代谢过程中相关氨基酸对高密度发酵生产腺苷蛋氨酸的影响

对清酒酵母高密度发酵生产S-腺苷-L-蛋氨酸（SAM）代谢过程中的相关氨基酸进行了考察。分别考察了十二种氨基酸对生物量和SAM产量的影响。实验发现L-胱氨酸、L-半胱氨酸、L-赖氨酸、L-组氨酸和L-蛋氨酸对SAM的积累有利,其中L-赖氨酸和L-组氨酸可以提高生物量,进而提高SAM的产量;L-胱氨酸、L-半胱氨酸和L-蛋氨酸可以提高SAM的含量,但是会抑制生物量的增长。通过3种补加方式的比较,得到最优的补加方式为：L-赖氨酸和L-组氨酸在培养基中加入,L-胱氨酸,L-半胱氨酸和L-蛋氨酸采取在发酵过程前24h流加。通过正交实验确定补加量为：L-赖氨酸为1g／L,L-组氨酸为1g／L,L-胱氨酸为1．5g／L,L-半胱氨酸为1g／L,L-蛋氨酸为1g／L。将此结果应用于5L发酵罐培养,SAM最高产量为5．53g/L,生物量为128g／L。     

苦皮藤试管苗生根培养研究

探讨了培养因子对诱导苦皮藤(Celastrus angulatus Maxim)试管苗生根的作用,采用正交试验设计法测试了苦皮藤生根关键因素多效唑(MET)、吲哚丁酸(IBA)、暗培养及培养基中盐浓度(简称培养基)的效应.方差分析结果显示,暗培养对苦皮藤生根作用极显著,因子作用大小依次为:暗培养＞培养基＞MET×IBA＞MET＞IBA×培养基＞IBA.诱导苦皮藤组培苗生根的最佳因素配比为:1/2 MS MET 3.0 mg·L-1 IBA 0.8 mg·L-1和1/2 MS MET 3.0 mg·L-1 IBA0.5 mg·L-1,暗培养12 d效果最好.     

苦皮藤试管苗生根培养研究

探讨了培养因子对诱导苦皮藤(Celastrus angulatus Maxim)试管苗生根的作用,采用正交试验设计法测试了苦皮藤生根关键因素多效唑(MET)、吲哚丁酸(IBA)、暗培养及培养基中盐浓度（简称培养基）的效应。方差分析结果显示,暗培养对苦皮藤生根作用极显著,因子作用大小依次为：暗培养＞培养基＞MET×IBA＞MET＞IBA×培养基＞IBA。诱导苦皮藤组培苗生根的最佳因素配比为：1/2 MS+MET 3.0 mg.L-1 + IBA 0.8 mg.L-1和1/2 MS +MET 3.0 mg.L-1 + IBA0.5 mg.L-1,暗培养12 d效果最好。     

贵州洪家渡水库水体不同形态汞的分布特征

通过分析贵州省乌江流域新建的洪家渡水库水体不同形态汞的浓度,探讨了水库水体各形态汞的分布特征,旨在进一步弄清新修建的水库水体汞的甲基化过程.结果表明,洪家渡水库总汞(THg)浓度变化范围在0.32～6.75 ng·L-1;溶解态汞(DHg)浓度变化范围在0.23～2.27 ng·L-1;颗粒态(PHg)汞浓度变化范围在0.03～4.51 ng·L-1;总甲基汞(TMeHg)浓度变化范围在0.04～0.18 ng·L-1;溶解态甲基汞(DMeHg)浓度变化范围在0.02～0.08 ng·L-1;颗粒态甲基汞(PMeHg)浓度变化范围在0.01～0.13 ng·L-1.洪家渡水库水体不同形态汞存在着季节性变化,水体THg、DHg和PHg的季节变化表现为夏、秋季大于春、冬季;水体TMeHg、DMeHg和PMeHg的季节变化表现为春、夏季大于秋、冬季.在空间上,水体甲基汞含量从表层到底层表现无规律性的增加趋势,从上游到下游的浓度也无明显的变化,与北美欧洲新建水库水体甲基汞浓度(0.01～6.6 ng·L-1)进行比较,发现洪家渡水库水体甲基汞浓度明显偏低,以上结果说明洪家渡水库水体没有显著的汞甲基化作用,这与乌江流域淹没土壤的贫瘠、有机质含量偏低有关.     

组织叶酸检测方法的建立及应用

目的:采用目前常用的化学发光法检测组织叶酸,探索不同叶酸前处理,寻求合适的检测组织叶酸的实验方法。并将优化好的方法应用于叶酸缺乏小鼠模型的检测。方法:通过化学发光法检测组织叶酸含量,首先将一定量的组织放入合适的buffer中,匀浆器匀浆后,通过超声或煮沸处理样本,离心后,得到上清样本,建立标曲和质控后,进行上机测定。该方法中我们通过探讨不同的buffer(包括PBS、Lysis和Tris-base盐溶液)处理组织叶酸,和不同的处理条件(包括是否煮沸及煮沸时间、样本处理时间等),探讨该方法检测组织叶酸最优化条件;并应用该方法进行小鼠叶酸缺乏模型中各组织叶酸含量的检测。结果:我们选择雄鼠肝组织进行叶酸检测,分别用PBS、Lysis和Tris-base盐溶液进行样本前处理,结果发现同样的处理条件下,如不煮沸条件下,Tris-base盐溶液(38.72 ng/mg)PBS(15.68 ng/mg)Lysis(11.9 ng/mg),提示Tris-base盐溶液可能有助于叶酸的稳定,防止降解。同时,我们探讨煮沸对叶酸检测结果的影响,结果发现同一种前处理下(如Tris-base),不煮沸(38.72 ng/mg)煮沸1分钟(36.36 ng/mg)煮沸3分钟(33.28 ng/mg)煮沸5分钟(30.72 ng/mg),说明叶酸含量随着煮沸时间的延长逐渐降低。此外,我们还对叶酸检测结果的重复性和稳定性进行了评估,结果发现不煮沸条件下,叶酸结果重复性很好,但随着时间延长稳定性会逐渐下降。因此,我们选择了Tris-base盐溶液进行前处理,条件选择为不煮沸和立即检测来减少因样本处理问题产生的误差。我们用该方法检测了叶酸缺乏小鼠模型的各组织叶酸含量,结果发现肝组织18.81 ng/mg降为8.46 ng/mg,脑组织从0.37 ng/mg下降为0.19 ng/mg,脾脏组织从1.53 ng/mg下降为0.26 ng/mg,肺组织从0.47 ng/mg下降为0.13 ng/mg,肾脏组织从2 ng/mg下降为0.9 ng/mg,心脏组织从0.33 ng/mg下降为0.06 ng/mg说明饮食叶酸是体内叶酸的主要来源,其缺乏可能导致多种组织叶酸代谢异常。结论:组织叶酸化学发光法检测条件优化为Tris-base盐溶液进行前处理,条件选择为不煮沸,并将样本立即进行检测。我们用该方法检测小鼠叶酸缺乏模型发现低叶酸饮食能显著降低各组织的叶酸含量,可能对生长发育造成潜在的影响。     

红叶椿的组织培养和快速繁殖

1植物名称臭椿(Ailanthus altissima)芽变品种"红叶椿". 2培养材料当年生枝条上的腋芽. 3培养条件芽萌动培养基:(1)MS 6-BA0.5 mg·L-1(单位下同) IBA 0.1;(2)MS 6-BA 1 IBA0.25.分化培养基:(3)MS 6-BA 1 IBA 0.1;(4)MS 6-BA 2 IBA 0.25.     

中华结缕草成熟胚的离体培养再生植株

1植物名称中华结缕草(Zoysia sinica). 2材料类别成熟种子. 3培养条件基本培养基为改良MS(MSm):MS无机盐 核黄素(VB2)1.0mg·L-1(单位下同) 盐酸噻胺(VB1)1.0 烟酸(Vpp)0.5 盐酸吡哆辛(VB6)0.5 肌醇100 酶水解酪蛋白(CH)300 蔗糖30 g.L-1 琼脂6.0 g·L-1.愈伤组织诱导培养基:(1)MSm4葡萄糖20 g·L-1 2,4-D 2.5 6-BA 0.25;愈伤组织继代培养基:(2)MSm 6-BA 0.1 2,4-D 1.6,(3)MSm 6-BA 0.1 2,4-D 2.0,(4)MSm 6-BA 0.1 2,4-D 2.4,(5)MSm 6-BA 0.1 2,4-D 2.8,(6)MSm 6-BA 0.1 2,4-D 3.2;分化培养基:(7)MSm.愈伤组织诱导和继代培养均为黑暗条件,分化培养过程中光照度为2000 lx,光照时间为12 h.d-1,培养温度(26±2)℃.     

血清游离精氨酸的快速检测

建立快速、准确的精氨酸定量检测方法。采用 6 30 0黄金系统氨基酸分析仪 ,在锂柱生理体液分析方法基础上 ,建立血清游离精氨酸 (ARG)快速测定方法。血清样本经磺基水杨酸沉淀蛋白后取上清液进行色谱分析 ,色谱柱为Beckman公司阳离子交换柱 (12cm× 4 .0mm) ;流动相为 2 0mmol·L- 1 柠檬酸锂水溶液 ,流速为 2 0ml·h- 1 ;比色波长 5 70nm。该法检测精氨酸浓度的线性范围为 5mg·L- 1 ～ 5 0mg·L- 1 ,相关系数 0 .99834,最低检测限 1mg·L- 1 ,重复性 :日内RSD 0 .4 0 % ,日间RSD 0 .5 5 % ,回收率 97.6 7%～ 10 0 .6 7% (平均值 99.0 7% ) ;整个实验过程耗时 2 8min。该法简便、快速、准确、可靠 ,适用于临床和科研工作。     

Hcy、ACA、FA联合检测在预测脑梗死发生中的临床价值

目的:探讨Hcy、ACA、FA联合检测在预测脑梗死发生中的临床价值.方法:采用EILSA及化学发光免疫分析技术,对66例脑梗死患者和30例健康对照者进行血清的ACA、Hcy及FA的检测.结果:脑梗死患者Hey浓度为[(22.68±12.70)μmol/L]明显高于健康对照组[(13.38+2.61)μmol/L],差异非常显著(P<0.01).叶酸[(3.87±2.02)ng/ml]水平明显低于健康对照组[(519.37±437.57)ng/mL](P<0.05);脑梗死患者ACA和高Hey血症的检出率为53.03%和68.18%与健康对照组(16.67%)比较,差异非常显著(P<0.01);ACA、Hcy及FA单独预测脑梗死的敏感性分别为87.50%、90.00%和96.3%;特异性为44.60%、54.40%和37.09%;在预测脑梗死发生中ACA、Hcy及FA联合检测优于单项检测.结论:高Hcy血症和ACA均是脑梗死发病的独立的危险因素.ACA可能参与脑血管病变的发病过程.ACA、Hcy及FA联合检测可较好地预测脑梗死的发生,其中以ACA和Hcy联合检测为最佳,对于预测脑梗死发生具有较高的临床价值.     

假俭草的组织培养与植株再生

1植物名称假俭草(Eremochloa ophiuroides),又名百足草. 2材料类别成熟种子. 3培养条件(1)诱导愈伤组织培养基:MS 6-BA Q1mg·L-1(单位下同) 2,4-D 4.5.(2)继代培养基:MS 6-BA 0.1 2,4-D 4.0 Vc 5.0.(3)芽分化培养基:MS 6-BA 2.0 NAA1.0 CoCl25.0 TDZ 0.5.(4)生根培养基:1/2MS NAA 0.5 IAA 0.5 MET 0.5 活性炭0.1%.     

Evidence for altered methionine methyl-group utilization in the diabetic rat's brain

The methionine (MET) derivative, S-adenosylmethionine (SAM), provides methyl-groups for methylation reactions in many neural processes. In rats made diabetic with streptozotocin (SZ), brain SAM levels were generally lower (10–20%) than in controls, with a constant decrease being observed five weeks after onset of diabetes. This decrease in SAM levels may be due to reduced precursor (MET) availability because greatly elevating plasma MET concentrations in SZ diabetic rats by dietary manipulation increased their neural SAM concentrations to be approximately or even greater than (5–20%) those of controls. In contrast, neural levels of SAM's demethylated product, S-adenosylhomocysteine (SAH), were reduced to a greater extent (17–44%) than SAM levels in all groups of SZ diabetic rats independent of their plasma MET concentrations or brain SAM levels. This indicates that the decrease in SAH levels is not simply due to substrate (SAM) restriction. These changes in MET metabolites appear to be a general effect of diabetes rather than a non-pancreatic side-effect of SZ, because genetically diabetic BB Wistar rats also exhibited reduced brain SAM (25%) and brain SAH (46%) levels. These results indicate that methyl-groups from MET are handled differently in the brain of the diabetic rat, which considering the variety and importance of neural methylation reactions, could have important consequences for the diabetic.Abbreviations MET methionine - SAM S-adenosylmethionine - SAH S-adenosylhomocysteine - SZ streptozotocin - BBW BB Wistar - LNAA large neutral amino acids - BCAA branchedchain amino acids - MET:BCAA methionine to branched-chain amino acid ratio - MET:LNAA methionine to large neutral amino acid ratio     

Methylation potential associated with diet,genotype, protein,and metabolite levels in the Delta Obesity Vitamin Study

Micronutrient research typically focuses on analyzing the effects of single or a few nutrients on health by analyzing a limited number of biomarkers. The observational study described here analyzed micronutrients, plasma proteins, dietary intakes, and genotype using a systems approach. Participants attended a community-based summer day program for 6–14 year old in 2 years. Genetic makeup, blood metabolite and protein levels, and dietary differences were measured in each individual. Twenty-four-hour dietary intakes, eight micronutrients (vitamins A, D, E, thiamin, folic acid, riboflavin, pyridoxal, and pyridoxine) and 3 one-carbon metabolites [homocysteine (Hcy), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH)], and 1,129 plasma proteins were analyzed as a function of diet at metabolite level, plasma protein level, age, and sex. Cluster analysis identified two groups differing in SAM/SAH and differing in dietary intake patterns indicating that SAM/SAH was a potential marker of nutritional status. The approach used to analyze genetic association with the SAM/SAH metabolites is called middle-out: SNPs in 275 genes involved in the one-carbon pathway (folate, pyridoxal/pyridoxine, thiamin) or were correlated with SAM/SAH (vitamin A, E, Hcy) were analyzed instead of the entire 1M SNP data set. This procedure identified 46 SNPs in 25 genes associated with SAM/SAH demonstrating a genetic contribution to the methylation potential. Individual plasma metabolites correlated with 99 plasma proteins. Fourteen proteins correlated with body mass index, 49 with group age, and 30 with sex. The analytical strategy described here identified subgroups for targeted nutritional interventions.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0403-9) contains supplementary material, which is available to authorized users.     

也门铁的组织培养技术研究

以4品种也门铁的茎段为外植体进行组织培养技术研究。结果表明,控制普通也门铁茎段褐化效果最理想的培养基为MS + 0.25 g·L-1 VC + 0.50 g·L-1 Na2S2O3;诱导也门铁不定芽萌动的最佳培养基为MS + 3.0 mg·L-1 6-BA + 0.2 mg·L-1 NAA + 0.1 mg·L-1 KT;诱导也门铁不定芽增殖的最佳培养基,因品种不同而异,普通也门铁和金心也门铁为MS + 2.0 mg·L-1 6-BA + 1.0 mg·L-1 NAA + 0.1 mg·L-1 GA3,扭纹铁和金心扭纹铁为MS + 1.0 mg·L-1 6-BA + 0.5 mg·L-1 NAA + 0.1 mg·L-1 GA3;也门铁壮苗的最佳培养基为MS + 0.05 mg·L-1 6-BA + 0.05 mg·L-1 NAA + l g·L-1AC;也门铁生根最优培养基为1/2MS + 0.5 mg·L-1 IBA。     

Application of liquid chromatography-tandem mass spectrometry for the determination of opioidmimetics in the brain dialysates from rats treated with opioidmimetics intraperitoneally

We have determined three opioidmimetics (compounds I-III) in the rat brain dialysates after intraperitoneal (i.p.) administration of compounds I-III using a liquid chromatography/mass spectrometry with tandem mass spectrometry (LC-MS/MS). The dialysate samples with methanol were directly analyzed by online column-switching liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 421 of m/z 657 for compound I, m/z 421 of m/z 643 for compound II, and m/z 407 of m/z 629 for compound III) on LC-MS/MS with electrospray ionization (ESI), opioidmimetics in rat brain dialysates were determined. Calibration curves of the method showed a good linearity in the range of 10-100 ng/ml for each compound. The limit of determination was estimated to be ca. 1 ng/ml for compounds II and III, and ca. 5 ng/ml for compound I, respectively. The precision of analysis showed coefficients of variation ranging from 4.7 to 10.4% at compound III concentration (10-100 ng/ml) in Ringer's solution. As a result, the procedure proved to be very suitable for routine analysis. The method was applied to the analysis of three opioidmimetics in the brain dialysate samples from rats treated with these compounds.     

Determination of 5-methyltetrahydrofolate (13C-labeled and unlabeled) in human plasma and urine by combined liquid chromatography mass spectrometry

The association of folates with the prevention of neural tube defects and reduced risk of other chronic diseases has stimulated interest in the development of techniques for the study of their bioavailability in humans. Stable isotope protocols differentiate between oral and/or intravenous test doses of folate and natural levels of folate already present in the body. An liquid chromatography/mass spectrometry (LC/MS) procedure is described that has been validated for the determination of [13C]5-methyltetrahydropteroyl monoglutamic acid ([13C]5-CH3H4PteGlu) in plasma and urine, following oral dosing of volunteers with different labeled folates. Folate binding protein affinity columns were used for sample purification prior to LC/MS determination. Chromatographic separation was achieved using a Superspher 100RP18 (4 microm) column and mobile phase of 0.1 mol/L acetic acid (pH 3.3):acetonitrile (90:10; 250 microL/min). Selected ion monitoring was conducted on the [M-H](-) ion: m/z 458 and 459 for analyzing 5-CH3H4PteGlu; m/z 464 [M+6-H](-) to determine 5-CH3H4PteGlu derived from the label dose; m/z 444 for analysis of 2H4PteGlu internal standard, and m/z 446 and 478 to confirm that there was no direct absorption of unmetabolized compounds. Calibration was linear over the range 0-9 x 10(-9) mol/L; the limits of detection and quantification were 0.2 x 10(-9) and 0.55 x 10(-9) mol/L, respectively. The mean coefficient of variation of the ratios (m/z 463/458) was 7.4%. The method has potential applications for other key folates involved in one-carbon metabolism.     

Simultaneous determination of erythromycin propionate and base in human plasma by high-performance liquid chromatography-electrospray mass spectrometry

An analytical method for simultaneous determination of erythromycin propionate and its active metabolite, erythromycin base, in human plasma by high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI-MS) was developed and validated. Roxithromycin was selected as the internal standard. The samples were directly injected after simple deproteinized procedure only. The separation was achieved on a Johnson Spherigel analytical column packed with 5 microm C18 silica, employing acetonitrile -0.1% formic acid aqueous solution (50:50) as mobile phase. The quantification of target compounds was obtained by using a selected ion monitoring (SIM) at m/z 790.7 for erythromycin propionate, m/z 734.7 for erythromycin base and m/z 837.8 for roxithromycin. The correlation coefficients of the calibration curves were better than 0.997 (n=6), in the ranges from 2 ng/ml to 1 microg/ml, and from 1 to 10 microg/ml for erythromycin propionate and base. The method can provide the necessary sensitivity, precision and accuracy to allow the simultaneous determination of both compounds in a patient's plasma following a single administration of erythromycin stinoprate capsule (500 mg erythromycin base equivalent).     

Dynamic contact forces on leukocyte microvilli and their penetration of the endothelial glycocalyx

We develop a theoretical model to examine the combined effect of gravity and microvillus length heterogeneity on tip contact force (F(m)(z)) during free rolling in vitro, including the initiation of L-, P-, and E-selectin tethers and the threshold behavior at low shear. F (m)(z) grows nonlinearly with shear. At shear stress of 1 dyn/cm(2), F(m)(z) is one to two orders of magnitude greater than the 0.1 pN force for gravitational settling without flow. At shear stresses > 0.2 dyn/cm(2) only the longest microvilli contact the substrate; hence at the shear threshold (0.4 dyn/cm(2) for L-selectin), only 5% of microvilli can initiate tethering interaction. The characteristic time for tip contact is surprisingly short, typically 0.1-1 ms. This model is then applied in vivo to explore the free-rolling interaction of leukocyte microvilli with endothelial glycocalyx and the necessary conditions for glycocalyx penetration to initiate cell rolling. The model predicts that for arteriolar capillaries even the longest microvilli cannot initiate rolling, except in regions of low shear or flow reversal. In postcapillary venules, where shear stress is approximately 2 dyn/cm(2), tethering interactions are highly likely, provided that there are some relatively long microvilli. Once tethering is initiated, rolling tends to ensue because F(m)(z) and contact duration will both increase substantially to facilitate glycocalyx penetration by the shorter microvilli.     

Simultaneous determination of benazepril hydrochloride and benazeprilat in plasma by high-performance liquid chromatography/electrospray-mass spectrometry

An analytical method for simultaneous determination of benazepril and its active metabolite, benazeprilat, in human plasma by high-performance liquid chromatography/electrospray-mass spectrometry was developed and validated. Rutaecarpine was selected as the internal standard. The separation was achieved on a C(18) column with acetonitrile and aqueous solution (0.1% formic acid) as mobile phase with a gradient mode. The quantification of target compounds was using a selective ionization recording at m/z 425.5 for benazepril, m/z 397.5 for benzeprilat and m/z 288.3 for rutaecarpine. The correlation coefficients of the calibration curves were better than 0.992 (n = 6), in the range of 6.67-666.67 ng/ml for benazepril and benazeprilat. The inter- and intra-day accuracy, precision, linear range had been investigated in detail. The method can be used to assess the bioavailability and pharmacokinetics of the drug.     

来自猪肠道芽胞杆菌培养液中氨基酸成分分析

从动物肠道分离得到112株芽胞杆菌,采用氨基酸全自动分析仪对分离得到的芽胞杆菌的上清进行氨基酸分析。结果表明,不同芽胞杆菌菌株上清中各氨基酸水平相差较大。其中,动物的几种限制性必需氨基酸中,赖氨酸的含量分布在0.18~0.32g·L~(-1)之间,蛋氨酸的含量分布在0.05~0.09g·L~(-1)之间。精氨酸的含量分布在0.004~0.02g·L~(-1)之间。所有氨基酸种类中,赖氨酸、亮氨酸、苯丙氨酸、异亮氨酸含量较为丰富,超过了0.1g·L~(-1),而天冬氨酸、苏氨酸、丝氨酸含量较少,不到0.01g·L~(-1)。这些结果为开发提高饲料氮利用效率的饲用益生菌提供参考。     

Determination of cabergoline and L-dopa in human plasma using liquid chromatography-tandem mass spectrometry

We determined cabergoline and L-dopa in human plasma using liquid chromatography-mass spectrometry with tandem mass spectrometry (LC-MS-MS). The deproteinized plasma samples with organic solvent or acid were analyzed directly by reversed-phase liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 381 of m/z 452 for cabergoline and m/z 152 of m/z 198 for L-dopa) on LC-MS-MS with electrospray ionization (ESI), cabergoline and L-dopa in human plasma were determined. Calibration curves of the method showed a good linearity in the range 5-250 pg/ml for cabergoline and 1-200 ng/ml for L-dopa, respectively. The limit of determination was estimated to be approximately 2 pg/ml for cabergoline and approximately 0.1 ng/ml for L-dopa, respectively. The method was applied to the analysis of cabergoline and L-dopa in plasma samples from patients treated with these drugs. The precision of analysis showed coefficients of variation ranging from 3.8% to 10.5% at cabergoline concentration of 13.8-26.2 pg/ml and from 2.9% to 8.9% at an L-dopa concentration of 302.5-522.1 ng/ml in patient plasma. As a result, the procedure proved to be very suitable for routine analysis.