A nutrição é um dos principais fatores que afetam a eficiência reprodutiva por influenciar o crescimento, maturação e capacidade ovulatória do folículo bem como o perfil e estado metabólico do animal, gerando cenários que prejudicam ou corroboram o desenvolvimento e estabelecimento da prenhez. Diferentes fontes energéticas utilizadas na nutrição são capazes de alterar os padrões de fermentação ruminal e causar respostas endócrinas distintas. A partir disso, os objetivos desse estudo foram avaliar de que forma duas fontes energéticas da dieta influenciam a produção embrionária, a expressão gênica de enzimas hepáticas que metabolizam progesterona (P4) e a insulinemia. Em um delineamento em crossover, 22 vacas holandesas não lactantes e não gestantes foram distribuídas em dois grupos: um recebendo milho e outro polpa cítrica como fonte de energia da dieta. A quantidade de alimento fornecida foi de 1,3% do peso corporal em matéria seca por dia. O estudo foi composto por dois períodos de 71 dias de duração e em cada um deles foram realizadas duas superovulações (aos 35 e aos 70 dias) e uma biópsia hepática (aos 71 dias). Amostras sanguíneas foram colhidas imediatamente antes do fornecimento do alimento e 4 horas após, em dias pré-determinados, para dosagem de glicose, insulina e P4. Ao final do estudo as vacas passaram por teste de tolerância à glicose (TTG). Foi quantificada a expressão gênica de enzimas que metabolizam a P4 por RT-qPCR. A análise estatística foi realizada por meio de regressão logística pelo Proc MIXED do SAS 9.3. Os dados de expressão gênica foram avaliados por meio de delta CT. Imediatamente antes do fornecimento do alimento, a insulina circulante foi maior para o grupo milho (P < 0,01) e a P4 circulante foi maior para o grupo polpa cítrica (P < 0,01). Quatro horas após a alimentação, a P4 foi igual entre os tratamentos. A relação da P4 circulante na hora 4 e 0 foi maior para o grupo milho (P < 0,01). Tanto a glicose basal como o Homa-IR (Homeostasis model assessment of insulin resistance) foram maiores para o grupo milho. No TTG, o grupo milho apresentou maior pico de glicose no momento 5 minutos, maior taxa de decaimento da glicose (P = 0,01) e menor tempo de meia vida da glicose (P = 0,05). Não houve efeito de tratamento na resposta superestimulatoria, superovulatória, produção de embriões e na expressão gênica das enzimas que metabolizam a P4, mas as superovulações realizadas aos 70 dias produziram embriões de qualidade inferior em relação às realizadas aos 35 dias, independente de tratamento. Conclui-se que, embora tenha sido possível alterar a insulina circulante através da dieta, a quantidade e qualidade de embriões produzidos não foram alteradas. O aumento pós-prandial da P4 circulante não foi relacionado a menor expressão gênica das enzimas hepáticas que metabolizam a P4. / Nutrition is one of the main factors affecting reproductive efficiency by influencing the growth, maturation and ovulatory capacity of the follicle and metabolic status of the animal, leading to scenarios that impair or corroborate the development and establishment of pregnancy. Different energy sources in the composition of the diet can alter ruminal fermentation patterns and cause different endocrine responses. The objectives of this study were to evaluate how two different energy sources in the diet can influence embryo production, gene expression of liver enzymes that metabolize progesterone (P4) and circulating insulin. In a crossover design, 22 non-lactating and non-pregnant Holstein cows were allocated into two groups: one receiving corn and other receiving citrus pulp as the energy supply of the diet. The amount of feed provided was 1.3% of body weight of dry matter per day. The study consisted of two 71-days periods and cows were superovulated twice on each period (at 35 and 70 days). Liver biopsy was performed after 71 days from the beginning of each replicate. Blood was sampled immediately before feeding and 4 hours later, at predetermined days, for measurement of glucose, insulin and P4. At the end of the study cows underwent a glucose tolerance test (GTT). Gene expression of liver enzymes that metabolize P4 was quantified by RT-qPCR. Statistical analysis was performed using logistic regression of Proc Mixed of SAS 9.3. Gene expression data were evaluated using delta CT. Immediately before feeding, the circulating insulin was greater for the corn group (P < 0.01) and circulating P4 was greater for the citrus pulp group (P < 0.01). Four hours after feeding, circulating P4 was similar. The relationship of circulating P4 between hour 4 and 0 was greater for the corn group (P < 0.01). Both basal circulating glucose and HOMA-IR (Homeostasis model assessment of insulin resistance) were greater for the corn group. The corn group had also greater glucose peak at the time 5 minutes of the GTT, greater glucose rate of decay (P = 0.01) and a shorter half-life of glucose (P = 0.05). There was no treatment effect on superstimulatory and superovulatory response, on embryo production and on gene expression of liver enzymes that metabolize P4. However, superovulations performed at 70 days produced lower embryo quality compared to those performed at 35 days, regardless of treatment. In conclusion, although it was possible to change circulating insulin by feeding different diets, the quantity and quality of embryos produced were not affected by the diets. The postprandial increase in circulating P4 was not associated with altered gene expression of hepatic enzymes that metabolize P4.

<p>Insulin as well as glucagon are important hormones in maintaining glucose homeostasis and regulating the metabolism in the body. Insulin receptors (IR) are transmembrane receptors that promote a signal transduction when activated by insulin. This can for example cause an increased influx of glucose into the cell performed by so called glucose transporters (GLUTs). These membrane proteins facilitate the transport of glucose from the blood into the cells, so the cell always has a constant supply of energy. Peroxisome proliferator-activated receptors (PPAR) are nuclear fatty acid receptors. They are activated by lipids and regulate fatty acid transcription. PPARδ/β is located in skeletal muscle and can promote fatty acid catabolism as well as cause a switch in fuel preference from glucose to fatty acids. It has been suggested that ligands for PPARδ could act as insulin sensitizers. The PPARγ coactivator-1α can increase mitochondrial content in skeletal muscle if over expressed. The same is true for endurance exercise.</p><p>Hormones released from adipose tissue can cause hyperphagia<strong> </strong>and obesity if over- or under expressed. They can also work in the opposite way by decreasing appetite with weight loss as an effect. Impaired signalling or dysfunctional receptor can cause insulin resistance, obesity and diabetes. Lipolysis occurs in adipose tissues and is conducted by three enzymes, namely adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and monoglyceride lipase (MGL). There are some factors that can increase lipolysis such as caffeine, a low glycemic index, high protein intake and training.</p><p>The enzyme PEPCK is involved in the gluconeogensis in the liver and kidney cortex, and also in the glyceroneogenesis in the liver, as well as in brown and white adipose tissue. When overexpressed in skeletal muscle the enzyme increases the muscle activity. The overexpression of the enzyme did promote the β-oxidation as energy source for the muscles during exercise, instead of muscle glycogen as fuel.</p><p>The processes of protein synthesis and breakdown are together called protein turnover. Muscle grows when synthesis is greater than breakdown, and withers if breakdown exceeds the level of synthesis. Acute effects of training is catabolic, but long time exercise causes however an increased protein synthesis. Leucine, an essential amino acid, has an important role in the initiation phase of translation. Glutamine is probably important in the regulation of muscle protein synthesis and breakdown. Together with glutamate, aspartate and asparagine, these are responsible for the amino acid metabolism that occurs in the muscles. Protein synthesis reaches its maximum in the recovery phase after intense training.</p>

Rationale: Eosinophils are “end cell” leucocytes, associated with allergy, asthma and helminthiasis. At sites of inflammation, eosinophils may modulate immune response through expression of the extra-hepatic tryptophan-catabolising enzyme, Indoleamine 2, 3-dioxygenase (IDO). Kynurenines, products of tryptophan cleavage, induce apoptosis of T-cells, including thymocytes. Eosinophils naturally home to the thymi in mammals. Thymus is a primary lymphoid organ, where T-cells develop and undergo selection. My hypothesis is that eosinophils homing to the thymi participate in T-cell development through their expression of IDO. Methods: Immunohistochemistry revealed eosinophils in thymic tissue. Immunocytochemistry and flow cytometry were used to locate IDO protein expression in the thymus particularly in thymic eosinophils. RT-PCR and real-time PCR determined the presence of IDO mRNA in the thymus. Results: thymic eosinophils express IDO and infiltrate compartments associated with negative selection. The highest IDO transcription correlated with the influx of eosinophils and prevalence of immature thymocytes. / Experimental Medicine

Insulin as well as glucagon are important hormones in maintaining glucose homeostasis and regulating the metabolism in the body. Insulin receptors (IR) are transmembrane receptors that promote a signal transduction when activated by insulin. This can for example cause an increased influx of glucose into the cell performed by so called glucose transporters (GLUTs). These membrane proteins facilitate the transport of glucose from the blood into the cells, so the cell always has a constant supply of energy. Peroxisome proliferator-activated receptors (PPAR) are nuclear fatty acid receptors. They are activated by lipids and regulate fatty acid transcription. PPARδ/β is located in skeletal muscle and can promote fatty acid catabolism as well as cause a switch in fuel preference from glucose to fatty acids. It has been suggested that ligands for PPARδ could act as insulin sensitizers. The PPARγ coactivator-1α can increase mitochondrial content in skeletal muscle if over expressed. The same is true for endurance exercise. Hormones released from adipose tissue can cause hyperphagia and obesity if over- or under expressed. They can also work in the opposite way by decreasing appetite with weight loss as an effect. Impaired signalling or dysfunctional receptor can cause insulin resistance, obesity and diabetes. Lipolysis occurs in adipose tissues and is conducted by three enzymes, namely adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and monoglyceride lipase (MGL). There are some factors that can increase lipolysis such as caffeine, a low glycemic index, high protein intake and training. The enzyme PEPCK is involved in the gluconeogensis in the liver and kidney cortex, and also in the glyceroneogenesis in the liver, as well as in brown and white adipose tissue. When overexpressed in skeletal muscle the enzyme increases the muscle activity. The overexpression of the enzyme did promote the β-oxidation as energy source for the muscles during exercise, instead of muscle glycogen as fuel. The processes of protein synthesis and breakdown are together called protein turnover. Muscle grows when synthesis is greater than breakdown, and withers if breakdown exceeds the level of synthesis. Acute effects of training is catabolic, but long time exercise causes however an increased protein synthesis. Leucine, an essential amino acid, has an important role in the initiation phase of translation. Glutamine is probably important in the regulation of muscle protein synthesis and breakdown. Together with glutamate, aspartate and asparagine, these are responsible for the amino acid metabolism that occurs in the muscles. Protein synthesis reaches its maximum in the recovery phase after intense training.

No description available.

Among multiple interconnected pathways for L-Lysine catabolism in pseudomonads, it has been reported that Pseudomonas aeruginosa PAO1 employs the decarboxylase and the transaminase pathways. However, knowledge of several genes involved in operation and regulation of these pathways was still missing. Transcriptome analyses coupled with promoter activity measurements and growth phenotype analyses led us to identify new members in L-Lys and D-Lys catabolism and regulation, including gcdR-gcdHG for glutarate utilization, dpkA, amaR-amaAB and PA2035 for D-Lys catabolism, lysR-lysXE for putative L-Lys efflux and lysP for putative L-Lys uptake. The amaAB operon is induced by L-Lys, D-Lys and pipecolate supporting the convergence of Lys catabolic pathways to pipecolate. Growth on pipecolate was retarded in the gcdG and gcdH mutants, suggesting the importance of glutarate in pipecolate and 2-aminoadipate utilization. Furthermore, this study indicated links in control of interconnected networks of lysine and arginine catabolism in P. aeruginosa. Effect of D-amino acids and the genes involved in their metabolism are of great interest in both bacteria and mammals. D-Arg utilization in PAO1 requires the coupled dehydrogenases DauB and DauA. In this study, DauB was found to use only L-Arg as its substrate unlike its partner dehydrogenase DauA with wide substrate specificity. However, evidence from this study and previous studies suggest that the coupled enzymes DauB and DauA are unique for D-Arg catabolism. The three D-amino acid dehydrogenases DguA, DadA and DauA were found to have somewhat limited in vivo substrate specificity compared to that found in vitro tested using purified enzymes. Many studies showed that D-amino acids are toxic to bacteria. The ΔdguA, ΔdadA and ΔdauA triple mutant had two-fold lower minimum inhibition concentration of carbenicillin and tetracycline compared to wild-type PAO1. Both in the wild-type PAO1 and the triple mutant, synergy was observed between gentamicin or tetracycline (at concentrations below the MIC) and D-amino acids resulting in growth inhibition or reduction, respectively. However, no special synergistic or antagonistic effects were observed specifically in the ΔdguA, ΔdadA and ΔdauA triple mutant as compared to the wild-type PAO1 when D-amino acids were given in combination with antibiotics.

Lactic acid bacteria are essential as flavor producers of cheese and fermented products. They are capable of catabolizing aromatic, branched chain, and sulfur amino acids to flavor compounds. During cheese ripening the numbers of lactococcal colonies decrease, but lactococci survive without replication in culture. This prompted an investigation into possible mechanisms of catabolism of branched chain amino acids into branched chain fatty acids and the physiological relevance of amino acid catabolism to the bacteria. We hypothesized that lactococci catabolize branched chain amino acids to branched chain fatty acids during nonculturability. Lactococci, lactobacilli, and brevibacteria catabolized both branched chain amino acids and keto acids into branched chain fatty acids. Lactococci survived carbohydrate-limited conditions for over 4 yrs. Their survival was represented by maintaining intracellular ATP, enzyme activity, membrane integrity, capability of ATP- and PMF-dependent substrate transport, transcription, and catabolism of amino acids to fatty acids. Assays conducted with NMR spectroscopy coupled with in silico analysis showed that branched chain substrates are catabolized via keto acids, HMG-CoA, and acetyl-CoA to branched chain fatty acids. A short list of candidate genes was identified for the pathway by gene expression analysis coupled to NMR analysis. The expression of these genes and the presence of the related catabolites were identified in long-term starved cultures of nonculturable lactococci. This verified that catabolism of branched chain amino acids to branched chain fatty acids occurred during the nonculturable state only and in conditions of carbohydrate deprivation. The pathway also facilitated fixation of carbon by lactococci, revealing the mechanism of survival of lactococci over 4 yrs in culture without the addition of external carbon sources. Between strains the availability of carbohydrate and acid stress played significant roles in modulating their ability to produce branched chain catabolites. The ability of lactococci to catabolize branched chain amino acids during sugar starvation represents a shift in carbon catabolic routes. The identified pathway also represented a balance between catabolism and anabolism, suggesting that the bacteria were in a homeostatic state during nonculturability. We accepted the hypothesis that nonculturable lactococci catabolized branched chain amino acids to branched chain fatty acids during starvation./p>

Amino acids derived from the degradation of casein in cheese serve as precursors for the generation of desirable and undesirable flavor compounds. Microbial degradation of aromatic amino acids is associated with the formation of aroma compounds that impart putrid-fecal, barny-utensil, and floral off-flavors in cheese, but pathways for their production had not been established. This study investigated Tyr and Phe catabolism by Lactobacillus casei and Lactobacillus helveticus cheese flavor adjuncts under simulated Cheddar cheese-ripening conditions (pH 5.2, 4% NaCl, 15°C, no sugar). Enzyme assays of cell-free extracts and micellar electrokinetic capillary chromatography of supernatants indicated that L. casei and L. helveticus strains catabolize Tyr and Phe by successive transamination and dehydrogenation reactions. Major products of Tyr and Phe catabolism included off-flavor compounds formed by chemical degradation of the α-keto acids, produced by transamination, and aromatic α-hydroxy acids derived from α-keto acids by α-hydroxy acid dehydrogenases. Action of Lacrococcus lactis aminotransferase enzymes on Trp, Tyr, and Phe also leads to the formation of α-keto acids, but unlike lactobacilli, the former bacteria do not express dehydrogenase activity under cheese-like conditions (pH 5.2, 4% NaCl, 15°C, no sugar). Since aromatic α-keto acids may degrade spontaneously into undesirable flavor compounds, α-hydroxy acid dehydrogenases may be useful in controlling off-flavor development via diversion of chemically labile α-keto acids to more stable a-hydroxy acids. To test this hypothesis, we investigated the effect of D-hydroxyisocaproate dehydrogenase overexpression by a L. casei adjunct on chemical and sensory properties of reduced-fat Cheddar cheese made with and without addition of 20 mM α-ketoglutarate. The D-hydroxyisocaproic acid dehydrogenase gene (D-HicDH) was cloned into a high copy number vector pTRKH2 and transformed into L. casei ATCC334. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter + L. casei ATCC334 with pTRKH2, and starter + L. casei ATCC334 with pTRKH2: D-HicDH, and then volatile analysis was performed by gas chromatography and mass spectrometry. Statistical analysis of volatile data after 3 mo of ripening at 7°C showed profiles of ketones, aldehydes, alcohols, esters, sulfur compounds, and benzaldehyde were significantly altered by culture treatments and α-ketoglutarate addition, and these treatments also affected sensory flavor attributes of experimental cheeses. Results also indicated overexpression of D-hydroxyisocaproic acid dehydrogenase can divert labile α-keto acids into more stable compounds, but the overall effect seemed to diminish both beneficial and detrimental flavor notes.

This thesis describes the chemical investigation of three marine sponges from Antarctica and the total syntheses of natural products erebusinone (12) and its derivative, erebusinonamine (52). Investigation of the yellow Antarctic marine sponge Isodictya setifera resulted in the isolation of two secondary metabolites, purine analog (32) and 3-hydroxykynurenine (24). Chemical investigation of Isodictya setifera led to the isolation of six secondary metabolites which included 5-methyl-2-deoxycytidine (25), uridine (28), 2-deoxycytidine (31), homarine (37), hydroxyquinoline (33), 3-hydroxykynurenine (24). The latter two compounds were found to be intermediates of tryptophan catabolism in crustaceans. From the Antarctic marine sponge Isodictya antractica ceramide analog (39) was isolated and its chemical structure was assigned by a combination of spectroscopic and chemical analyses. Stereochemistry was determined by modified Mosher's method. Erebusinone (12), a yellow pigment isolated from the Antarctic marine sponge Isodictya erinacea has been implicated in molt inhibition and mortality against the Antarctic crustacean amphipod, Orchomene plebs, possibly serving as a precursor of a xanthurenic acid analog. Thought to act as a 3-hydroxykynurenine 24 mimic, erebusinone (12) may be involved chemical defense. This appears to be the first example in the marine realm of an organism utilizing tryptophan catabolism to modulate molting as a defensive mechanism. To further investigate the bioactivity and ecological role of erebusinone (12), the synthesis of this pigment was carried out in an overall yield of 44% involving seven steps which were economical and convenient. Erebusinonamine (52) was also similarly synthesized in eight steps with an overall yield of 45%.

Arginine utilization in Pseudomonas aeruginosa with multiple catabolic pathways represents one of the best examples of metabolic versatility of this organism. To identify genes of this complex arginine network, we employed DNA microarray to analyze the transcriptional profiles of this organism in response to L-arginine. While most genes in arginine uptake, regulation and metabolism have been identified as members of the ArgR regulon in our previous study, eighteen putative transcriptional units of 38 genes including the two known genes of the arginine dehydrogenase (ADH) pathway, kauB and gbuA, were found inducible by exogenous L-arginine but independent of ArgR. The potential physiological functions of those candidate genes in L-arginine utilization were studied by growth phenotype analysis in knockout mutants. The insertion mutation of aruH encoding an L-arginine:pyruvate transaminase abolished the capability to grow on L-arginine of an aruF mutant devoid of a functional arginine succinyltransferase (AST) pathway, the major route of arginine utilization. The aruH gene was cloned and over-expressed in E. coli. Taking L-arginine and pyruvate as the substrates, the reaction products of recombinant enzyme were identified by MS and HPLC as 2-ketoarginine and L-alanine. Lineweaver-Burk plots of the data revealed a series of parallel lines characteristic of ping-pong kinetics mechanism, and the apparent Km and catalytic efficiency (Kcat/Km) were 1.6 ± 0.1 mM and 24.1 mM-1 s-1 for pyruvate and 13.9 ± 0.8 mM and 2.8 mM-1 s-1 for L-arginine. Recombinant AruH showed an optimal pH at 9.0 and substrate specificity with an order of preference being Arg > Lys > Met > Leu > Orn > Gln. These data led us to propose the arginine transaminase (ATA) pathway that removes the α-amino group of L-arginine via transamination instead of oxidative deamination by dehydrogenase or oxidase as originally proposed. In the same genetic locus, we also identified a two-component system, AruRS, for the regulation of arginine-responsive induction of the ATA pathway. Our latest DNA microarray experiments under D-arginine conditions also revealed PA3863 as the candidate gene encoding D-arginine dehydrogenase which might lead to the recognition of a wider network of arginine metabolism than we previously recognized.