fiber. There was evidence that when pH drops during fermentation, as was the case for protein-FOS mixtures, it played a significant role in suppressing protein fermentation. Ammonia production was not largely affected by increasing the proportion of dietary fiber. A significant reduction did not occur until FOS made up at least 50% of the protein-fiber substrate; for Pectin, WB+PS, and Even Mix fibers, 75% inclusion was required for a significant decrease in ammonia. Interestingly, protein was butyrogenic. Protein as the sole substrate produced more butyrate than either Pectin or Even Mix as the sole substrates, and in fact, addition of Pectin to protein significantly reduced butyrate concentrations. However, the possible benefits of butyrate produced via protein fermentation needs to be tempered by the production of potentially toxic compounds and the association between protein fermentation and colorectal cancer. Overall, the thesis findings showed protein fermentation to be relatively stable and not easily influenced by increasing the availability of dietary fiber, and no clear evidence of microbial preference for carbohydrates over protein was found.