大黄鱼稚鱼L-蛋氨酸需要量的研究

研究了1242日龄大黄鱼（Pseudosciaena crocea R.）稚鱼蛋氨酸需要量。以白鱼粉、磷虾粉和乌贼粉作蛋白源,通过添加L-晶体氨基酸使饲料与大黄鱼卵的必需氨基酸组成一致（蛋氨酸除外）,制成6种等氮（8.8%）等能（16.65 kJ/g）的微黏合饲料。L-蛋氨酸梯度依次为饲料的1.19%、1.62%、2.18%、2.65%、3.13%和3.66%,或饲料蛋白的2.17%、2.95%、3.95%、4.81%、5.70%和6.65%。以生物饵料（丰年虫无节幼体和桡足类）作对照组。每处理设3个重复,每桶（180 L）内随机放3500尾初始体重为（1.930.11） mg 的12日龄大黄鱼稚鱼。实验为期30d。结果显示,稚鱼的成活率随饲料蛋氨酸水平的升高而升高,在2.18%蛋氨酸水平时达到最高,之后无显著变化。特定生长率（SGR）随饲料蛋氨酸水平的升高而升高,在2.18%蛋氨酸水平时达到最高,之后则呈下降趋势。对照组稚鱼的成活率和SGR最高,均显著高于蛋氨酸组（P0.05）。各组间稚鱼体脂肪和灰分差异不显著,但体蛋白随饲料蛋氨酸水平的升高而升高,在2.65%蛋氨酸水平时达到最高,之后则稍微下降;对照组鱼体蛋白和各必需氨基酸含量均显著高于其他组（P0.05）。经二次多项式模型分析,1242日龄大黄鱼稚鱼的蛋氨酸需要量为饲料的2.58%或饲料蛋白的4.69%。       

The effect of different krill meals fed to laboratory rats on their blood indices.

1. In 102 laboratory rats fed with (a) the krill standardized meal, (b) the krill meal with low chitin content, (c) the casein diet with D,L-methionine, (d) the casein diet with D,L-methionine supplemented with the krill carapace meal, (e) the casein diet with D,L-methionine supplemented with ash from the krill standardized meal and (f) the control diet--"Murigran" standard pelleted feed; the different blood indices were investigated. 2. The mean values of following indices: the number of erythrocytes and leucocytes, the percentage of leucocytes, the corpuscular haemoglobin concentration and red blood cell diameter were similar in all experimental and control groups. 3. The mean values of haematocrit and haemoglobin levels, the mean corpuscular thickness and volume were lower in rats fed with the casein diet with D,L-methionine supplemented with the krill carapace meal than in other groups. 4. All kinds of investigated indices were similar in rats fed with krill meal with low chitin contents, whose parents received the standardized krill meal and no sex differences have been shown here.     

Taurine levels in blood and urine of vitamin B6-deficient and estrogen-treated rats

The objects of the present study were to determine whether a L-methionine or a L-cysteine test-load was more effective in evaluating the effects of estrogen on vitamin B6 deficiency in rats, and to determine whether urine or blood was the better test fluid to measure taurine levels. In animals fed a vitamin B6-supplemented diet, urinary excretion of taurine was twofold higher after administration of a L-cysteine test-load compared to a L-methionine test-load. Greater percentage reductions in the quantity of excreted taurine were observed in the vitamin B6-deficient rats when L-cysteine was utilized as the test-load than when L-methionine (2 mmole) was utilized. Treatment with estrogen demonstrated no significant differences in urinary taurine excretion. Vitamin B6 deficiency without estrogen treatment raised the levels of taurine in the blood of the animals treated with a L-methionine test-load. No changes in the blood taurine levels were observed in the animals administered a L-cysteine test-load.     

Studies on the earthy-musty odours in natural water (IV). Mechanism of earthy-musty odour production of actinomycetes

Many reservoirs for water supply have been troubled with earthy-musty odour compounds--2-metylisoborneol (2-MIB) and geosmin. Both of these compounds are terpenoid and related to the metabolite of L-methionine. An experiment using [CD3]methionine and [14CH3]methionine showed that the C-2 methyl group in 2-MIB originate from L-methionine. In the incubation experiment with 10-1000 mg/l of L-methionine, 2-MIB and geosmin appeared in the earlier stages, in greater amounts than in the control. The maximum production of 2-MIB and geosmin increased considerably in the experiments with 10 and 100 mg/l of L-methionine. The effective time for L-methionine addition was after 1 d. Additions after 3 and 5 d were similar to the control. In the incubation experiment with 10-1000 mg/l of folic acid, 2-MIB and geosmin increased only during the 1000 mg/l addition. There seems to be little doubt that L-methionine takes part in the metabolism of 2-MIB and geosmin.     

High protein diet in pregnancy. Effects on neonatal metabolism

The effect of a high protein diet (20% casein + D,L-methionine) administered to Wistar rats during pregnancy on some aspects of cellular growth and RNA metabolism of progeny has been studied. Comparisons were made with well-nourished (10% casein + D,L-methionine) controls. Newborns individual weight, litter weight and number of newborns per litter were unmodified. However, neonate protein content dropped significantly when compared with controls. Both rate of DNA and number of nuclei were unchanged. Protein/DNA ratio (cellular size relative to protein) decreased, which might have led to an atrophy phenomenon, even if the newborn weight/number of nuclei ratio was not modified. Acid DNase activity rose, bringing about DNA breakdown. Total RNA content together with RNase activity fell in newborn from rats suffering high protein diet. Moreover, protein synthesis capacity (RNA/protein ratio) did not change. These results suggest that the administration of a high protein diet to pregnant rats lead to changes in newborn protein rate and nucleic acid turnover by modulating specific nuclease activity.     

Depletion of delta 9 desaturase (EC 1.14.99.5) activity in lactating rat during protein restriction

The effects of protein restriction on the activity of delta9 desaturase (EC 1.14.99.5) were investigated in lactating rats. A control group was fed a balanced diet (20% casein) for 14 days, whereas the experimental groups were fed a low-protein diet (8% casein), supplemented with or without L-methionine (0.4%), for 14 days. The enzyme activity was measured by incubations of hepatic microsomal pellets with (1-14C) stearic acid. Results showed a decreased delta9 desaturase activity, after 2,7 and 14 days of depleted diet, of -50, -40 and -33% respectively, compared with control. The supplementation of the low-protein diet with 0.4% methionine, which favours food consumption as well as growth, did not improve the altered delta9 desaturase activity. Our data evidenced that delta9 desaturase activity is depleted by protein restriction during lactation, when the protein needs are high for the biosynthesis of animal tissues. This change has to be considered as a sign of depressed delta9 desaturase biosynthesis or modifications of enzymatic properties, or both.     

Brain protein synthesis in awake rats: equilibrium of L-methionine between plasma and direct precursor pool

After 1 hr. of continuous infusion of L-(35S)methionine the specific activities of L-methionine in plasma and tissue-free and tRNA-bound L-methionine in brain were in the same range. This result indicates that, under steady-state conditions, dilution of the precursor pool for protein synthesis (L-methionyl-tRNA) by L-methionine derived from a source other than plasma can be considered as negligible.     

Transcriptional analysis of L-methionine catabolism in Brevibacterium linens ATCC9175

The expression of genes possibly involved in L-methionine and lactate catabolic pathways were performed in Brevibacterium linens (ATCC9175) in the presence or absence of added L-methionine. The expression of 27 genes of 39 selected genes differed significantly in L-methionine-enriched cultures. The expression of the gene encoding L-methionine gamma-lyase (MGL) is high in L-methionine-enriched cultures and is accompanied by a dramatic increase in volatile sulfur compounds (VSC) biosynthesis. Several genes encoding alpha-ketoacid dehydrogenase and one gene encoding an acetolactate synthase were also up-regulated by L-methionine, and are probably involved in the catabolism of alpha-ketobutyrate, the primary degradation product of L-methionine to methanethiol. Gene expression profiles together with biochemical data were used to propose catabolic pathways for L-methionine in B. linens and their possible regulation by L-methionine.     

Changes in the metabolism of muscle RNA in rats given a high protein diet

The effect of a high protein diet (20% casein + D,L-methionine) administered to adult Wistar rats on some aspects of muscle RNA metabolism has been studied. Body weight increased in spite of lower intake. However, gastrocnemius muscle remained unmodified, although protein content increased. Total RNA decreased in the whole muscle although RNA/DNA ratio did not change. Protein synthesis capacity diminished 81% relative to controls in spite the fact that an excessive amount of available amino acids exists. RNA loss might depend on a high catabolism, since acid RNase activity increased over control values. Therefore, it may be concluded that a high protein diet leads to a lower protein synthesis capacity through an elevated RNA breakdown.     

Identification and functional analysis of the gene encoding methionine-gamma-lyase in Brevibacterium linens

The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-methionine-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B. linens.     

L-蛋氨酸及甲醇浓度对毕赤酵母摇瓶发酵生产S-腺苷蛋氨酸的影响

利用甲醇传感器及高效液相色谱检测毕赤酵母摇瓶发酵过程的甲醇浓度及S-腺苷蛋氨酸(SAM)浓度,发现L-蛋氨酸浓度及甲醇浓度对毕赤酵母细胞生长及合成S-腺苷蛋氨酸具有影响,据此对摇瓶发酵过程的L-蛋氨酸浓度及甲醇浓度进行优化。优化结果表明:当L-蛋氨酸浓度为7.5 g/L时,最适于SAM积累,产量达到0.83 g/L;进而利用甲醇传感器对发酵过程的甲醇浓度进行检测及控制,考察不同甲醇浓度对SAM产量的影响,毕赤酵母产SAM的最佳甲醇浓度为15 g/L,在此浓度下SAM的产量达到1.41 g/L,比对照实验增加了21%。     

The physiology of L-methionine catabolism to the secondary metabolite ethylene by Escherichia coli

Catabolism of L-methionine by Escherichia coli strain B SPAO led to the formation of ethylene as a secondary metabolite (ethylenogenesis). Methionine was initially deaminated by a transamination reaction to the 2-oxo acid 2-oxo-4-methylthiobutyric acid (KMBA) which was then converted to ethylene. The utilization of L-methionine as an additional nitrogen source was investigated by examining ethylene synthesis under different nitrogen supply conditions. Ethylene formation in batch culture was unaffected by the concentration of the precursor L-methionine in the medium although increasing concentrations of NH4Cl resulted in progressively less ethylene formation. Cultures grown without L-methionine did not produce ethylene but were able to synthesize ethylene when L-methionine or KMBA was provided. Addition of L-tyrosine to batch cultures reduced the yield of ethylene after 42 h by 54%. Under these conditions the maximum transient level of KMBA was reduced by 32% and occurred later compared to when L-methionine was the only amino acid supplement. Continuous cultures grown under ammonia limitation produced both ethylene and KMBA. In contrast, when glucose was limiting, neither of these metabolites were produced. Cells harvested from continuous cultures grown under glucose or ammonia limitation were able to synthesize ethylene from either L-methionine or KMBA although their capacity for ethylene synthesis (ethylenogenic capacity) was optimal under ammonia limitation (C:N ratio = 20).     

补加前体L-蛋氨酸对高密度发酵生产S-腺苷-L-蛋氨酸的影响

将高密度发酵技术成功应用于S-腺苷-L-蛋氨酸的生产。考察了补加前体L-蛋氨酸的量以及补加策略对酿酒酵母G14发酵生产S-腺苷-L-蛋氨酸的影响。实验发现补加前体L-蛋氨酸能明显促进S-腺苷-L-蛋氨酸的积累。同时还发现不同的补加策略对菌体浓度以及S-腺苷-L-蛋氨酸的产量和浓度有不同的影响。确定了补加L-蛋氨酸不应低于0.7g/10g菌体干重。比较了五种不同的补加前体L-蛋氨酸的方式。结果表明在菌体干重达到高密度的情况下(120g/L)补加前体L-蛋氨酸进行转化生产S-腺苷-L-蛋氨酸能达到比较好的效果一次性补加9g L-蛋氨酸,SAM的积累量在补加后的18h达到最高,为4.31g/L;采取流加方式补加L-蛋氨酸,流加速率为2g/h,共流加5h,流加结束28h后SAM达到最高积累量后者达到4.98g/L。两者最终的生物量均可达到130g/L以上。     

Comparison of 13C NMR chemical shifts of 2-aminovaleric acid,L-methionine and DL-selenomethionine and their interactions with aurothiomalate in aqueous solution

The¹³C NMR chemical shifts of DL-selenomethionine were measured and compared with L-methionine and 2-aminovaleric acid in neutral and basic aqueous solutions. The Cγ and Cδ carbons which are directly attached to the sulphur atom of L-methionine experience a shielding effect compared to the Cγ and Cδ of 2-amonivaleric acid resonances. However, shielding effects were observed on Cγ and Cδ resonances when S was substituted by Se, i.e., on going from L-methionine to DL-selenomethionine. The interaction of L-methionine and DL-selenomethionine with aurothiomalate was also studied. The results show that L-methionine does not bind to gold(I) at any pH. However, there is a weak binding observed with DL-selenomethionine in basic aqueous solutions, as seen by ¹³C NMR spectroscopy.     

Cell growth of skeletal muscle as an indicator of protein requirements of the adult rat

Skeletal muscle growth, muscle nucleic acids and muscle protein synthesis capacity, were measured to evaluate the protein requirement of adult rats. Wistar rats were fed on diets containing 4%, 10% or 20% casein + D,L-methionine. All diets were provided for 21 days beginning at 90 days of age. Body weight, food efficiency and net weight change increased as the casein content of the diet increased. Muscle DNA, RNA and RNA/protein were lost, but protein and protein/DNA increased on the 4% and 20% protein diet. This fact involves an aplasia phenomenon although the hypertrophic growth is maintained. Alterations of the insulin and GH plasma levels were observed. These findings indicate that for adult rats the 4% and 20% protein diets are not adequate for the period of adult maintenance.     

Is L-methionine a trigger factor for Alzheimer’s-like neurodegeneration?: Changes in Aβ oligomers, <Emphasis Type="Italic">tau</Emphasis> phosphorylation,synaptic proteins, <Emphasis Type="Italic">Wnt</Emphasis> signaling and behavioral impairment in wild-type mice

Background

L-methionine, the principal sulfur-containing amino acid in proteins, plays critical roles in cell physiology as an antioxidant and in the breakdown of fats and heavy metals. Previous studies suggesting the use of L-methionine as a treatment for depression and other diseases indicate that it might also improve memory and propose a role in brain function. However, some evidence indicates that an excess of methionine can be harmful and can increase the risk of developing Type-2 diabetes, heart diseases, certain types of cancer, brain alterations such as schizophrenia, and memory impairment.

Results

Here, we report the effects of an L-methionine-enriched diet in wild-type mice and emphasize changes in brain structure and function. The animals in our studypresented 1) higher levels of phosphorylated tau protein, 2) increased levels of amyloid-β (Aβ)-peptides, including the formation of Aβ oligomers, 3) increased levels of inflammatory response,4) increased oxidative stress, 5) decreased level of synaptic proteins, and 6) memory impairment and loss. We also observed dysfunction of the Wnt signaling pathway.

Conclusion

Taken together, the results of our study indicate that an L-methionine-enriched diet causes neurotoxic effects in vivo and might contribute to the appearance of Alzheimer’s-like neurodegeneration.

     

Ornithine decarboxylase activity is inhibited by the polyamine precursor amino acids at the protein stability level in Caco-2 cells

High concentrations of certain amino acids are known to affect hormonal secretion, immune function, electrolyte balance or metabolic functions. However, there is a lack of knowledge regarding the molecular mechanisms responsible for these effects. We showed that, as well as spermidine transport, the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis, is decreased in human colon adenocarcinoma cells, Caco-2, following a 4-h supplementation with one of the two polyamine precursor amino acids, L-arginine or L-methionine. Dose-response assays indicated that the inhibitory effect of supplemental L-methionine was stronger than that of supplemental L-arginine. However, it was transient, being even replaced by ODC induction after 8 h, whereas the inhibitory effect of L-arginine lasted for at least 8 h. Unlike L-cysteine, neither L-methionine nor L-arginine could inhibit ODC activity in a crude acellular preparation of the enzyme. The inhibition of ODC activity in cells exposed to L-methionine or L-arginine was due to a decreased abundance of ODC protein without change at the mRNA level and each of these amino acids could counteract ODC induction by a glycine supplement. Contrary to the latter, supplemental L-methionine or L-arginine induced a marked decrease in ODC half-life, concomitantly with an increase in the activity of antizyme, an ODC inhibitory protein. Thus, depending on their nature, amino acids can up- or downregulate ODC activity at the protein stability level.     

New polar constituents in the pupae of the silkworm Bombyx mori L. (II): developmental changes of the constituents

A correlation between the quantitative changes in L-methionine analogs, the ratio of D-serine/L-serine during the pupal stage, and metamorphosis was observed. The glycoside appearing at low blood sugar values during the pupal stage was isolated and characterized as D-glucosyl-L-tyrosine. (1)H-NMR indicated the appearance and increase of this glycoside, and Mirrorcle Ray CV4 equipment was used to take X-ray pictures of the pupal bodies. The results indicate that γ-cyclic di-L-glutamate and L-methionine sulfone might be concerned with ammonia assimilation in the pupae, and that D-glucosyl-L-tyrosine served as a switch for the fatty acid (pupal oil) dissimilation hybrid system.     

Assay method for antitumor L-methionine gamma-lyase: comprehensive kinetic analysis of the complex reaction with L-methionine

L-Methionine gamma-lyase (EC 4.4.1.11) is a pyridoxal 5'-phosphate-dependent multifunctional enzyme. Measuring the initial velocity of alpha-ketobutyrate production by alpha,gamma-elimination of L-methionine catalyzed by L-methionine gamma-lyase is not very feasible, because the enzyme simultaneously catalyzes both gamma-replacement and alpha,gamma-elimination. To develop an accurate enzyme assay, the comprehensive enzyme kinetics needed to be elucidated by progress curve analysis on the basis of a reaction model for conversion of L-methionine to alpha-ketobutyrate, methanethiol, and ammonia with pyridoxal 5'-phosphate as a cofactor. Kinetic parameters were determined by linear transformation using an approximation of a Maclaurin series from the whole velocity of alpha-ketobutyrate production including alpha,gamma-elimination and gamma-replacement. The significance of gamma-replacement was revealed both theoretically and practically by the kinetic analysis. The enzyme activity was standardized and represented as the Vmax value taking into consideration gamma-replacement in the presence of L-methionine at 37 degrees C and pH 8.0. The novel method that we proposed is accurate, sensitive, reproducible, and linear over a wide range for the determination of L-methionine gamma-lyase activity.