Two feeding trials were conducted to study the requirements of juvenile cobia for dietary methionine and lysine. Experiment I compared both fishmeal-based (fish meal and soybean meal as protein sources) and casein-based (casein and gelatin as protein sources) diets. All diets were isonitrogenous and isoenergetic. Crystalline amino acids was pre-coated with carboxymethylcellulose (CMC) and supplemented to simulate the amino acid pattern of juvenile cobia muscle protein except methionine. Five methionine levels of 0.45, 0.70, 0.95, 1.20 and 1.45% (1.07, 1.66, 2.26, 2.86 and 3.45% of protein) were studied. The test diets contained 0.16% cystine. Juvenile cobia (initial weight 35g) were fed test diets for 8 weeks. The results showed that the cobia used fish meal more effectively than casein. Final weight, percent weight gain (PWG), feed conversion ratio(FCR), hepatosomatic index (HSI), condition factor (CF), protein efficiency ratio (PER), protein retention (PR), carcass proximate composition and free methionine concentration in the serum were significantly enhanced (p<0.05) by increasing dietary methionine level. But profile of essential amino acids in the muscle and serum superoxidase and lysozyme activity were not significantly affected. Weight gain of the cobia fed the fishmeal-based diet containing 1.2% methionine was significantly higher than the other dietary groups. Methionine content of 0.45% resulted in reduced growth. No significant difference in weight gain was found in the casein-containing diets when methionine was 0.7% and over. The cobia fed diet containing 0.45% methionine started show apparent skin lesions in their heads on the sixth week of the growth trial and significant mortalities were observed since the seventh week (p<0.05). The optimum dietary level of methionine for cobia, estimated by the broken-line regression analysis on weight gain, was 1.10% (2.62% of protein) based on the fishmeal-based diet results and was 0.70% (1.67% of protein) based on the casein-based diet results. Experiment II used fish meal and wheat gluten as the protein sources. Pre-coated crystalline amino acids were supplemented to simulate the amino acid pattern of juvenile cobia muscle protein except lysine. Lysine levels including 1.49, 1.75, 2.00, 2.25, 2.50 and 2.75% (3.54, 4.16, 4.76, 5.35, 5.95 and 6.54%) were studied. Juvenile cobia (initially 12g) were fed test diets for 8 weeks. The results showed that the final weight, PWG, FCR, CF, PER, PR, carcass proximate composition and free lysine concentration in the serum were significantly enhanced (p<0.05) by increasing lysine levels. But HSI and essential amino acid profile of fish in the muscle were not significantly affected. Final weight and percent weight gain of the cobia fed diets containing 1.75% lysine or over were significantly higher than fish fed diet contained 1.49% lysine. FCR was lower for cobia fed diets containing 1.75% lysine or over than the 1.49% lysine group. The optimum dietary level of lysine, estimated by the broken-line regression analysis on weight gain was 1.76% (4.19% of protein), and was 1.95% (4.64% of protein) on serum free lysine concentration. There was no overt deficiency symptom, such as caudal fin rot in rainbow trout, in the cobia during the growth trial.

The present study used Escherichia coli¡]BL21¡^that contained pET-23a-bpGH plasmids, to express black porgy growth hormone ¡]bpGH¡^. The bpGH was refolded at pH 11.3 in the presence of catalytic amounts of cysteine and purified by ion exchanger chromatography and gel filtration chromatography. The purified bpGH is a monomer and has a molecular weight of 22 kDa. Using the bpGH, the effects of the growth hormone on growth, essential amino acid deposition and nonspecific immunity in black porgy were studied. The experiment was a 4*3 ¡]diet*GH¡^ factorial design. Four experimental diets were formulated in that fish meal was replaced by the mixture of soya protein and gelatin so that fish meal / soya mixture = 100 / 0, 75 / 25, 50 / 50 and 25 / 75, respectively. GH treatments included non-injection, once per 3 days and once per 12 days. GH was injected intramuscularly at a dosage of 0.05 &#x00B5;g / g wet body weight. The growth trial lasted for 72 days. The results showed that GH administration significantly enhanced weight gain, feed efficiency, protein efficiency ratio and muscle methionine concentration of the fish. GH injection improved the growth performance of the fish fed low protein quality diets to a level equals to the groups fed high protein quality diets. These results indicate that GH injections enhanced the perferential absorption and deposition of the first limiting amino acid methionine from the diets. In addition, GH administration enhanced alternative complement activity and increased serum lysozyme concentration, implicating the enhancement of the immunity.

DL-Methionine (Met) has been conventionally used in swine diets with assumption of similar bioefficacy with L-Met. However, because L-Met is the form that is utilized by animals for protein synthesis, L-Met could, theoretically, be more available. Four experiments were conducted to evaluate L-Met bioavailability in nursery pigs with 21-day growth trials. A total of 105,105,112 and 84 crossbred pigs were used in Exp. 1, 2, 3 and 4, respectively. Each experiment had a low Met basal diet and 3 levels of the Met sources (DL-Met and L-Met). In addition to the basal diet, supplementation levels were 0.053%, 0.107% and 0.160% in Exp. 1, 0.040%, 0.080% and 0.120% in Exp. 2, 0.033%, 0.067% and 0.100% in Exp.3, 0.040%, 0.080% and 0.120% in Exp. 4. Body weight (BW), average daily gain (ADG), average daily feed intake (ADFI), gain: feed (G:F) were measured and plasma urea nitrogen (PUN) was analyzed in blood samples weekly. In Exp. 3 and 4, preference studies were conducted with the basal diet and the second highest level of each Met source. When additional DL-Met or L-Met were supplemented to the basal diet, BW, ADG, ADFI, and G:F ratio increased (P < 0.05). In the comparison between the DL-Met and L-Met diets in Exp. 1, pigs in the L-Met group had greater ADG and G:F ratios in the d 0-7 (P < 0.05) period than those in the DL-Met group. However, there were no differences for the overall experimental period. In Exp. 2, pigs in the DL-Met group had greater BW (P < 0.05), ADG (P < 0.05) and ADFI (P < 0.05) than those in the L-Met group for the overall period whereas no differences were observed in G:F ratios and PUN concentrations. In Exp. 3 and 4, there were no differences in BW, ADG, ADFI, G:F ratios or PUN concentrations between L-Met and DL-Met groups for the overall period. There was no preference exhibited for either the DL-Met or L-Met diet. In the results of relative bioavailability of L-Met to DL-Met, the values was 111.1% for d 0-14 based on the estimation by ADG in Exp. 1; L-Met bioavailability was lower than DL-Met based on all response measures in Exp. 2. However, in Exp. 3, relative bioavailability of L-Met to DL-Met was 100.4, 147.3, and 104.1% for d 0-14 ADG, G:F ratio and PUN concentrations. In Exp 4, the relative bioavailability of L-Met was 92.9, 139.4 and 70.4% for d 0-14 ADG, G:F ratio and PUN concentrations. In conclusion, using L-Met in the nursery diet demonstrated no consistent beneficial effect on ADG, G:F ratio or relative bioavailability compared to conventional DL-Met.

Resolving the genetic and mechanistic bases of complex biological behaviors remains a central challenge in the post-genomic era. Among these is the emergence of interspecies cooperation, a feature common across levels of biological organization. Of the numerous examples afforded by nature, microbes arguably provide the greatest ability to connect underlying genotypes to cooperative phenotypes.

No description available.

CueO is a multicopper oxidase and catalyses the four-electron reduction of dioxygen to water and functions to protect Escherichia coli against copper-induced toxicity. The mechanism of oxygen reduction in multicopper oxidases has been well studied, but the key structures of the reaction intermediates are not known. A combination of kinetic measurements, mutagenesis and X-ray crystallographic studies were conducted to entrap and structurally characterize the reaction states in CueO. CueO has a methionine-rich insert and a labile copper binding site, two features found only in multicopper oxidases involved in copper detoxification. The role of these features in CueO activity has been investigated. In a separate study, a simple mathematical model based on infectivity amongst clustered receptors was developed to explain the chemotactic sensitivity, response range and other key features of chemotaxis.This study describes the successful entrapment of three out of four functional states in CueO. The crystal structures of these reaction states are presented. Using single-turnover oxygen reduction kinetics that were measured using a stopped-flow device, the optical absorption features of three different fully oxidized forms of CueO were captured: the native intermediate, the resting oxidized state and another intermediate lying between them. Stopped-flow studies combined with electron transfer kinetic measurements revealed a role of the conserved residue, E506, in either the protonation of the native intermediate or the release of water molecules formed as a product of the reaction.Cu(I) and Ag(I) bound crystal structures of CueO were determined revealing three binding sites along the methionine-rich helix used by both metal ions. The labile, regulatory copper site in CueO was shown to be a Cu(I) susbtrate oxidation site. Ag(I) was shown to be a potent inhibitor of all CueO activities in vitro and copper detoxification by the cue system in vivo. The cus system was discovered to be necessary for removing Ag(I) inhibition of copper detoxification by the cue system. These results provide further insights into the role of CueO in copper detoxification and the effect of silver on the detoxification mechanism.

Several of the inborn errors of vitamin B$ sb{12}$ (cobalamin, Cbl) metabolism (cblC, cblD, cblE, cblF, cblG) are associated with homocystinuria and hypomethioninemia due to a functional deficiency of the cytoplasmic enzyme methionine synthase which requires methylcobalamin (MeCbl) as a cofactor. We compared the growth of cultured fibroblasts from controls with those from patients with a selective deficiency of MeCbl (cblE and cblG) and with those from patients with a defect in both MeCbl and adenosylcobalamin (AdoCbl) (cblC, cblD and cblF). Cells were grown in methionine and folic acid free media and in fully supplemented medium. Control cells were able to grow in the deficient medium supplied with homocysteine, cobalamin and folate, while mutant cells were not, due to their inability to synthesize methionine from its immediate metabolic precursor, homocysteine. This differential growth is useful for screening for genetic defects of methionine biosynthesis. Moreover, by correcting methionine auxotrophy in these cells, it may be possible to isolate genes which code for the products that are deficient in these disorders.

Except for tyrosine, the amino acid requirement of parenterally fed (PN) human neonates has not been derived. Methionine and cysteine are indispensable and dispensable sulphur amino acids respectively. Cysteine is synthesized from methionine. Cysteine is unstable in solution, and is left out or added in very small amounts to amino acid solutions. Methionine is added to compensate for the lack of cysteine, assuming that the neonate will convert methionine to cysteine to meet the body’s metabolic demand. Methionine is hepatotoxic and there is evidence that the neonate has limited ability for its conversion to cysteine. To determine the requirement of the neonate for methionine, PN-fed, stable, post-surgical neonates received graded intakes of methionine. The mean methionine requirement was estimated to be 49 mg.kg-1.day-1, which is 48 to 90% of the methionine content of current commercial amino acid solutions. Because cysteine is the rate limiting substrate for glutathione (GSH) synthesis and current methods of determining amino acid requirement measure requirement for protein synthesis, SAA requirements for maintenance of GSH status was deleniated in healthy adult males and in PN-fed human neonates. GSH kinetics was measured in healthy men receiving the mean methionine requirement and graded intakes of cysteine. GSH synthesis did not change with the addition of cysteine. Additionally, PN-fed post-surgical neonates recieved a methionine-adequate cysteine-free PN followed by cysteine supplemented PN for two 3-day periods and GSH kinetics measured on days 3 and 6. There was no change in GSH synthesis in response to cysteine supplementation. It is concluded that the PN-fed human neonate is capable of synthesizing enough cysteine from methionine not only for protein synthesis but for GSH synthesis. For both healthy men and stable post-surgical neonates, the requirement for GSH synthesis is met at the sulphur amino acid requirement derived using the indicator amino acid technique

Except for tyrosine, the amino acid requirement of parenterally fed (PN) human neonates has not been derived. Methionine and cysteine are indispensable and dispensable sulphur amino acids respectively. Cysteine is synthesized from methionine. Cysteine is unstable in solution, and is left out or added in very small amounts to amino acid solutions. Methionine is added to compensate for the lack of cysteine, assuming that the neonate will convert methionine to cysteine to meet the body’s metabolic demand. Methionine is hepatotoxic and there is evidence that the neonate has limited ability for its conversion to cysteine. To determine the requirement of the neonate for methionine, PN-fed, stable, post-surgical neonates received graded intakes of methionine. The mean methionine requirement was estimated to be 49 mg.kg-1.day-1, which is 48 to 90% of the methionine content of current commercial amino acid solutions. Because cysteine is the rate limiting substrate for glutathione (GSH) synthesis and current methods of determining amino acid requirement measure requirement for protein synthesis, SAA requirements for maintenance of GSH status was deleniated in healthy adult males and in PN-fed human neonates. GSH kinetics was measured in healthy men receiving the mean methionine requirement and graded intakes of cysteine. GSH synthesis did not change with the addition of cysteine. Additionally, PN-fed post-surgical neonates recieved a methionine-adequate cysteine-free PN followed by cysteine supplemented PN for two 3-day periods and GSH kinetics measured on days 3 and 6. There was no change in GSH synthesis in response to cysteine supplementation. It is concluded that the PN-fed human neonate is capable of synthesizing enough cysteine from methionine not only for protein synthesis but for GSH synthesis. For both healthy men and stable post-surgical neonates, the requirement for GSH synthesis is met at the sulphur amino acid requirement derived using the indicator amino acid technique

The thesis consists of two parts targeting the characterization of chromatographic behavior of linear tryptic and cyclic peptides containing oxidized methionine (Met) in reversed-phased chromatography. The retention order of methionine-containing peptide analogues was observed to be the same in both studies: Met oxide < Met dioxide < Met. For linear tryptic peptides, the magnitude of the retention time shift may vary dramatically: from –9 % to +0.36 % acetonitrile. Particularly, large negative retention time shifts are found mostly associated with methionine being in the hydrophobic face of an amphipathic helix. Contrary to previously reported observations, I demonstrate for the first time that methionine oxidation may increase peptide hydrophobicity, this occurs only when methionine is in the N3 position of the N-capping stabilization motif preceding an amphipathic helix. In the second study, the effect of peak splitting was observed for some Met oxide-containing cyclolinopeptides, which most likely appear due to diastereomerization.