家蚕体内维生素B6的存在形态和转换代谢

采用不含桑叶粉末、以去维生素牛乳酪蛋白为蛋白源的准合成饲料饲育家蚕Bombyx mori幼虫,探讨了家蚕体内维生素B（VB₆）化合物的存在形态和转换代谢途经。随饲料中盐酸吡哆醇（PN-HCl）添加量的增加,幼虫体内吡哆醇(PN)含量相应变化,其次是吡哆醛(PL);而辅酶型磷酸吡哆醛(PLP)和磷酸吡哆胺(PMP)含量存在稳定性。饲料中的吡哆醇以单纯扩散的形式进入体液;体液中的吡哆醇被各种组织吸收后,在各自的吡哆醛激酶和PNP/磷酸吡哆胺氧化酶的作用下,转变成辅酶型磷酸吡哆醛。家蚕不同于哺乳动物,没有特定的辅酶型磷酸吡哆醛形成组织和辅酶型磷酸吡哆醛的转送机制。同时家蚕体内缺乏具储存VB₆功能的辅酶型磷酸吡哆醛结合蛋白,推测这是用缺乏VB₆的饲料饲育各龄起蚕,幼虫当龄死亡的主要原因。     

苯酚对多刺裸腹溞糖及蛋白质代谢酶的影响

采用急性毒性方法,研究了苯酚对多刺裸腹溞(Moina macrocopa)糖及蛋白质代谢的影响。实验设对照组和5个苯酚处理组,苯酚浓度分别为0.25、0.75、1.25、1.75、2.25 mg/L。分别在苯酚处理24 h和48 h后用分光光度法测定丙酮酸激酶(PK)、乳酸脱氢酶(LDH)、琥珀酸脱氢酶(SDH)、谷草转氨酶(GOT)和谷丙转氨酶(GPT)的活力。结果显示,随着苯酚浓度的增加和处理时间的延长,LDH活力呈现升高趋势,PK和SDH活力与对照组相比没有显著性差异。GOT活力在处理24 h后先升后降,处理48 h后活力降低;GPT活力在处理24 h、48 h后活力均升高。结论是:苯酚中毒需要高水平的代谢能量,导致裸腹溞代谢发生重大改变,诱导能量代谢一定程度上由碳水化合物分解代谢向蛋白分解代谢转变。     

支链氨基酸及代谢中间产物对胰岛素抵抗的调控及其机制

支链氨基酸作为必需氨基酸,可用于合成含氮化合物,也可充当信号分子调节物质代谢。研究表明,支链氨基酸水平升高与胰岛素抵抗和2型糖尿病发生密切相关,其可通过激活哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路来影响胰岛素信号转导,还可通过损害脂质代谢和影响线粒体功能来调控胰岛素抵抗。此外,支链氨基酸分解代谢异常会导致代谢中间产物(如支链α-酮酸、3-羟基异丁酸和β-氨基异丁酸等)积累,其中支链α-酮酸和3-羟基异丁酸可通过影响胰岛素信号通路、损害脂质代谢等来诱发胰岛素抵抗,而β-氨基异丁酸可通过减少脂质积聚和炎症反应、增强脂肪酸氧化等来改善胰岛素抵抗。本文系统综述了支链氨基酸及其代谢中间产物对胰岛素抵抗的影响及调控机制,以期为胰岛素抵抗和2型糖尿病的防治提供新方向。     

紫薇响应盐胁迫和碱胁迫的代谢组分析

为探究‘红叶’紫薇(Lagerstroemia indica ‘Pink Velour’)对盐胁迫和碱胁迫的代谢响应机制,对‘红叶’紫薇1年生无性扦插苗分别进行盐胁迫(NaCl,pH=7.02)和碱胁迫(NaHCO₃和Na₂CO₃的Na⁺摩尔比为2:1,pH=9.52)处理,采用液相色谱质谱(LC-MS)分析叶片代谢组学变化,并比较2个处理组与对照(CK)之间的代谢差异。结果表明：与CK相比,盐处理组共筛选出156个差异代谢物,碱处理组共筛选出176个差异代谢物,2个对比组共有23个差异代谢物,其余均为特有差异代谢物。KEGG富集分析表明,次生代谢产物生物合成、氨基酸代谢、糖类代谢、脂肪酸代谢和植物激素合成是响应盐胁迫和碱胁迫的主要代谢通路;甘氨酸、丝氨酸和苏氨酸合成代谢、ABC转运蛋白和维生素B₆合成代谢是盐胁迫组的特有代谢通路;缬氨酸、亮氨酸、异亮氨酸、精氨酸和脯氨酸生物合成代谢、叶酸合成代谢,以及戊糖和葡萄糖醛酸相互转化和抗坏血酸合成代谢是碱胁迫组的特有代谢通路。这些差异...     

球形芽孢杆菌对致倦库蚊的后致死作用

研究了球形芽孢杆菌Bacillus sphaericus C3-41菌株对致倦库蚊Culex quinquefasciatus幼虫的毒力及其后致死作用。生物测定表明,该菌株对目标蚊幼虫具有很高的毒力,其丙酮粉剂对3～4龄幼虫48 h的半致死浓度(LC₅₀)为(6.92±0.22) μg/L。用不同亚致死浓度处理2～3龄致倦库蚊幼虫,存活幼虫在后期发育中存在明显的延续死亡和损伤现象,经LC₃₀、LC₅₀、LC₇₀、LC₉₀和LC₉₈剂量的C3.41粉剂处理的致倦库蚊羽化前的总死亡率分别为84%、91%、95%、97%和100%,同时存活的幼虫、蛹和成蚊的发育和行为也受到一定的影响。这种后致死作用随处理浓度的升高而增强,可能同球形芽孢杆菌毒素蛋白对处理期间蚊幼虫中肠上皮细胞造成的损伤相关。     

食物中多不饱和脂肪酸在家蝇幼虫体内的富集与代谢及对其生长的影响

【目的】阐明家蝇 Musca domestica 幼虫对食物中各种多不饱和脂肪酸的富集能力以及代谢转化情况,并探究各种多不饱和脂肪酸对家蝇幼虫生长的影响。【方法】在基础饲料中添加不同浓度(3%, 6%和12%)的多不饱和脂肪酸(亚油酸、α-亚麻酸、花生四烯酸和二十二碳六烯酸)饲养经过脱脂传代培养的家蝇幼虫;提取家蝇幼虫的总脂肪酸,利用气相色谱仪进行检测和分析;测定统计幼虫体重,以分析多不饱和脂肪酸对家蝇幼虫生长的影响。【结果】亚油酸、α-亚麻酸和花生四烯酸在家蝇幼虫体内均能被富集,且它们的富集程度随着食物中多不饱和脂肪酸的添加浓度的升高而增加,其中亚油酸、α-亚麻酸和花生四烯酸在幼虫体内富集的最高含量(占体内总脂肪酸的比例)分别为21.93%, 16.13%和9.68%,而二十二碳六烯酸不能在家蝇幼虫体内富集,提示家蝇幼虫食物中添加的各种多不饱和脂肪酸经过代谢后并没有在其体内产生新的脂肪酸,而食物中添加的二十二碳六烯酸在家蝇幼虫体内被分解代谢后消除。饲喂α-亚麻酸及花生四烯酸后家蝇幼虫体重增长较为明显,其中6%α-亚麻酸添加组的幼虫体重显著高于对照组(取食脱脂饲料)和3%和12%α-亚麻酸添加组,3%和6%花生四烯酸添加组的幼虫体重显著高于对照组和12%花生四烯酸添加组。【结论】家蝇幼虫体内能够从食物中富集部分多不饱和脂肪酸,多不饱和脂肪酸碳链越长其富集程度越低直至不能富集,富集的多不饱和脂肪酸对家蝇幼虫生长有不同程度的影响。     

长爪沙鼠冷驯化过程中甲状腺激素的变化

22℃室温中的长爪沙鼠血清T₃和T₄浓度较低,肝脏和褐色脂肪组织（BAT）的T₄5'-脱碘酶活力也较低。冷暴露（4℃）1d 后,血清T₃与T₄浓度迅猛增长,随后1-4周的冷驯化中,T₃缓慢上升,T₄逐渐下降,肝脏和BAT 的T₄5'-脱碘酶活力变化与血清T₃和T₃\T₄变化一致。表明冷驯化激活了甲状腺功能,激活了外周组织中的甲状腺激素代谢。     

家蚕吡哆醛激酶基因定点突变及突变体功能

【目的】吡哆醛激酶（pyridoxal kinase,PLK）是维生素B₆关键代谢酶。前期研究克隆出家蚕Bombyx mori吡哆醛激酶cDNA,经序列比对发现几个重要且保守的氨基酸残基在此蛋白中被替换。为明确家蚕吡哆醛激酶分子若干特定位置上氨基酸残基在酶功能上的作用进行本研究。【方法】采用重叠延伸法对家蚕吡哆醛激酶Thr⁴⁷,Asn¹²¹, Ile⁵⁴, Arg⁸⁸和Trp²³⁰氨基酸残基进行定点突变, 构建表达载体pET-22b（+）-PLK并转入大肠杆菌Escherichia coli Rosetta中进行诱导表达,经亲和层析对重组蛋白进行纯化, 通过酶活性检测进行功能鉴定。【结果】家蚕吡哆醛激酶Thr⁴⁷,Ile⁵⁴和Arg⁸⁸氨基酸突变后酶的催化活力分别下降82%, 58%和85%;Asn¹²¹突变对酶的催化活力几乎没有影响;而Trp²³⁰突变导致酶丧失催化活性。【结论】本研究明确了选定氨基酸侧链基团在家蚕吡哆醛激酶催化功能上的意义。     

饥饿胁迫对多刺裸腹溞糖与蛋白质代谢的影响

研究了饥饿24 h和48 h对多刺裸腹溞Moina macrocopa糖和蛋白质代谢中间代谢产物以及主要代谢酶的影响.结果显示,饥饿能够显著影响多刺裸腹溞机体内葡萄糖、丙酮酸、总町溶性蛋白和总氨基酸含量;同时对琥珀酸脱氢酶(SDH)、丙酮酸激酶(PK)、谷丙转氨酶(GPT)和谷草转氨酶(GOT)的活力也有不同程度的影响.饥饿24 h和48 h,葡萄糖含量分别降低了32.17%和37.55%;丙酮酸含量分别降低了46.04%和38.53%,与对照组相比.发生了显著性变化(P<0.05).饥饿24 h可溶性蛋白含量降低了32.14%(P<0.05);饥饿24 h和48 h总氨基酸含量呈极显著下降趋势(P<0.01),分别下降了61.47%和65.38%.实验证明,饥饿胁迫可诱导SDH活力增加,48 h时活力极显著升高(P<0.01);而PK活力变化不显著(P>0.05);GPT和GOT两种转氨酶活性在饥饿胁迫下呈上升趋势,但是与对照组相比无显著性差异(P>0.05).     

杆状病毒的垂直传播及绿色荧光蛋白在棉铃虫幼虫中的表达

用转座穿梭系统构建了携带绿色荧光蛋白基因（gfp）的重组棉铃虫核型多角体病毒rHa-FGP,以其多角体添食感染棉铃虫3龄幼虫,室内饲养3代,各代均可见自然光下发绿色荧光的棉铃虫幼虫,其中子代不再重复感染。F₀、F₁、F₂代发绿色荧光的棉铃虫幼虫所占百分比分别为34%、20%、8%。提取虫体内的病毒多角体DNA,以PCR和斑点杂交鉴定表明,gfp不仅在亲代棉铃虫体内正常表达,而且在子代幼虫中表达,HaNPV通过卵实现了垂直传播。     

Catabolism of leucine to branched-chain fatty acids in Staphylococcus xylosus

AIMS: Staphylococcus xylosus is an important starter culture in the production of flavours from the branched-chain amino acids leucine, valine and isoleucine in fermented meat products. The sensorially most important flavour compounds are the branched-chain aldehydes and acids derived from the corresponding amino acids and this paper intends to perspectivate these flavour compounds in the context of leucine metabolism. METHODS AND RESULTS: GC and GC/MS analysis combined with stable isotope labelling was used to study leucine catabolism. This amino acid together with valine and isoleucine was used as precursors for the production of branched-chain fatty acids for cell membrane biosynthesis during growth. A 83.3% of the cellular fatty acids were branched. The dominating fatty acid was anteiso-C(15:0) that constituted 55% of the fatty acids. A pyridoxal 5'-phosphate and alpha-ketoacid dependent reaction catalysed the deamination of leucine, valine and isoleucine into their corresponding alpha-ketoacids. As alpha-amino group acceptor alpha-keto-beta-methylvaleric acid and alpha-ketoisovaleric acid was much more efficient than alpha-ketoglutarate. The sensorially and metabolic key intermediate on the pathway to the branched-chain fatty acids, 3-methylbutanoic acid was produced from leucine at the onset of the stationary growth phase and then, when the growth medium became scarce in leucine, from the oxidation of glucose via pyruvate. CONCLUSIONS: This paper demonstrates that the sensorially important branched-chain aldehydes and acids are important intermediates on the metabolic route leading to branched-chain fatty acids for cell membrane biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: The metabolic information obtained is extremely important in connection with a future biotechnological design of starter cultures for production of fermented meat.     

Use of the flux model of amino acid metabolism of Escherichia coli

A program implementing a flux model of Escherichia coli metabolism was used to analyze the effects of the addition of amino acids (tryptophan, tyrosine, phenylalanine, leucine, isoleucine, valine, histidine, lysine, threonine, cysteine, methionine, arginine, proline) to minimal medium or media lacking nitrogen, carbon, or both. The overall response of the metabolic system to the addition of various amino acids to the minimal medium is similar. Glycolysis and the synthesis of pyruvate with its subsequent degradation to acetate via acetyl-CoA become more efficient, whereas the fluxes through the pentose phosphate pathway and the TCA cycle decrease. If amino acids are used as the sole source of carbon, nitrogen, or both, the changes in the flux distribution are determined mainly by the carbon limitation. The phosphoenolpyruvate to glucose-6-phosphate flux increases; the flux through the pentose phosphate path is directed towards ribulose-5-phosphate. Other changes are determined by the compounds that are the primary products of catabolism of the added amino acid.     

The dissimilation of leucine, isoleucine and valine to volatile fatty acids by adult Fasciola hepatica

The dissimilation of leucine, isoleucine and valine to volatile fatty acids was determined in Fasciola hepatica and the degradation of (U−¹⁴C) branched amino acids to the volatile fatty acids end products demonstrated. F. hepatica was found to metabolize leucine, isoleucine and valine to isovaleric, 2-methylbutyric and isobutyric acid respectively. The rate of formation of isobutyrate, isovalerate and 2-methylbutyrate was found to be positively related to the rate of propionic acid production with air or nitrogen as the gas phase. However, under 95% O₂/5% CO₂ the formation of the branched chain acids was independent of propionic acid production. The production of isobutyrate, isovalerate and 2-methylbutyrate caused a simultaneous reduction in the rate of acetate formation. The role of propionate formation in regulating metabolism is discussed.     

Fatty acid synthesis from amino acids in sheep adipose tissue

The rates of incorporation of 14C from 14C labelled acetate, glucose, alanine, leucine, isoleucine and valine into fatty acids has been measured in perirenal adipose tissue from foetal lambs and 8-month-old sheep, and into both fatty acids and acylglycerol glycerol in adipose tissue from 3-year-old sheep and 220-240 g female rats. Rates of incorporation of 14C from amino acids into fatty acids were much lower in adipose tissue from sheep (at all three ages) than from rats, whereas rates of incorporation of 14C into acylglycerol glycerol were either greater in sheep adipose tissue or the same as in rat adipose tissue. The rate of incorporation of 14C from amino acids into fatty acids decreased in the order leucine greater than alanine greater than isoleucine greater than valine in adipose tissue from rats and foetal lambs, and in the order leucine greater than alanine = isoleucine greater than valine in adipose tissue from 8-month- and 3-year-old sheep. Amino acids make a very small contribution to fatty acid synthesis in adipose tissue from sheep at all stages of development examined while fatty acids are a minor product of amino acid metabolism in sheep adipose tissue. The study provides further evidence for an important role for ATP-citrate lyase in restricting the utilization of acetyl-CoA generated in the mitochondria for fatty acid synthesis.     

Branched-chain amino acid metabolism controls membrane phospholipid structure in Staphylococcus aureus

Branched-chain amino acids (primarily isoleucine) are important regulators of virulence and are converted to precursor molecules used to initiate fatty acid synthesis in Staphylococcus aureus. Defining how bacteria control their membrane phospholipid composition is key to understanding their adaptation to different environments. Here, we used mass tracing experiments to show that extracellular isoleucine is preferentially metabolized by the branched-chain ketoacid dehydrogenase complex, in contrast to valine, which is not efficiently converted to isobutyryl-CoA. This selectivity creates a ratio of anteiso:iso C₅-CoAs that matches the anteiso:iso ratio in membrane phospholipids, indicating indiscriminate utilization of these precursors by the initiation condensing enzyme FabH. Lipidomics analysis showed that removal of isoleucine and leucine from the medium led to the replacement of phospholipid molecular species containing anteiso/iso 17- and 19-carbon fatty acids with 18- and 20-carbon straight-chain fatty acids. This compositional change is driven by an increase in the acetyl-CoA:C₅-CoA ratio, enhancing the utilization of acetyl-CoA by FabH. The acyl carrier protein (ACP) pool normally consists of odd carbon acyl-ACP intermediates, but when branched-chain amino acids are absent from the environment, there was a large increase in even carbon acyl-ACP pathway intermediates. The high substrate selectivity of PlsC ensures that, in the presence or the absence of extracellular Ile/Leu, the 2-position is occupied by a branched-chain 15-carbon fatty acid. These metabolomic measurements show how the metabolism of isoleucine and leucine, rather than the selectivity of FabH, control the structure of membrane phospholipids.     

Some Effects of Protein Deficiency in Young Growing Pigs. II. Blood Plasma Free Amino Acids

The influence of protein deficiency, rehabilitation and total starvation on the free amino acid levels in the blood plasma of pigs has been investigated. It was found that the concentration of most amino acids was reduced during protein deficiency. The levels of leucine, isoleucine and valine were diminished by the greatest proportion, followed by threonine, tyrosine and citrulline. During the first few weeks of protein deficiency the levels of lysine, histidine and arginine were slightly increased, but later decreased below control values. Concentrations of glycine and alanine were altered in a similar way except that the initial increase was much more pronounced. The concentrations of most of these amino acids returned to control levels after rehabilitation. Total starvation led to an increase in concentration of leucine, isoleucine, valine, threonine and to a smaller extent phenylalanine, lysine, citrulline and arginine. The concentration of glycine, alanine and glutamic acid were very much reduced. The level of urea in the circulation dropped reversibly during protein deficiency and increased very much during total starvation.     

Effects of amino acids on Thiobacillus acidophilus. I. Growth studies with special reference to valine.

The heterotrophic growth of Thiobacillus acidophilus was inhibited by branched-chain amino acids; valine, isoleucine, and leucine. The inhibition by valine and leucine were partially reversed by isoleucine, and the inhibition by isoleucine was partially reversed by valine. Inhibitions by methionine or threonine were partially reversed when both amino acids were present in the growth medium. Inhibition by tyrosine was increased by phenylalanine or tryptophan. Cystine completely inhibited growth. Other amino acids tested produced little or no inhibition. Acetohydroxy acid synthetase (AHAS) activity was demonstrated in crude extracts of T. acidophilus. In crude extracts the optimum pH was 8.5 with a shift to 9.0 in the presence of valine. Valine was the only branched-chain amino acid which inhibited the AHAS activity. The presence of only one peak of AHAS activity upon centrifugation in linear glycerol density gradients demonstrated that the AHAS activity sediments as one component.     

Quantitative analysis of free amino acids and carbohydrates in spring barley anthers at different stages of maturity

The content of the carbohydrates glucose, fructose and sucrose was determined in spring barley anthers at different stages of maturity. During maturation the sucrose content of the anthers increased markedly. The following 17 free amino acids were detected in anthers of different stages of maturity: aspartic acid, glutamic acid, serine, alanine, arginine, leucine, isoleucine, lysine, α-aminobutyric acid, glutamine, proline, tyrosine, phenylalanine, valine, threonine, cystine and glycine. Quantitative analysis was only carried out in amino acids present in higher concentrations in the analysed samples. These were: aspartic acid, glutamic acid, α-aminobutyric acid, proline, serine, valine and glutamine, and a mixture of amino acids (leucine, isoleucine, valine and phenylalanine). The total content of free amino acids increased with increasing maturity of the anthers. However, not all amino acids followed contributed to this increase, but only proline, glutamic acid, aspartic acid and glutamine. A small difference was found in the variety Gopal in which the aspartic acid content did not increase significantly, but the content of the mixture of amino acids and serine did. With the exception of green anthers of the variety Firlbecks Union, proline was present in the highest concentration in all samples analysed.     

Maple syrup urine disease: Branched-chain amino acid concentrations and metabolism in cultured human lymphoblasts

The intracellular concentration of free leucine, isoleucine, and valine and their metabolism were studied in lymphoblast cultures established from peripheral blood of an individual with maple syrup urine disease (MSUD) and a control subject. Branched-chain -keto acid decarboxylase activity in the MSUD cells was 10% or less of the control value as measured by the ability of the cells to release ¹⁴CO₂ from the corresponding [1-¹⁴C]labeled branched-chain amino acid. The intracellular concentrations of free leucine and isoleucine were increased three-fold in MSUD lymphoblasts as compared to control cells. Free valine was present in only trace amounts of less than 0.1 mMin both cell lines. Exposure of normal and mutant cells to a 10 mMload of leucine, isoleucine, and valine resulted in a comparable concentration within cells after 24 hr. Concentrations returned to base values in normal cells 12 hr after removal of load, but leucine remained elevated in MSUD cells after 3 days. Leucine and its keto acid, -ketoisocaproic acid, added to the culture medium gave significant growth inhibition of MSUD lymphoblasts but not of normal cells, in the millimolar range. Isoleucine, valine, and their keto acids had no effect.This investigation was supported in part by Grants AM-13622, AM-05646, and GM-17702 from the United States Public Health Service, Veterans Administration Grant M.R.I.S. No. 3181 to Dr. Nathan Gochman, and grants from the National Foundation and the Kroc Foundation. S. D. S. is a Postdoctoral Research Fellow supported by United States Public Health Service Training Grant AM-05646.     

Isoleucine or valine deprivation stimulates fat loss via increasing energy expenditure and regulating lipid metabolism in WAT

There has been a growing interest in controlling body weight by increasing dietary levels of leucine recently. By contrast, we have focused on studying the effect of deficiency of branched-chain amino acids (BCAAs) leucine on lipid metabolism. We previously have shown that mice fed a leucine-deficient diet for 7 days exhibit significant changes in lipid metabolism as demonstrated by suppressed lipogenesis in the liver and increased fat mobilization in white adipose tissue, the latter of which was found to be caused by increased lipolysis in WAT and uncoupling protein 1 expression in brown adipose tissue. The goal of our current study is to investigate whether the above effects of leucine deficiency can be generalized to the deficiency of other BCAAs including valine and isoleucine. In our current study, we show that valine or isoleucine deficiency has similar effects on reducing fat mass to leucine deprivation, in a similar manner as those observed during leucine deprivation.