<p>Methionine (Met) is an essential amino acid (EAA) and is often considered one of the first limiting amino acids (AA) for milk production in dairy cattle. In diets of lactating dairy cattle, in the United States (U.S.), Met is considered limiting due to its low abundance in commonly fed ingredients like soybean and corn and its high degradability in the rumen. To circumvent this problem, rumen-protected Met (RPM) products were produced and are currently supplemented to dairy cattle. Supplemental RPM products protect Met from degradation in the rumen and deliver Met to the intestine to be absorbed into circulation, increasing metabolizable Met supply. Due to Met important roles in protein synthesis and as a methyl donor, dairy producers have supplemented RPM for decades. The primary benefit for supplementing RPM is improved milk protein concentration, which is important as a majority of dairy farms in the U.S. are paid based on milk components. </p> <p>Research is focused on finding a more economically viable RPM product that provides similar or better bioavailability of Met compared to commercially available products. This has led to the development of a novel rumen-protected Met product (EMT 3.4) in which Met is added to the soluble portion of distillers and incorporated with distillers grains. The result is distillers grains with solubles (DDGS) with elevated Met concentration. Incorporating Met into the solubles portion of DDGS has shown to protect Met from degradation when analyzed in vitro but has not been assessed in vivo. Thus, the primary objective of this study was to evaluate the bioavailability of EMT 3.4 via dilution of selenomethionine (SeMet) in milk protein and secondarily its impact on milk production and body composition. Dietary selenium (Se) yeast will deliver SeMet to the intestines and subsequently be absorbed. After adequate time feeding Se yeast, SeMet will label the different pools of Met similar to feeding radiolabeled nitrogen (N). Once pools of Met are labeled with SeMet, milk Se, a proxy for SeMet, can be measured in the milk. Concentrations of milk Se will fluctuate based on entry of Met, and as Met increases in the milk, the concentration of Se will decrease. To asses bioavailability milk Se is divided by milk N (Se:N) and the ratios of the treatments can be compared.</p> <p>The objective of this study was to evaluate the bioavailability of a novel rumen protected Met product and its effect on milk production and body composition compared to positive and negative controls. Dietary treatments consisted of a negative control treatment (NCT), not supplemented with RPM, a positive control treatment (PCT), supplemented Met via Smartamine M, and the experimental Met treatment (EMT), supplemented Met via EMT 3.4. Cattle fed PCT and EMT exhibited improved bioavailability indicated by a reduction in milk Se to milk N ratio when compared to NCT cows. Using milk Se concentration as an indicator of entry rate and amount of Met in the milk pool, PCT cows had a significantly lower milk Se concentration compared to NCT. Lower milk Se concentration of PCT cows indicates greater amount of Met in milk and subsequently a significantly greater milk protein yield was observed in PCT. Although EMT had similar bioavailability to PCT, the EMT cows had an intermediate response in milk Se but no production responses. The lack of production responses in EMT was observed alongside an elevated milk urea nitrogen (MUN) and a negative change in longissimus dorsi depth (LDD). A negative change in body weight (BW) was observed in NCT cows. Changes in body composition, as the ones observed in this study, are not usually observed unless cattle are energy or protein deficient. Given that diets of this study were formulated to meet requirements for producing 40 kg/day of milk and on average cattle were producing less, it is unlikely that cattle were energy or protein deficient. Results demonstrated that EMT provided similar Met bioavailability as Smartamine M in PCT demonstrated by responses in milk Se:N. The PCT tended to increase milk yield and increased milk protein, whereas EMT provided no observable production responses in this study.</p> <p><br></p>

No description available.

Purified brush border and basolateral membranes were isolated from homogenized intestinal enterocytes of Holstein steers by divalent cation precipitation followed by differential and sucrose density gradient centrifugation. Membrane marker enzymes were used to determine the effectiveness of the fractionation procedure. Alkaline phosphatase and sodium-potassium adenosine triphosphatase served as the marker enzymes for the brush border and basolateral membranes, respectively. The brush border fraction was enriched 5.1-fold over the cellular homogenate. Purification of 10.1-fold over cellular homogenate was obtained for the basolateral membrane fraction. Electronmicrographs and osmotic response data were used to confirm the vesicular nature of the membrane preparations. Brush border membrane vesicles from bovine jejunal and ileal tissue were used to evaluate lysine (LYS) and methionine (MET) transport. Total transport of LYS and MET was divided into mediated and diffusion components. Mediated uptake was further divided into sodium-dependent (Na⁺) and sodium independent (Na⁻) systems. Total LYS and MET uptake by ileal tissue tended to be higher than jejunal tissue at all concentrations evaluated but differences were significant (P<.O5) at 2.5 and 7.5 mM for LYS and 5, 12.5 and 15 mM for MET. The greater capacity of ileal tissue appeared to be due to the Na⁺ component of LYS uptake and the diffusion component of MET uptake. Methionine transporters had lower affinities and higher capacities than the corresponding LYS transporters in both ileal and jejunal tissue. Methionine transport was greater (P<.O5) than LYS transport in both ileal and jejunal tissue when initial amino acid concentration was 7.5 mM. When initial amino acid concentration was 1.25 mM, MET uptake was greater (P<.13) than LYS uptake in jejunal but not ileal tissue. The relative contribution of mediated and diffusion uptake systems to total MET and LYS uptake was found to be dependent of substrate concentration. / Ph. D.

Characteristics of methionine uptake by brush border membrane vesicles of bovine small intestine were investigated. Alkaline phosphatase marked the brush border membrane fraction obtained through differential centrifugation followed by a sucrose gradient. This preparation yielded a 10-fold enrichment of activity over homogenate. Methionine uptake was found to be into an osmotically active space. A binding constant of 75.4 pmol/mg membrane protein was determined. A significant (p<.05) sodium stimulation of methionine uptake was observed. This indicated active (energy-dependent) transport in addition to the diffusive component of intravesicular mention accumulation. Decreasing the pH of buffer medium significantly (p<.05) depresses methionine transport. A K_m = .114 mM and V_MAX = 56.5 pmol/s/mg membrane protein were ascertained for methionine. / M.S.

Two experiments were conducted with a total of 1,728 Large White turkeys to determine the relative potencies of four methionine compounds on an equimolar basis. A 28% protein basal diet composed primarily of ground yellow corn, dehulled soybean meal, and meat and bone meal was supplemented with DL-methionine (DL), sodium salt of DL-methionine (MENA), methionine hydroxy analogue calcium salt (MHAC), or methionine hydroxy analogue free acid (MHAA) at the .06, .12, .18, .24, or .3090 level of added methionine. Each of these 20 diets was fed to two pens of poults of each sex (9 birds per pen), and the basal diet was fed to eight pens of each sex from one day to sevenweeks of age in each experiment. From the combined six-week body weight data, MENA was 104.7 (86.1, 123.3), MHAC 101.8 (83.5, 120.1), and MHAA 92.590 (74.9, 110.1) as potent as DL (with 9590 fiducial limits) by the slope ratio procedure. No differences were detectable among the methionine products. The extra one degree of freedom associated with the nonlinear procedure accounted for 4.8 and 10. 7% of the residual variation in Experiments 1 and 2, respectively. Relative potencies were obtained for MENA of 110.3 and 115.6, MHAC of 100.4 and 88.1, and MHAA of 89.7 and 112.390 in Experiments 1 and 2, respectively. Two additional experiments were conducted using a total of 1,680 poults to study the relationships of methionine, choline, and sulfate in practical-type diets. A 6 x 2 x 2 factorial design was used involving increments of .0690 DL-methionine from 0 to .3090, 0 or .20% choline chloride, and 0 or . 10% potassium sulfate. The variables were added to a 21 90 protein basal diet containing 61% ground yellow corn and 32% dehulled soybean meal to which no supplemental choline or sulfate was added. Each of the 24 diets was fed to two pens of medium-type turkeys of each sex (9 birds per pen) from four to eight-weeks of age in the first experiment, and to two pens of Large White turkeys of each sex (9 and 8 birds per pen for males and females, respectively) from three to seven-weeks of age in the second experiment. From the addition of .06% and .12% DL-methionine, body weight gains were increased 11.0 and 16.5% in the first experiment, and 8.9 and 13.690 in the second experiment, respectively. In addition, methionine increased feed consumption about 1/3 these amounts, and feed efficiencies 1/2 these amounts. The addition of choline or sulfate failed to significantly increase these parameters, even in the absence of methionine. No significant interactions were observed among the dietary variables in either experiment. / M. S.

Duodenal, jejunal and ileal sections from sheep were used to study <i>in vitro</i> absorption of valine, threonine and methionine. Everted sacs were incubated for 45 minutes at 39 C under an atmosphere of 95% O₂ - 5% CO₂ in Krebs-Ringer bicarbonate buffer (mucosal and serosal fluid) containing 5 μmoles per ml of the test amino acid. The hydrolyzed tissue and mucosal and serosal fluids were analyzed for amino acid content. The duodenum absorbed smaller quantities of amino acids from the mucosal fluid than the jejunum and significantly (P<.01) less than the ileum. The release of amino acids from duodenal tissue was small for threonine and methionine and negative for valine. Accumulation of amino acids by duodenal tissue was not significantly different from the ileum, but this represented a larger part of the amount absorbed from the mucosal fluid. The duodenum appears to be of limited importance as a site of amino acid absorption. The jejunum readily absorbed valine and methionine, but absorbed only a small amount of threonine from the mucosal fluid. The release of amino acids into the serosal fluid by the jejunum was very slight in comparison to the ileum (P<.01). Tissue accumulation of amino acids was significantly less (P<.05) than in either the duodenum or ileum. While the jejunum generally absorbed large amounts of amino acids from the mucosal fluid, it did not release or accumulate amino acids in large amounts. The ileum maximized movement of all amino acids. It absorbed significantly more (P<.01) amino acids from the mucosal fluid and released significantly more (P<.01) amino acids into the serosal fluid. The accumulation of valine and threonine by ileal tissue was significantly greater (P<.05) than the jejunum. The ileum, therefore, appears to be the most active and efficient site of valine, threonine and methionine absorption in the sheep. / Master of Science

La découverte récente de nouveaux gènes, de mécanismes d’épissage alternatif et d’interactions entre les protéines du métabolisme intracellulaire de la Cbl suggère que de nouvelles interactions protéiques peuvent prendre part aux mécanismes de régulation de ce métabolisme. Nos données confirment, dans les cellules humaines des HepG2 et des fibroblastes de patients CblC et cblG, diverses interactions qui ont été jusqu'à présent que décrites in vitro ou chez les bactéries : MS avec méthionine synthase réductase (MSR), MS avec MMACHC et MMACHC avec MMADHC. Nos données révèlent également de nouvelles interactions : MMADHC avec MS, MMADHC avec MSR, MSR avec MMACHC et MS avec les isoformes de MAT. De plus, l'absence de MS ou MMACHC perturbe les interactions impliquant les autres partenaires protéiques de l'interactome MS. En conclusion, cette étude suggère que les différentes étapes du métabolisme intracellulaire de Cbl pourraient se produire dans un grand complexe multiprotéique composé d'au moins de MS, MSR, MMACHC, MMADHC et les isoformes de MAT, et qui contribuerait à protéger la Cbl du milieu cytoplasmique / The recent discovery of new genes, alternative splicing and protein-protein interactions between intracellular processing of vitamin B12 or cobalamin (Cbl) highlights the importance of an MS interactome. The goal of this PhD project is to further characterize the interactions of MS with other potential partners in a so-called MS interactome. Our data confirm for the first time in human cells (HepG2 cells and fibroblasts from cblC and cblG patients) various interactions that were so far only described in vitro or in bacteria: MS with methionine synthase reductase (MSR), MS with MMACHC, and MMACHC with MMADHC. Our data also reveal novel interactions: MMADHC with MTR, MMADHC with MSR, MSR with MMACHC and MS with MAT isoforms. Moreover, our data show that the absence of MS or MMACHC disturbs the interactions involving the other members of the MS interactome. In summary, this study suggests that different steps of the intracellular processing of Cbl could occur in a large multiprotein complex composed of at least MS, MSR, MMACHC, MMADHC and MAT isoforms that would contribute to protect the rare and highly reactive Cbl from the cytoplasmic milieu

Plusieurs facteurs conduisent à la génération de radicaux libres oxydants dans les organismes qui endommagent les biomolécules et en particulier les protéines du vivant. L’une des cibles de l’oxydation dans les protéines est la méthionine (Met). Son oxydation provoque des effets très dommageables, comme la maladie d’Alzheimer ou les maladies à prion. Nous avons étudié la réaction des radicaux hydroxyle (•OH) et de l’état triplet de la 3-carboxybenzophénone (3CB*) avec ces peptides. Le but de ce travail était de caractériser les espèces transitoires et les produits stables formés après oxydation radiolytique et photolytique de peptides contenant la Met (les dipeptides contenant de la Met et les pepetides plus longs tels que la bradykinine, une hormone humaine importante impliquée dans la diminution de la pression artérielle). Nous avons utilisé la photolyse éclair laser (LFP) et la radiolyse pulsée (PR) (pour les espèces transitoires de courte durée de vie), tandis que la radiolyse gamma et la photolyse continue (pour obtenir une caractérisation des produits stables). Les modifications structurelles ont été caractérisées par des techniques de HPLC et par couplage de la spectrométrie de masse (MS) et la détection par spectrométrie infrarouge couplée à la MS (IRMPD, CLIO laser à électrons libres). En outre, il nous a paru intéressant d’étudier deux autres dérivés de la benzophénone (BP), l’oxybenzone (OXB) et le sulisobenzone (SB), qui sont largement utilisés dans les produits de protection solaire commerciaux. En effet, l’application d’écrans solaires est controversée car certaines études épidémiologiques ont indiqué un risque accru de mélanome malin pour leurs utilisateurs. L’oxydation de dipeptides contenant Met par les radicaux •OH ou photosensibilisée par la ³(CB)* a conduit à la formation de radicaux cations centrés sur le soufre de la Met (>S•⁺) qui ont été en outre stabilisés par la formation de liaison deux centres à trois électrons (S∴Y)⁺, Y étant un atome possédant un doublet libre, ou qui ont subi une déprotonation donnant les radicaux contrés sur le carbone en α (α-S). L’oxydation des dipeptides par •OH a abouti à la formation de sulfoxyde de Met (MetSO) en tant que produit principal. Sans aucun doute, l’identification et la caractérisation des MetSO en solutions désoxygénées contenant la catalase est une étape importante dans la quête de produits stables. Toutefois, dans certains cas, d’autres produits ont été identifiés. En ce qui concerne, les produits stables de photolyse, ce sont des adduits avec le groupement 3CB, probablement résultant de la réaction de recombinaison radical-radical. Un autre produit formé au cours de la photolyse était 3CB-3CB résultant d’une photo-addition, qui a une structure similaire à celle du produit d’irradiations de la BP. Tous les produits identifiés (MetSO et la photo-adduits) ont été formés à partir des radicaux α-S par l’intermédiaire d’une dismutation ou une réaction avec 3CBH•/3CB•⁻. L’oxydation de la Met-Lys-bradykinine (MKBR) a abouti à la formation de photo-adduits similaires par réaction sensibilisée avec 3CB. L’oxydation induite de MKBR par •OH a abouti à plusieurs produits, en accord avec la non sélectivité des radicaux •OH. L’un des principaux produits est le MetSO et la phénylalanine hydroxylée. Notons que l’arginine n’est pas oxydée. Enfin, la photolyse de SB et OXB a été étudiée à l’aide de photolyse éclair au laser femto-et nanoseconde, ainsi que l’oxydation à un électron de ces molécules par radicaux •OH ont été réalisées en PR. Les résultats obtenus ont été comparés à ceux d’autres dérivés de la BP. L’état singulet excité subit un quenching à 100 % par transfert de proton intraomléculaire à l’état excité (ESIPT) en milieu aprotique et en milieu non polaire. Dans le cas d’un solvant polaire, la formation de radicaux phénoxyles a été identifiée. La réactivité des filtres solaires UV-excité vers dérivés simples de méthionine est également en cours d’étude. / Several factors (radiation, metabolism, pollutants) lead to the generation of oxidizing free radicals in living organisms that damage all biomolecules and especially proteins. One of the protein targets is Methionine (Met). Its oxidation causes highly damaging effects, such as Alzheimer’s or prion disease. The aim of this work was to investigate the transient species and the stable products formed after radiolytic and photolytic oxidation of Met-containing peptides. The reaction of hydroxyl radicals (•OH) and 3-carboxybenzophenone triplet state with Met-residue in peptides was investigated for model compounds (Met-dipeptides) and for longer peptides (e.g. Bradykinin). Laser flash photolysis and pulse radiolysis were used to characterize short-lived transient species, while gamma radiolysis and steady-state photolysis were used for quantitative and qualitative characterization of stable products. The structural modifications induced by oxidation have been characterized by the HPLC coupled with mass spectrometry and Infrared Multi Photon Dissociation Spectroscopy (IRMPD, CLIO Free electron laser). The oxidation of investigated Met-containing compounds by •OH or 3CB* led to the formation of S-centered radical cation >S•+ on Met-residue, that were further stabilized by formation of two-centered three-electron bond (S∴Y)+ or underwent the deprotonation reaction yielding the α-(alkylthio)alkyl radicals (α-S). The oxidation of Met-containing dipeptides by •OH radicals yielded the formation of Met sulfoxide (MetSO) as a main product. Undoubtedly, the identification and characterization of MetSO in deoxygenated solutions containing catalase was a milestone in investigation of stable products. However, in some cases, other products were identified. The stable products of photolysis were adducts with 3-carboxybenzophenone moiety, resulting from radical recombination reaction. Another identified product formed during photolysis was 3CB-3CB benzpinacol photoadduct, which has similar structure to the product of BP irradiations. Identified products (MetSO and the photo-adduct) were formed from the α-S via disproportionation or reaction with 3CBH•/3CBH•⁻. The oxidation of Met-Lys-Bradykinin (MKBR) yielded formation of similar photo-adducts via sensitized reaction with the 3CB*. The •OH induced oxidation of MKBR yielded several products, e.g. the sulfoxide and hydroxylated phenylalanine. In addition, other derivatives of benzophenone (oxybenzone (OXB) and sulisobenzone (SB)) were investigated due. They are widely used in commercial sun-protecting products dp to their unique photophysical properties. However the application of sunscreens awakes controversies because some epidemiological studies indicated an increased risk of malignant melanoma for their users. Photo-instability of sunscreen filters would result in reduced protection and may produce reactive free radicals or mutagens. In addition, the reactions of the sunscreens with oxygen free radicals e.g. hydroxyl radicals are likely to arise and they were not yet sufficiently documented. Finally, the radiolytic and photolytic properties of SB and OXB were investigated using femto-and nanosecond laser flash photolysis. Pulse radiolysis studies of the oxidation of those molecules by •OH radicals were performed. The results obtained for SB and OXB were compared to several other benzophenone derivatives. The results shown the formation of excited singlet state that was deactivated efficiently via the Excited State Intramolecular Proton Transfer (ESIPT). In case of polar solvent, the formation of trace amounts phenoxyl radicals was identified, while for nonpolar media those radicals were not observed. The reactivity of UV-excited sun filters towards simple derivatives of Met was also investigated, however, this topic requires further and more detailed investigations.

To expand the understanding of aging in the model organism Caenorhabditis elegans, global quantification of metabolite and protein levels in young and aged nematodes was performed using mass spectrometry. With age, there was a decreased abundance of proteins functioning in transcription termination, mRNA degradation, mRNA stability, protein synthesis, and proteasomal function. Furthermore, there was altered S-adenosyl methionine metabolism as well as a decreased abundance of the S-adenosyl methionine synthetase (SAMS-1) protein. Other aging-related changes included alterations in free fatty acid levels and composition, decreased levels of ribosomal proteins, decreased levels of NADP-dependent isocitrate dehydrogenase (IDH1), a shift in the cellular redox state, an increase in sorbitol content, alterations in free amino acid levels, and indications of altered muscle function and sarcoplasmic reticulum Ca2+ homeostasis. There were also decreases in pyrimidine and purine metabolite levels, most markedly nitrogenous bases. Supplementing the culture medium with cytidine (a pyrimidine nucleoside) or hypoxanthine (a purine base) increased lifespan slightly, suggesting that aging-induced alterations in ribonucleotide metabolism affect lifespan. An age-related increase in body size, lipotoxicity from ectopic yolk lipoprotein accumulation, a decline in NAD+ levels, and mitochondrial electron transport chain dysfunction may explain many of these changes. In addition, dietary restriction in aged worms resulting from sarcopenia of the pharyngeal pump likely decreases the abundance of SAMS-1, possibly leading to decreased phosphatidylcholine levels, larger lipid droplets, and ER and mitochondrial stress. The complementary use of proteomics and metabolomics yielded unique insights into the molecular processes altered with age in C. elegans.

S-Adenosyl-L-methionine (SAM) is an essential metabolite for all domains of life. SAM- dependent reactions result in three major metabolites: S-adenosyl-L-homocysteine (SAH), methylthioadenosine (MTA), and 5'-deoxyadenosine (5'-dA). Each of these has been demonstrated to be feedback inhibitors of SAM dependent enzymes. Thus, each metabolite has a pathway to prevent inhibition through the salvage of nucleoside and ribose moieties. However, these salvage pathways are not universally conserved. In the anaerobic archaeal organism Methanocaldococcus jannaschii, the salvage of SAH, MTA, and 5'-dA, proceeds first via deamination to S-inosylhomocysteine (SIH), methylthioinosine (MTI), and 5'-deoxyinosine (5'-dI). The annotated SAH hydrolase from M. jannaschii is specific for SIH and the hydrolyzed product homocysteine is then methylated to methionine. The salvage of MTA is known to proceed through the methionine salvage pathway, however, an anaerobic route for the salvage of MTA is still mostly unknown. Only two enzymes from the methionine salvage pathway are annotated in M. jannaschii's proteome, a methylthioinosine phosphorylase (MTIP) and methylthioribose 1-phosphate isomerase (MTRI). These enzymes were shown to produce methylthioribulose 1-phosphate from MTI. Unfortunately, how MTI is converted to either 2-keto-(4-methylthio)butyrate or methionine remains unknown. The two enzymes involved in the salvage of MTI have also been demonstrated to be involved in the salvage of 5'-dI. Interestingly, there is little information on how 5'-dA or 5'-dI is recycled and it is proposed here to be the source of deoxysugars for the production methylglyoxal, a precursor for aromatic amino acids. MTIP and MTRI were demonstrated to produce 5-deoxyribulose 1-phosphate from 5'-dI. Additionally, two enzymes annotated as part of the pentose phosphate pathway, ribulose 5-phosphate 3-epimerase and transketolase, were able to convert 5-deoxyribulose 1-phosphate to lactaldehyde. Lactaldehyde was then reduced to methylglyoxal by an essential enzyme in methanogenesis, N5, N10-methylenetetahydromethanopterin reductase with NADPH. These results further demonstrate a novel route for the biosynthesis of methylglyoxal. Lastly, hypoxanthine produced from phosphorolysis of inosine, MTI, and 5'-dI was demonstrated to be reincorporated through the hypoxanthine/guanine phosphoribosyltransferase (Hpt) to IMP. Together these reactions represent novel pathways for the salvage of the SAM nucleoside and ribose moieties in M. jannaschii. / Ph. D.