EFFECT OF METHIONINE AND METHIONINE SULPHOXIMINE ON RAT BRAIN S-ADENOSYL METHIONINE LEVELS

Rat brain SAM levels were markedly increased after methionine administration, whereas the convulsant, L-methionine-dl-sulphoximine (MSO), produced a 35 per cent decrease in whole brain content of S-adenosyl-L-methionine (SAM). When methionine was given in combination with MSO, SAM levels were not decreased. Studies on the regional distribution of SAM revealed only a small variation between regions (from 24 nmol/g in midbrain to 49-5 nmol/g in striatum). SAM levels were reduced by about 50 per cent in the cerebellum, striatum, cortex and hippocampus 3 and 6 h after MSO. It is proposed that abberant cerebral methylation processes may be involved in the genesis of the MSO seizure.     

ADAPTATION OF A METHIONINE AUXOTROPH ESCHERICHIA COLI GROWTH ASSAY TO MICROTITER PLATES FOR QUANTITATING METHIONINE

A rapid microtiter methionine assay was developed using a methionine auxotroph E. coli strain. The bacterial strain was first grown on rich media to promote extensive bacterial growth and the cells were depleted to exhaust all endogenous methionine. After depletion, cells were transferred to minimal media with increasing concentrations of methionine and microtiter plates were incubated at 37C for 6 h. Methionine microtiter standard curves yielded linear growth responses to increasing concentrations of methionine in the range of 0 to 26.8 μM. Addition of different antibiotic and antifungal agents to the media did not significantly alter the linear growth response observed in the microtiter assay. This microtiter plate E. coli methionine assay has potential as a rapid in vitro assay method for quantifying methionine.     

ASSESSMENT OF AN ESCHERICHIA COLI METHIONINE AUXOTROPH GROWTH ASSAY FOR QUANTIFYING CRYSTALLINE METHIONINE SUPPLEMENTED IN POULTRY FEEDS

Estimating availability of methionine is relevant to feed formulation since diets can be supplemented with crystalline methionine to meet the minimum requirements of rapidly growing birds. Bacterial assays have been developed to measure the bioavailable levels of several essential amino acids in feeds, including methionine. The E. coli methionine auxotroph strain used in this study exhibited a linear extent of growth response to increasing concentrations of methionine added to the minimal test media, in the range of 0 to 4 μg/mL. In addition the growth rates of the E. coli auxotroph were significantly (P < 0.01) different when the methionine concentrations were varied (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 μg/mL) in minimal media. To assay feeds, feed grade methionine was added to poultry feed mixtures and samples were diluted with M9 media. Using this assay for estimating crystalline methionine added to feed, the extent of growth of the methionine auxotroph was correlated with the levels of crystalline methionine supplemented in the feed (R²= 0.9873). For all supplementation levels methionine recovery percentages ranged from 71 to 80% indicating that the bacterial assay response to crystalline methionine was relatively constant in the presence of the feed matrix. The overall results indicate that the rapid detection of crystalline methionine added to feeds is possible using this E. coli methionine auxotroph growth-based assay.     

稚鳖蛋氨酸的营养需要量

将 90只中华稚鳖随机平均分为六组 ,分别饲喂含蛋氨酸 0 .82 %、1.12 %、1.32 %、1.5 2 %、1.72 %、2 .0 2 %的等能等氮半合成饵料 (胱氨酸为 0 .5 6 % )。结果表明 :蛋氨酸水平对特殊体重生长率 (SGR)、摄饵量 (FI)、饵料系数(FCR)、肝重 (LW)、肝重 /体重 (LW/BW)、体脂肪含量 (BFC)、体蛋氨酸含量 (BMC)、体蛋白沉积率 (PRR)、蛋氨酸沉积率 (MRR)有极显著或显著影响 (P <0 .0 1或P <0 .0 5 ) ,但是对体蛋白含量 (BPC)没有显著影响 (P >0 .0 5 )。在饵料胱氨酸为 0 .5 6 %时 ,稚鳖蛋氨酸需要量为 1.2 8% (风干日粮基础 )或 3.0 7% (蛋白基础 )。     

CONSTRUCTION AND GROWTH KINETICS OF A BIOLUMINESCENT METHIONINE AUXOTROPH ESCHERICHIA COLI STRAIN FOR POTENTIAL USE IN A METHIONINE BIOASSAY

Methionine is one of the essential and first limiting amino acids in animal nutrition. In this study, an Escherichia coli methionine auxotroph bacterial strain that exhibits a linear growth response to methionine concentrations was transformed with a plasmid containing genes encoding ampicillin resistance and bioluminescence in order to develop a microbiological technique for methionine quantitation. Transformants were selected based on antibiotic resistance and plasmid containing candidates were confirmed by restriction enzyme digestion and gel electrophoresis. To confirm the bioluminescent phenotype, video imaging of the strain using long exposure photography yielded colonies exhibiting bioluminescence. The strain was also tested in the presence of ampicillin supplemented media with increasing methionine concentrations and growth response (measured as optical density, OD), growth rates and methionine affinities were compared before and after transformation. Although the transformed E. coli methionine auxotroph exhibited somewhat different growth kinetic responses than the nontransformed strain, the standard curves used for estimating methionine concentrations were not different. Based on the results in this study the transformed bioluminescent strain could be used as an OD-based assay if bioluminescence equipment and materials are not available.     

蛋氨酸高产酵母突变株的推理筛选

以啤酒酵母（Saccharomycescerevisiae）菌株Ⅰ为出发株,经紫外线处理后,在含可完全抑制出发株生长的乙硫氨酸（浓度为2g/L）的基本培养从上筛出14株生长良好的突变株,其中I－2,I－9株还能在无硫基本培养基上良好生长。经氨基酸自动分析仪检测,I－2株菌体蛋白中的蛋氨酸含量（质量由分比）由I株的1．93增年9．28。     

红豆草抗甲硫氨酸变异体的筛选

用ＮａＮ３ 诱变过的红豆草（Ｏｎｏｂｒｙｃｈｉｓｖｉｃｉａｅｆｏｌｉａ Ｓｃｏｐ．）愈伤组织筛选得到了能抗８０ ｍ ｍ ｏｌ／Ｌ甲硫氨酸的变异细胞系——ＯＮｍ ｅｔｒ,并得到了再生植株。ＯＮｍ ｅｔｒ 脱离选择压力６ 个月后,其抗性仍比野生型的高５．６ 倍。同时还表现出对乙硫氨酸的交叉抗性,其抗性是野生型的６．５ 倍, 表明该抗性系抗性表达稳定。ＯＮｍ ｅｔｒ 愈伤组织中甲硫氨酸、赖氨酸、苏氨酸含量分别是野生型的４．００、１．０９、１．５０ 倍。ＯＮｍ ｅｔｒ再生植株中甲硫氨酸、赖氨酸、苏氨酸、异亮氨酸含量分别为野生型的２．０、３．５、３．５、２．５ 倍。在ＯＮｍ ｅｔｒ 愈伤组织可溶性蛋白ＳＤＳ－ＰＡＧＥ图谱中,出现了两条新多肽（３０ ｋＤ、２６ ｋＤ）。ＯＮｍ ｅｔｒ 细胞系过氧化物酶同工酶谱中亦出现了两条新酶带。这些变化表明该变异系已经产生变化了的基因产物     

ISOLATION OF NITROSOGUANIDINE-INDUCED CHLORELLA VULGARIS MUTANTS WITH HIGH METHIONINE CONTENT1

Mutants of Chlorella vulgaris induced by N-methyl-N′-nitro-N-nitrosoguanidine (NG), and, selected for the resistance to either ethionine or 6-methylpurine, were tested for the relative rate of incorporation into protein of ³H-methionine and ¹⁴C-leucine. A highly significant, correlation between the ³H-to-¹⁴C ratio in the protein and its methionine content was found. 6-Methylpurine proved to be more effective than ethionine as a screening agent for high methionine strains. Screening for 6-methylpurine resistance, followed by a second screening for the highest methionine-to-leucine incorporation ratio, led to isolation of the mutants with a content up to 45% higher in methionine and up to 3 times higher in cysteine with respect to the wild strain.     

POTENTIAL RAPID BIOASSAY FOR ALIMET® USING A METHIONINE ESCHERICHIA COLI AUXOTROPH

A potential rapid bioassay for methionine hydroxy analog (MHA) feed additive (ALIMET®) was examined using a methionine auxotroph E. coli strain. Bacterial cells were grown in minimal media containing a concentration range of 0 to 26.8 μM of either L-methionine or MHA as ALIMET®. Increasing either methionine or MHA concentration increased the growth rate of the methionine auxotroph. The estimated substrate affinities for methionine compared to MHA were not significantly different (P > 0.13) and the maximum growth rate estimates were also similar (P > 0.34). Methionine and MHA standard curves yielded linear responses (R²= 0.96) to increasing concentrations of the respective substrate. Based on these results it appears that the E. coli methionine auxotroph would have potential utility for further development of a rapid bioassay of ALIMET®.     

PHYSIOLOGICAL DEPENDENCE OF BRAIN METHIONINE AND S-ADENOSYLMETHIONINE CONCENTRATIONS ON SERUM AMINO ACID PATTERN

Abstract— Animals maintained on rat chow and water ad libitum in quarters illuminated for 12 h/day show diurnal rhythms in serum methionine and brain S -adenosylmethionine (SAM) concentrations. Brain methionine exhibits no such variation, nor does the ratio of serum methionine to the serum concentrations of six neutral amino acids which are believed to compete with methionine for uptake into brain. Administration of methionine to rats in doses that elevate serum methionine, but keep it within the daily physiological range, significantly increases brain concentrations of both methionine and SAM. The acute feeding of either a protein-free or a 40% casein meal also increases brain methionine and SAM, but does not affect serum methionine; however, both diets also increase the ratio of serum methionine to tyrosine, an amino acid whose postprandial concentration is indicative of the concentrations of the other amino acids that compete with methionine for transport into brain. These findings suggest that brain methionine levels increase physiologically after eating as a result of changes in the serum amino acid pattern. Furthermore, such naturally occurring increases in brain methionine appear to be associated with elevations in brain SAM.     

METHIONINE PROXIMITY ASSAY,A NOVEL METHOD FOR EXPLORING PEPTIDE LIGAND–RECEPTOR INTERACTION

ABSTRACT

Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms. Most studies of peptidergic GPCRs have been done with photolabeling peptides containing the benzophenone moiety as a reputedly non-selective probe. However our recent results are now showing that p-benzoylphenylalanine (Bpa) has some selectivity for Met residues in the receptor protein, reducing the accuracy of this method. Turning a problem into an asset, modified analogues of Bpa, e.g. p,?p″-nitrobenzoylphenylalanine (NO₂Bpa), display increased selectivity for such Met residues. It means a photoprobe containing such modified benzophenone-moieties does not label a receptor protein unless a Met residue is in the immediate vicinity. This unique property allows us to propose and show the feasibility and utility of a new method for scanning the contact areas of peptidergic GPCRs, the Methionine Proximity Assay (MPA). Putative contact residues of the receptor are exchanged to Met residues by site-directed mutagenesis and are subjected to photoaffinity labeling with such modified benzophenone-containing peptides. Successful incorporation indicates physical proximity of those residues. This principle is established and explored with benzophenone-containing analogues of angiotensin II and the two known human angiotensin II receptors AT₁ and AT₂, determining contact points in both receptors. This approach has several important advantages over other scanning approaches, e.g., the SCAM procedure, since the MPA-method can be used in the hydrophobic core of receptors.     

DISTRIBUTION OF THE MITOCHONDRIAL DEVIANT GENETIC CODE AUA FOR METHIONINE IN HETEROKONT ALGAE

The DNA sequence of the cytochrome oxidase subunit I ( COX I) gene (1059 bp), was determined in a number of heterokont algae, including five species of the Phaeophyceae [ Chorda filum (Linnaeus) Stackhouse, Colpomenia bullosa (Saunders) Yamada, Ectocarpus sp., Pseudochorda nagaii (Tokida) Inagaki, Undaria pinnatifida (Harvey) Suringar], and a member of the Raphidophyceae [ Chattonella antiqua (Hada) Ono]. The distribution of a deviant mitochondrial code, the AUA codon for methionine (AUA/Met), which was previously reported in the Xanthophyceae, was inferred from these COX I sequences. Comparative analyses of these sequences revealed that all the algae described above bear the universal genetic code, including the assignment for the AUA codon. A phylogenetic tree was constructed using the obtained sequences along with already-published COX I sequences of various heterokont algae. The clusters of the Xanthophyceae and the Phaeophyceae were resolved as sister groups with high bootstrap support, excluding a bacillariophycean species, a raphidophycean species, and three species of the Eustigmatophyceae. Taking the distribution of the deviant code and the COX I phylogenetic tree together, the genetic code change most probably occurred in an ancestor of the Xanthophyceae after it had branched off from the Phaeophyceae.     

EFFECTS OF METHIONINE AND S-ADENOSYLMETHIONINE ON PRODUCTION OF 1-METHYLADENINE IN STARFISH FOLLICLE CELLS

Incubation of follicle cells of the starfish ( Asterias amurensis ) with a gonad-stimulating hormonal peptide (GSS) leads to the production of 1-methyladenine. the trigger of oocyte maturation. Addition of L-methionine to the incubation medium promotes the production of 1-methyladenine. This study shows that S -adenosylmethionine also enhances the production of 1-methyladenine in such an incubation mixture. However, in the absence of GSS, addition of S -adenosylmethionine failed to produce an appreciable amount of 1-methyladenine. When an homogenate of isolated follicle cells was incubated, a certain amount of 1-methyladenine was produced, whether or not GSS was present. Addition of S -adenosylmethionine to the incubation mixture of follicle homogenate enhanced the production of 1-methyladenine. Although addition of adenosine triphosphate (ATP) to such an incubation mixture had little effect in producing 1-methyladenine, it exerted a promoting effect on 1-methyladenine production when S -adenosyl-methionine was present. These results suggest that methionine, through its active form, S -adenosylmethionine, acts as a donor of methyl group in the formation of 1-methyladenine.     

外源性蛋氨酸对鲤鱼离体消化率及酶解速度的影响

蛋氨酸属于含硫氨基酸,参与体内甲基转移、磷的代谢以及肾上腺素、胆碱和肌酸的合成,是鱼类饲料中典型的限制性氨基酸,大部分鱼类蛋氨酸的需要量为蛋白质的2%―3%。但是随着氨基酸平衡性最好的蛋白质原料—鱼粉价格大幅上涨,其他廉价蛋白源在饲料中大量使用,使得限制性氨基酸—蛋氨酸的缺乏更加严重。要使饲料蛋白质中的必需氨基酸组成趋于平衡,就要求我们能够有效的补充限制性氨基酸。现阶段,已经有越来越多的水产     

鹰嘴紫云英甲硫氨酸抗性系原生质体培养及植株再生

本研究建立了鹰嘴紫云英(AstragaluscicerL.)甲硫氨酸抗性系原生质体再生植株的实验体系。以茎切段诱导的松软愈伤组织为材料,通过酶法游离出大量有活力的原生质体。原生质体经培养持续细胞分裂形成了愈伤组织,并分化出再生苗。比较了不同培养基、培养密度对原生质体形成细胞分裂和再生的影响。结果表明,原生质体以2×105个/ml的植板密度,在附加2.0mg/L2,4-二氯苯氧乙酸(2,4-D)、0.2mg/L6-苄氨基嘌呤(6-BA)、200mg/L水解酪蛋白、2%蔗糖和0.3mol/L甘露醇DPD培养基中培养后,其分裂频率达38.3%。原生质体培养形成的愈伤组织仍具有对甲硫氨酸的抗性。转移到附加10mg/LKT、0.5mg/LNAA的MS分化培养基上,获得大量的再生苗。