In vivo conversion of 18- and 20-C essential fatty acids in rats using the multiple simultaneous stable isotope method

An important question for mammalian nutrition is the relative efficiency of C18 versus C20 essential fatty acids (EFAs) for supporting the tissue composition of n-3 and n-6 pathway end products. One specific question is whether C22 EFAs are made available to tissues more effectively by dietary alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) or by dietary eicosapentaenoic acid (20:5n-3) and dihomo-gamma-linolenic acid (20:3n-6). To address this question in a direct manner, four stable isotope compounds were given simultaneously in a novel paradigm. A single oral dose of a mixture of 2H5-18:3n-3, 13C-U-20:5n-3, 13C-U-18:2n-6, and 2H5-20:3n-6 was administered to rats given a defined diet. There was a preferential in vivo conversion of arachidonic acid (20:4n-6) to docosatetraenoic acid (22:4n-6) and of 22:4n-6 to n-6 docosapentaenoic acid (22:5n-6) when the substrates originated from the C18 precursors. However, when the end products docosahexaenoic acid (22:6n-3) or 22:5n-6 were expressed as the total amount in the plasma compartment divided by the dosage, this parameter was 11-fold greater for 20:5n-3 than for 18:3n-3 and 14-fold greater for 20:3n-6 than for 18:2n-6. Thus, on a per dosage basis, the total amounts of n-3 and n-6 end products accreted in plasma were considerably greater for C20 EFA precursors relative to C18.     

The Metabolism and Distribution of Docosapentaenoic Acid (n-6) in the Liver and Testis of Growing Rats

To investigate the metabolism and distribution of docosapentaenoic acid (22:5n-6, DPA) in the liver and testis of growing rats, 22:5n-6 was administered to their dams. Newborn rats with a low hepatic arachidonic acid (20:4n-6, AA) level were generated by administrating a diet rich in docosahexaenoic acid (22:6n-3, DHA) but n-6 fatty acid (FA) free to pregnant dams. After parturition, 22:5n-6 or linoleic acid (18:2n-6, LA) was administered with a high level of 22:6n-3 to the dams until weaning. At weaning, the hepatic 20:4n-6 level was significantly highest in the DPA-DHA but not LA-DHA diet-fed animals. The hepatic delta-6 desaturase (D6D) mRNA abundance was significantly lower in both the LA-DHA and DPA-DHA diet-fed animals, connoted with the 20:4n-6 content recovered by 22:5n-6 that did not involve D6D and supporting the occurrence of retroconversion in the liver of the growing rats. The low D6D level in the 3-week-old testis was not in proportion to the elevated 22:5n-6 level, implying that early testicular 22:5n-6 accumulation might require supply from the circulation system.     

Studies on the involvement of prostaglandins and their precursors in the rejection of Nippostrongylus brasiliensis from the rat

In vitro incubation of 6-day Nippo-strongylus brasiliensis in the presence of PGE₁ at 1000 ng/ml and PGE₂ at 500–10,000 ng/ml of medium did not affect worm motility nor in vivo survival of worms implanted into the small intestine of recipient rats. The intraduodenal injection of 250 and 500 μg PGE₁ or PGE₂ did not lead to expulsion of worms from infected rats. An in vitro exposure to precursor fatty acids of PGE₁ and PGE₂, dihomo-γ-linolenic acid and arachidonic acid, respectively, at concentrations of 1000–15,000 ng/ml of medium also failed to inhibit worm motility and in vivo worm survival. These results are at variance with some earlier reports and do not suggest that prostaglandins are directly involved in the immune rejection of N. brasiliensis. No prostaglandins could be demonstrated in worm homogenates.     

Production of L-DOPA(3,4-dihydroxyphenyl-L-alanine) from benzene by using a hybrid pathway

As an alternative approach to the production of L-DOPA from a cheap raw material, we constructed a hybrid pathway consisting of toluene dioxygenase, toluene cis-glycol dehydrogenase, and tyrosine phenol-lyase. In this pathway, catechol is formed from benzene through the sequential action of toluene dioxygenase and toluene cis-glycol dehydrogenase, and L-DOPA is synthesized from the resulting catechol in the presence of pyruvate and ammonia by tyrosine phenol-lyase cloned from Citrobacter freundii. When the hybrid pathway was expressed in E. coli, production of L-DOPA was as low as 3 mM in 4 h due to the toxic effect of benzene on the cells. In order to reduce lysis of cells, Pseudomonas aeruginosa was employed as an alternative, which resulted in accumulation of about 14 mM L-DOPA in 9 h, showing a stronger resistance to benzene.     

Prevention of UVB-Induced Production of the Inflammatory Mediator in Human Keratinocytes by Lactic Acid Derivatives Generated from Aromatic Amino Acids

The anti-inflammatory effects of lactic acid derivatives were investigated on ultraviolet B (UVB)-irradiated HaCaT human keratinocytes. A pretreatment with indole-3-lactic acid (ILA) and 4-hydroxyphenyllactic acid (HPLA) inhibited the UVB-induced production of interlekin-6 (IL-6). The inhibitory effect of L-HPLA was equivalent to that of a corresponding racemic mixture (DL-HPLA), suggesting that optical isomerism did not affect the anti-inflammatory activity of HPLA.     

Influence of testosterone on polyunsaturated fatty acid biosynthesis in Sertoli cells in culture

Sertoli cells play a central role in spermatogenesis, its development and regulation. They are target cells for androgen action in the seminiferous tubules. The Sertoli cell is considered to be the most important cell type in the testis with regard to essential fatty acid metabolism. We studied the response to testosterone of cultured Sertoli cells from immature rats by determining the fatty acid composition of total cellular lipids as well as by the biosynthesis of polyunsaturated fatty acids. Fatty acid methyl esters were analysed by gas liquid chromatography and radiochromatography. Two doses of testosterone were tested (150 and 300 ng ml(-1)). Significant differences were found in fatty acids derived from total cellular lipids after 8 days in culture in the presence of testosterone (300 ng ml(-1), for 48 h). Compared to controls, the hormone produced a significant increase of 16:1 and 18:1 n-9, and of 18:2 n-6, and a decrease of 20:4 and 22:5 n-6 in total cellular lipids. The decrease in the n-6 fatty acid ratios 20:4/20:3, 20:4/18:2 and 24:5/24:4, and the increase in 18:1n-9/18:0 and 16:1n-9/16:0 ratios were taken as an indirect signal of testosterone effects on Delta5, Delta6 and Delta9 desaturase activities. The drop in Delta5 and Delta6 desaturase activities was corroborated by analysing the transformation of [1-14C]20:3 n-6 into its higher homologues. We concluded that testosterone modifies the fatty acid pattern of cultured Sertoli cells, and this hormone is involved in polyunsaturated fatty acid biosynthesis, modulating Delta5 and Delta6 desaturases activity.     

The simple and rapid quantification method for L-3,4-dihydroxyphenylalanine (L-DOPA) from plant sprout using liquid chromatography-mass spectrometry

L-3,4-dihydroxyphenylalanine (L-DOPA) is one of the important secondary metabolites of plants and has been used for various purposes, such as in clinical treatment for Parkinson’s disease and dopamine-responsive dystonia. In plants, L-DOPA is a precursor of many alkaloids, catecholamines, and melanin; the L-DOPA synthesis pathway is similar to that in mammals. L-DOPA acts as an allelochemical, has an important role in several biological processes, such as stress response and metabolism, in plants. L-DOPA is widely used in the clinical treatment as well as a dietary supplement or psychotropic drug, understanding of biosynthesis of L-DOPA in plant could lead to a stable supply of L-DOPA. This paper describes an improved method for simple and rapid quantification of L-DOPA content using liquid chromatography-tandem mass spectrometry. The standard quantitative methods for L-DOPA require multiple purification steps or relatively large amounts of plant material. In our improved method, quantification of L-DOPA was possible with extract of one–two pieces of cotyledon without any partitioning or column for purification. The endogenous L-DOPA (approximately 4,000 µg g⁻¹ FW (fresh weight)) could be detected from the one pieces of cotyledon of the faba bean sprout using this method. This method was also effective for samples with low endogenous amounts of L-DOPA such as broccoli, Japanese white radish, pea, and red cabbage sprouts. Therefore, this improved method will allow to measurement of L-DOPA content easily and accurately from a small amount of plant tissue and contribute to understanding biosynthesis, catabolism, and transport of L-DOPA.     

脱落酸的发酵法生产及应用

脱落酸是重要的植物激素之一,可以增强作物对环境逆因子如干旱、寒冷等的抵抗能力,在抵抗农业自然灾害、植树造林、生态植被建设、城市园林绿化等领域有广阔的应用前景.施用脱落酸可减少化学农药的使用,保护自然环境.本文概述了脱落酸生产和应用方面的研究进展,包括产生菌的筛选、诱变、外施脱落酸在提高作物抗逆性上的应用,以及脱落酸抗肿...     

Activation of 5-HT(1A) but not 5-HT(1B) receptors attenuates an increase in extracellular dopamine derived from exogenously administered L-DOPA in the striatum with nigrostriatal denervation

In order to determine whether L-DOPA-derived extracellular dopamine (DA) in the striatum with dopaminergic denervation is affected by activation of serotonin autoreceptors (5-HT(1A) and 5-HT(1B) receptors), we applied in vivo brain microdialysis technique to 6-hydroxydopamine-lesioned rats and examined the effects of the selective 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and the selective 5-HT(1B) receptor agonist CGS-12066 A on L-DOPA-derived extracellular DA levels. Single L-DOPA injection (50 mg/kg i.p.) caused a rapid increase and a following decrease of extracellular DA, with a peak value at 100 min after L-DOPA injection. Pretreatment with both 0.3 mg/kg and 1 mg/kg 8-OH-DPAT (i.p.) significantly attenuated an increase in L-DOPA-derived extracellular DA and the times of peak DA levels were prolonged to 150 min and 225 min after L-DOPA injection, respectively. These 8-OH-DPAT-induced changes in L-DOPA-derived extracellular DA were antagonized by further pretreatment with WAY-100635, a selective 5-HT(1A) antagonist. In contrast, intrastriatal perfusion with the 5-HT(1B) agonist CGS-12066 A (10 nM and 100 nM) did not induce any changes in L-DOPA-derived extracellular DA. Thus, stimulation of 5-HT(1A) but not 5-HT(1B) receptors attenuated an increase in extracellular DA derived from exogenous L-DOPA. These results support the hypothesis that serotonergic neurons are primarily responsible for the storage and release of DA derived from exogenous L-DOPA in the absence of dopaminergic neurons.     

Abnormal n-6 fatty acid metabolism in cystic fibrosis is caused by activation of AMP-activated protein kinase

Cystic fibrosis (CF) patients and model systems exhibit consistent abnormalities in PUFA metabolism, including increased metabolism of linoleate to arachidonate. Recent studies have connected these abnormalities to increased expression and activity of the Δ6- and Δ5-desaturase enzymes. However, the mechanism connecting these changes to the CF transmembrane conductance regulator (CFTR) mutations responsible for CF is unknown. This study tests the hypothesis that increased activity of AMP-activated protein kinase (AMPK), previously described in CF bronchial epithelial cells, causes these changes in fatty acid metabolism by driving desaturase expression. Using CF bronchial epithelial cell culture models, we confirm elevated activity of AMPK in CF cells and show that it is due to increased phosphorylation of AMPK by Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ). We also show that inhibition of AMPK or CaMKKβ reduces desaturase expression and reverses the metabolic alterations seen in CF cells. These results signify a novel AMPK-dependent mechanism linking the genetic defect in CF to alterations in PUFA metabolism.     

Downstream processing and purification of eicosapentaenoic (20:5n-3) and arachidonic acids (20:4n-6) from the microalga Porphyridium cruentum

Eicosapentaenoic acid (FPA, 20:5n-3) and arachidonic acid (AA, 20:4n-3)were obtained from the microalga Porphyridium cruentum by a three-stepprocess: fatty acid extraction by direct saponification of biomass,polyunsaturated fatty acid (PUFA) concentration by urea inclusion complexingand EPA isolation by high-performance liquid chromatography (HPLC). Twosolvents were tested for direct saponification of lipids in biomass. Themost efficient solvent, ethanol (96% v/v), extracted 75% ofthe fatty acids. PUFAs concentration by urea inclusion employed a urea/fattyacid ratio of 4:1 wt/wt at the crystallization temperatures of 4°C and28°C. Concentration factors were similar at both temperatures, but theEPA and AA recoveries were higher at 28°C (67.7% and 61.8%for the two acids, respectively). EPA and AA were purified from this PUFAconcentrate using analytical scale HPLC and the best results of thisseparation were scaled up to preparative level (4.7 i. d. × 30 cmcompression radial cartridge). A 94.3% pure EPA fraction and a81.4% pure AA fraction were obtained. Suitability of severalmicroalgae (Porphyridium cruentum, Phaeodactylum tricornutum and Isochrysisgalbana) and cod liver oil as sources of highly pure PUFAs, mainly EPA, wascompared.     

Placental lipid droplet composition: Effect of a lifestyle intervention (UPBEAT) in obese pregnant women

Maternal obesity is associated with adverse outcomes. Placental lipid droplets (LD) have been implicated in maternal-fetal lipid transfer but it is not known whether placental LD fat composition is modifiable. We evaluated the effects of a diet and physical activity intervention in obese pregnant women compared to routine antenatal care (UPBEAT study) on placental LD composition. LD were isolated by ultracentrifugation. Total FAs and phospholipids (phosphatidylcholines, PCs; sphingomyelins, SMs and lyso-phosphatidylcholines, Lyso-PCs) were analyzed by LC-MS/MS. Placenta MFSD2a expression was assessed by western blot. Placental LDs from obese women were comprised of predominantly saturated and monounsaturated FAs. TG and Chol composition was similar between intervention (n?=?20) and control (n?=?23) groups. PCs containing dihomo-?-linolenic acid in LD were positively associated with gestational weight gain (P?<?0.007), and lowered by the intervention. In the whole sample, PCs carrying DHA and arachidonic acid were inversely associated with placental weight. Placenta MFSD2a expression was associated with DHA cord blood metabolites and relationships were observed between LD lipids, especially DHA carrying species, and cord blood metabolites. We describe placenta LD composition for the first time and demonstrate modest, potentially beneficial effects of a lifestyle intervention on LD FAs in obese pregnant women.     

Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration

Delta-6 desaturase (D6D) catalyzes the first step in the synthesis of highly unsaturated fatty acids (HUFA) such as arachidonic (AA), docosapentaenoic (DPAn-6), and docosahexaenoic (DHA) acids, as well as the last desaturation of DPAn-6 and DHA. We created D6D-null mice (−/−), which enabled us to study HUFA deficiency without depleting their precursors. In −/−, no in vivo AA synthesis was detected after administration of [U-¹³C]linoleic acid (LA), indicating absence of D6D isozyme. Unexpectedly, all of the −/− developed ulcerative dermatitis when fed a purified diet lacking D6D products but containing ample LA. The −/− also exhibited splenomegaly and ulceration in duodenum and ileocecal junction. Male −/− lacked normal spermatozoa with a severe impairment of spermiogenesis. Tissue HUFAs in −/− declined differentially: liver AA and DHA by 95%, and a smaller decrease in brain and testes. Dietary AA completely prevented dermatitis and intestinal ulcers in −/−. DPAn-6 was absent in −/− brain under AA supplementation, indicating absence of D6D isozyme for DPAn-6 synthesis from AA. This study demonstrated a distinct advantage of the D6D-null mice (−/−) to elucidate (1) AA function without complication of LA deprivation and (2) DHA function in the nervous system without AA depletion or DPAn-6 replacement seen in traditional models.—Stroud, C. K., T. Y. Nara, M. Roqueta-Rivera, E. C. Radlowski, P. Lawrence, Y. Zhang, B. H. Cho, M. Segre, R. A. Hess, J. T. Brenna, W. M. Haschek, and M. T. Nakamura. Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration.     

Precursors of Antifungal Substances from Cherry Leaves (Prunus yedoensis Matsumura)

A glycosidic fraction was extracted from fresh cherry leaves and treated with commercial β-glucosidase and the acetone powder from cherry leaves. After enzymatic hydrolysis, the formation of moderately antifungal benzaldehyde, benzyl alcohol, 2-phenylethanol and coumarin was confirmed by GLC and GC-MS. Thus, it was expected that the precursors of antifungal substances existed as glycosides in the leaves and would be hydrolyzed by the endogenous hydrolytic enzymes when the leaves were damaged. A survey of the constituents in the glycosidic fraction revealed the presence of benzyl p-D-glucoside and 2-phenylethyl β-D-glucoside, and of mandelonitrile β-D-glucosides, sambunigrin and prunasin.     

外源5-氨基乙酰丙酸对设施番茄果实发育及糖酸品质的影响北大核心CSCD

番茄果实糖酸类物质的含量及比例直接影响其风味品质,前期研究表明,适宜浓度的外源5-氨基乙酰丙酸(ALA)能够促进果实的成熟并提高其芳香品质。该试验为探究外源ALA对番茄果实发育及其糖酸品质的影响,以番茄‘原味1号’(Solanum lycopersicum cv.Yuanwei No.1)品种为试材,于第4穗果授粉后10 d果实表面喷施0、100和200 mg·L^(-1)的ALA溶液,分析ALA对番茄果实形态、果皮色泽及果实不同部位组织中糖、酸类物质组分及含量的影响。结果表明:(1)外源ALA溶液能显著促进番茄果实横径、纵径的增加,提高果实单果重,还显著降低果实硬度,促进果实软化,提升果实口感,并提高了果实V_(C)和可溶性固形物含量。(2)果实不同部位组织(包括果肉、小柱和隔膜)糖类物质组分含量测定结果显示,外源ALA处理能够显著提高果实可溶性总糖含量(包括果糖、葡萄糖和蔗糖),并有利于糖类物质向果肉中积累。(3)在有机酸类物质中,除酒石酸含量增加外,外源ALA处理均能不同程度地降低果实各部位组织中酸类物质含量,从而显著提高番茄果实果肉部位糖酸比,提升果实糖酸品质。研究发现,在番茄果实发育过程中外源施用200 mg·L^(-1) ALA不仅能够促进果实发育及着色,提高单果重,提升果实的外观品质,还有利于果实糖酸品质的形成。     

Preventive effects of ZPDC glycoprotein (24 kDa) on hepatotoxicity induced by mercury chloride in vitro and in vivo

Mercury is a potent environmental contaminant that exerts toxic effect on various vital organs in the human body. Recently, we isolated glycoprotein from Zanthoxylum piperitum DC (ZPDC), which has antioxidant and anticancer effects. In the present study, we determined the preventive effects of ZPDC glycoprotein on hepatic damage induced by mercury chloride (HgCl₂). We evaluated the activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)], extracellular signal‐regulated kinase (ERK)1/2, p38 mitogen‐activated protein kinase (MAPK), cyclo‐oxygenase (COX‐2), inducible nitric oxide synthetase (iNOS), and activator protein (AP‐1) and the quantitative expressions of nuclear factor E2‐related factor (Nrf2), heme oxygenase (HO‐1), metallothionein (MT) and reduced glutathione (GSH) in mercury‐chloride‐exposed (50 μM and 10 mg/kg body weight) primary cultured hepatocytes and ICR mice, using biochemical assays, radioactivity and immunoblot analysis. The results demonstrated that ZPDC glycoprotein decreased the levels of LDH, ALT, HO‐1 and MT, whereas it increased the activities of hepatic antioxidant enzymes (SOD, CAT and GPx) and reduced GSH in mercury‐chloride‐exposed primary cultured hepatocytes. Also, it suppressed arachidonic acid release and expression of ERK, p38 MAPK, COX‐2, iNOS, AP‐1 and Nrf‐2 in primary cultured hepatocytes and ICR mice exposed to mercury chloride. Collectively, ZPDC glycoprotein may have potential applications to prevent hepatotoxicity induced by mercury chloride. Copyright © 2014 John Wiley & Sons, Ltd.     

大豆硬脂酰-ACP Δ^9脱氢酶(GmSAD)基因家族的鉴定及功能分析

硬脂酰-ACPΔ~9脱氢酶(Stearoyl-acyl carrier proteinΔ~9 desaturase,SAD)在质体中催化单不饱和油酸或棕榈油酸的合成,是控制植物细胞饱和脂肪酸与不饱和脂肪酸比例的关键酶。为解析大豆油酸合成积累调控机制,文中对大豆Glycine max GmSAD家族成员进行全基因组鉴定和保守功能域及理化性质等分析。应用qRT-PCR检测GmSAD各成员的时空表达谱,构建表达载体并通过农杆菌介导烟草Nicotiana tabacum瞬时表达和油酸缺陷型酵母Saccharomyces cerevisiae突变株BY4389遗传转化测试GmSAD酶活性和生物学功能。结果表明,大豆基因组含有5个GmSADs家族成员,其编码酶蛋白均具有二铁中心和SAD酶特有的2个保守组氨酸富集基序(EENRHG和DEKRHE),预测其活性酶蛋白为同源二聚体。系统进化分析显示5个GmSAD分成2个亚组,分别与拟南芥AtSSI2和AtSAD6亲缘关系较近。GmSAD各成员在大豆根、茎、叶、花和不同发育时期种子等组织中表达谱差异明显,其中GmSAD5在发育种子中、晚期高量表达,与油脂富集时期相吻合。烟草叶片瞬时表达GmSAD5可使叶片组织中油酸和总油脂含量分别提高5.56%和2.73%,而硬脂酸含量相应降低2.46%。缺陷型酵母遗传转化测试显示,过表达GmSAD5能恢复缺陷酵母合成单不饱和油酸的能力和促进油脂积累。总之,大豆GmSAD5对硬脂酸底物选择性较强,能高效催化单不饱和油酸的生物合成,为大豆种子油酸和总油脂积累机制的研究奠定了基础,也可作为油脂品质遗传改良的优异靶标。     

Genetic manipulation of gamma-linolenic acid (GLA) synthesis in a commercial variety of evening primrose (Oenothera sp.)

A robust Agrobacterium-mediated transformation procedure was developed for Rigel, a commercial cultivar of evening primrose, and used to deliver a cDNA encoding a Delta(6)-desaturase from borage under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Analysis of the transformed plants demonstrated an altered profile of polyunsaturated fatty acids, with an increase in gamma-linolenic acid and octadecatetraenoic acid in leaf tissues when compared with control lines.