The Reversed Feto-Maternal Bile Acid Gradient in Intrahepatic Cholestasis of Pregnancy Is Corrected by Ursodeoxycholic Acid

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder associated with an increased risk of adverse fetal outcomes. It is characterised by raised maternal serum bile acids, which are believed to cause the adverse outcomes. ICP is commonly treated with ursodeoxycholic acid (UDCA). This study aimed to determine the fetal and maternal bile acid profiles in normal and ICP pregnancies, and to examine the effect of UDCA treatment. Matched maternal and umbilical cord serum samples were collected from untreated ICP (n = 18), UDCA-treated ICP (n = 46) and uncomplicated pregnancy (n = 15) cases at the time of delivery. Nineteen individual bile acids were measured using HPLC-MS/MS. Maternal and fetal serum bile acids are significantly raised in ICP compared with normal pregnancy (p = <0.0001 and <0.05, respectively), predominantly due to increased levels of conjugated cholic and chenodeoxycholic acid. There are no differences between the umbilical cord artery and cord vein levels of the major bile acid species. The feto-maternal gradient of bile acids is reversed in ICP. Treatment with UDCA significantly reduces serum bile acids in the maternal compartment (p = <0.0001), thereby reducing the feto-maternal transplacental gradient. UDCA-treatment does not cause a clinically important increase in lithocholic acid (LCA) concentrations. ICP is associated with significant quantitative and qualitative changes in the maternal and fetal bile acid pools. Treatment with UDCA reduces the level of bile acids in both compartments and reverses the qualitative changes. We have not found evidence to support the suggestion that UDCA treatment increases fetal LCA concentrations to deleterious levels.     

Callus Induction by (2-Chloroethyl) Phosphonic Acid on Cultured Cotton Ovules

The response of cultured cotton (Gossypium hirsutum L.) ovules to the ethylene releasing growth regulator, (2-chloroethyl) phosphonic acid (ethephon), was measured. Control ovules grown on a complete liquid medium without hormones continued the differentiation normal for cotton, although at a slower rate than in vivo. When ethephon was added to the medium, normal organ and fiber development was inhibited but callus was induced from the micropylar end of the ovule. The callus was extremely friable and produced many free cells in the culture medium. Dry weight of the callused ovules increased over 4-fold and total cell number increased 56% over the controls. Apparently ethylene released from the ethephon stimulated both cell division and cell expansion in forming the callus.     

Light-dependent Induction of Polyunsaturated Fatty Acid Biosynthesis in Greening Cucumber Cotyledons

Greening cucumber (Cucumis sativus L.) cotyledons exhibited dramatic increases in the ability to desaturate exogenously added [1-¹⁴C]oleic acid and [1-¹⁴C]linoleic acid within 2 to 3 hours of illumination. These increases were effectively inhibited by 10 micrograms per milliliter cycloheximide. Oleate desaturation remained at a high level in constant light for 5 to 6 days after induction and then declined by about 50%; when returned to the dark, the tissue showed a sharp decrease in conversion of [¹⁴C]oleate to [¹⁴C]linoleate. Linoleate desaturation reached a maximum about 15 hours after induction and declined immediately thereafter while the tissue still was in the light; after induction had peaked return of the tissue to the dark showed a dramatic fall of linoleate desaturation. The changes in desaturation were correlated with the conversion of the principal fatty acid in the etiolated cotyledons, linoleate, to α-linolenate, and with the assembly of the chlorophyll-containing photosynthetic membranes. The incorporation of [1-¹⁴C]acetate into lipids showed no significant light stimulation. The role of light in the regulation of certain aspects of plant metabolism during development is discussed.     

Reduction of Acid Tolerance by Tetracycline in Escherichia coli Expressing tetA(C) Is Reversed by Cations

When tetracycline was present, tetA(C) reduced acid tolerance, suppressed rpoS expression, and increased the concentration of total soluble proteins in stationary-phase Escherichia coli. The suppression of acid tolerance was reversed by 85 mM sodium, potassium, magnesium, and calcium ions but not by 85 mM sucrose. Implications for using TetA(C) are discussed.     

Biosynthesis of 5-aminolevulinic Acid via the Shemin Pathway in a Green Sugar Beet Callus

5-Aminolevulinic acid synthase (ALAS) has been detected in a normal (auxin- and cytokinin-dependent) green sugar beet callus under light and under darkness. ALAS activity was lower when the callus was grown under light. The supply of precursors of the Shemin pathway (glycine and succinate) to dark-grown callus enhanced considerably the capacity of the 5-aminolevulinic acid (ALA) formation. Glutamate, -aminobutyrate or -ketoglutarate also increased ALA accumulation. Such an accumulation was also obtained after inhibition of polyamine synthesis. The results show that glutamate or its derivatives might feed the Shemin pathway in conditions preventing glutamate to be used through the Beale pathway.     

A Novel Red Clover Hydroxycinnamoyl Transferase Has Enzymatic Activities Consistent with a Role in Phaselic Acid Biosynthesis

Red clover (Trifolium pratense) leaves accumulate several μmol g⁻¹ fresh weight of phaselic acid [2-O-(caffeoyl)-l-malate]. Postharvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases prevents breakdown of forage protein during storage. Forage crops like alfalfa (Medicago sativa) lack both polyphenol oxidase and o-diphenols, and breakdown of their protein upon harvest and storage results in economic losses and release of excess nitrogen into the environment. Understanding how red clover synthesizes o-diphenols such as phaselic acid will help in the development of forage crops utilizing this natural system of protein protection. A possible pathway for phaselic acid biosynthesis predicts a hydroxycinnamoyl transferase (HCT) capable of forming caffeoyl and/or p-coumaroyl esters with malate. Genes encoding two distinct HCTs were identified in red clover. HCT1 shares more than 75% amino acid identity with a number of well-characterized shikimate O-HCTs implicated in monolignol biosynthesis. HCT2 shares only 34% amino acid sequence identity with HCT1 and has limited sequence identity to any previously identified HCT. Expression analyses indicate that HCT1 mRNA accumulates to 4-fold higher levels in stems than in leaves, whereas HCT2 mRNA accumulates to 10-fold higher levels in leaves than in stems. Activity assays of HCT1 and HCT2 proteins expressed in Escherichia coli indicate that HCT1 transfers caffeoyl or p-coumaroyl moieties from a coenzyme A-thiolester to shikimate but not malate, whereas HCT2 transfers caffeoyl or p-coumaroyl moieties from a coenzyme A-thiolester to malate but not shikimate. Together, these results indicate that HCT1 is involved in monolignol biosynthesis and HCT2 is a novel transferase likely involved in phaselic acid biosynthesis.In contrast to many other forage legumes (e.g. alfalfa [Medicago sativa]; Jones et al., 1995), red clover (Trifolium pratense) accumulates relatively high levels of the phenylpropanoid o-diphenol phaselic acid [2-O-(caffeoyl)-l-malic acid; hereafter referred to as caffeoyl-malate or phaselic acid] in its leaves (Hatfield and Muck, 1999; Winters et al., 2008). In red clover, upon cellular disruption, phaselic acid and other o-diphenols are readily oxidized by a soluble polyphenol oxidase (PPO) to produce their corresponding o-quinones (Hatfield and Muck, 1999; Sullivan et al., 2004). The formation of such o-quinones by PPO, and the subsequent secondary reactions of these quinones, are most often associated with browning of fresh fruits and vegetables (Steffens et al., 1994), which has a negative impact on perceived quality. When preserved by ensiling, however, oxidation of o-diphenols by PPO in red clover prevents degradation of protein during storage (Sullivan et al., 2004; Sullivan and Hatfield, 2006). Although alfalfa lacks significant levels of both PPO activity and o-diphenol compounds in its leaves, red clover''s natural system of protein protection has been transferred to this forage legume by expressing a red clover PPO transgene in alfalfa and exogenously adding o-diphenol PPO substrates to the resulting tissues or tissue extracts (Sullivan et al., 2004; Sullivan and Hatfield, 2006). Because ruminant animals poorly utilize degraded protein, adaptation of the PPO system to alfalfa and other forage crops would have substantial positive economic and environmental impacts (Sullivan and Hatfield, 2006). Unfortunately, lack of system components in these forage crops, especially the o-diphenol PPO substrates, presents a challenge to practical adaptation of this natural system of protein preservation. Consequently, understanding how red clover is able to accumulate o-diphenols such as phaselic acid will be a key step to adapt the PPO/o-diphenol system to a wide range of economically important forage crops.The biosynthetic pathways whereby red clover synthesizes and accumulates phaselic acid and other o-diphenols have not been defined. However, in the Brassicaceae, hydroxycinnamoyl esters with malic acid can be made via the action of sinapoyl-Glc:malate sinapoyltransferase (SMT; EC 2.3.1), which is capable of transferring a hydroxycinnamoyl moiety from a hydroxycinnamoyl-Glc ester to a malic acid acceptor. In Arabidopsis (Arabidopsis thaliana), SNG1 (for sinapoylglucose accumulator 1), which encodes the enzyme, has been shown to be responsible for the accumulation of sinapoylmalate in seeds and leaves (Lehfeldt et al., 2000). An SMT from radish (Raphanus sativus), presumably the homolog of the Arabidopsis SNG1 gene product, has been purified to apparent homogeneity and characterized (Grawe et al., 1992). The purified enzyme is capable of utilizing sinapoyl-, feruloyl-, caffeoyl-, and to a lesser extent p-coumaroyl-Glc esters to form the corresponding malic acid esters, suggesting that it is responsible for the accumulation of these esters in vivo. In contrast, in many plants, formation of certain hydroxycinnamoyl esters is often mediated by a member of the BAHD transferase family (D''Auria, 2006) that utilize a CoA thiolester hydroxycinnamoyl donor. Some of the best characterized of these hydroxycinnamoyl transferases (HCTs) are those associated with the biosynthesis of monolignols (Hoffmann et al., 2003, 2004; Shadle et al., 2007). These are capable of transferring p-coumaroyl or caffeoyl moieties from the respective CoA thiolesters to form 5-O-esters with shikimic acid or, to a lesser extent, 3-O-esters with quinic acid. Separable enzymatic activities capable of transferring a p-coumaroyl moiety to either shikimate/quinate or to 4′-hydroxyphenyllactate in basil (Ocimum basilicum) peltate gland extracts have been identified, although genes encoding these activities have not been cloned (Gang et al., 2002). Niggeweg et al. (2004) used gene-silencing experiments to definitively demonstrate that a hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) is responsible for chlorogenic acid accumulation in the Solanaceae. Although phaselic acid biosynthesis in red clover could be via a pathway utilizing SMT, lack of an apparent SNG1 homolog in a collection of red clover EST sequences derived from leaves and young plants suggests otherwise (see “Discussion”). Therefore, pathways in red clover for the biosynthesis of phaselic acid utilizing one or more BAHD family transferase (Fig. 1) should be considered. In these proposed pathways, Phe would be converted to p-coumaroyl-CoA by the sequential action of Phe ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), and 4-coumarate:CoA ligase (4CL). The action of one or more specific HCTs and one or more p-coumarate 3′-hydroxylases (C3Hs) would then result in the formation of phaselic acid.Open in a separate windowFigure 1.Possible pathways for phaselic acid biosynthesis in red clover. Proposed pathway enzymes for the production of phaselic acid include PAL, 4CL, hydroxycinnamoyl:shikimate transferase (HCT-S), hydroxycinnamoyl:malate transferase (HCT-M), and C3H. The branch point at p-coumaroyl-CoA represents two alternative pathways. For simplicity, not all reactants and products are shown.Existing literature suggests that C3H enzymes, which are cytochrome P450 enzymes (CYP98A subfamily), do not directly hydroxylate p-coumaric acid to caffeic acid but rather act on p-coumaroyl ester derivatives. For example, the enzyme from Arabidopsis hydroxylates shikimic and quinic acid esters of p-coumaric acid but only poorly or not at all p-coumaric acid or its Glc or CoA esters (Schoch et al., 2001; Franke et al., 2002). Thus, one model of phaselic acid biosynthesis is the formation of 2-O-(p-coumaroyl)-l-malic acid (hereafter referred to as p-coumaroyl-malate) by a HCT and its subsequent hydroxylation by a C3H enzyme capable of utilizing the malic acid ester as a substrate (Fig. 1, bottom, red pathway). An alternative model would require at least two HCT activities for phaselic acid biosynthesis (Fig. 1, top, blue pathway). The first activity would form a substrate suitable for hydroxylation (e.g. p-coumaroyl-shikimate, since several characterized C3H enzymes appear to favor this substrate [Schoch et al., 2001; Franke et al., 2002; Gang et al., 2002; Morant et al., 2007]). Following hydroxylation to the caffeoyl derivative by a C3H, the first HCT activity could synthesize caffeoyl-CoA via its reverse reaction (Hoffmann et al., 2003; Niggeweg et al., 2004). A second HCT activity would then transfer the caffeoyl moiety to malic acid to form phaselic acid. Both pathways predict a transferase capable of transferring a hydroxycinnamoyl moiety (either p-coumaroyl or caffeoyl) to malic acid. Also, these pathways are consistent with the observation that, at least in vitro, several characterized HCT enzymes are capable of transfer reactions utilizing either p-coumaroyl- or caffeoyl-CoA (Hoffmann et al., 2003; Niggeweg et al., 2004). The identification and characterization of two distinct HCTs from red clover, one of which has properties consistent with a role in phaselic acid biosynthesis, are reported here.     

Biosynthesis of d-Erythroascorbic Acid by Candida

Epigallocatechin 3-gallate (EGCG) has cytotoxic effects in many cancer cells. It has been reported that A549 lung cancer cells are markedly resistant to cell death induced by EGCG. In the present study, the effects of EGCG on A549 lung cancer cell growth and angiogenesis were studied. We found that EGCG dose-dependently suppressed A549 cell growth, while A549 cells were markedly resistant to cell death in vitro. Next we found that EGCG increased endostatin expression and suppressed vascular endothelial growth factor (VEGF) expression. We further studied to determine whether EGCG would suppress A549 tumor growth in nude mouse and angiogenesis. EGCG in drinking water significantly suppressed A549 tumor growth in nude mice. Histological analysis revealed that the number of CD34 positive vessels had a tendency to decrease in the tumor. In sum, EGCG had anti-proliferative effects of A549 on tumor growth and showed a tendency to suppress angiogenesis.     

para-Aminobenzoic Acid Is a Precursor in Coenzyme Q6 Biosynthesis in Saccharomyces cerevisiae

Coenzyme Q (ubiquinone or Q) is a crucial mitochondrial lipid required for respiratory electron transport in eukaryotes. 4-Hydroxybenozoate (4HB) is an aromatic ring precursor that forms the benzoquinone ring of Q and is used extensively to examine Q biosynthesis. However, the direct precursor compounds and enzymatic steps for synthesis of 4HB in yeast are unknown. Here we show that para-aminobenzoic acid (pABA), a well known precursor of folate, also functions as a precursor for Q biosynthesis. A hexaprenylated form of pABA (prenyl-pABA) is normally present in wild-type yeast crude lipid extracts but is absent in yeast abz1 mutants starved for pABA. A stable ¹³C₆-isotope of pABA (p- amino[aromatic-¹³C₆]benzoic acid ([¹³C₆]pABA)), is prenylated in either wild-type or abz1 mutant yeast to form prenyl-[¹³C₆]pABA. We demonstrate by HPLC and mass spectrometry that yeast incubated with either [¹³C₆]pABA or [¹³C₆]4HB generate both ¹³C₆-demethoxy-Q (DMQ), a late stage Q biosynthetic intermediate, as well as the final product ¹³C₆-coenzyme Q. Pulse-labeling analyses show that formation of prenyl-pABA occurs within minutes and precedes the synthesis of Q. Yeast utilizing pABA as a ring precursor produce another nitrogen containing intermediate, 4-imino-DMQ₆. This intermediate is produced in small quantities in wild-type yeast cultured in standard media and in abz1 mutants supplemented with pABA. We suggest a mechanism where Schiff base-mediated deimination forms DMQ₆ quinone, thereby eliminating the nitrogen contributed by pABA. This scheme results in the convergence of the 4HB and pABA pathways in eukaryotic Q biosynthesis and has implications regarding the action of pABA-based antifolates.     

杂景天愈伤组织诱导及红景天苷测定

以杂景天的子叶、胚轴为外植体,接种在附加不同激素组合的MS培养基上诱导愈伤组织产生,愈伤组织经继代培养后,用高效液相色谱检测红景天苷含量.结果显示,杂景天的子叶是诱导愈伤组织的理想外植体,子叶在MS 1 mg/L BA 0.5 mg/L 2,4-D和MS 1 mg/L BA 0.5 mg/L NAA培养基上的愈伤组织诱导率较高(81.8%、80%).愈伤组织有红、绿2种类型.HPLC检测显示红色愈伤组织不含红景天苷,绿色愈伤组织红景天苷含量为0.288 6%.表明利用组织培养生产红景天苷是可行的.     

Sucrose Induction of Anthocyanin Biosynthesis Mediated by DELLA

Dear Editor, While they affect all aspects of plant life, the exact molecu- lar mechanisms involved in sugar sensing and signaling are still mostly unknown. However, using the induction of antho- cyanin biosynthesis as a convenient readout and tool, the DELLA proteins can be identified as key positive regulators in sucrose-specific signaling and a point of integration of diverse metabolic and hormonal signals.     

抗生素对大豆愈伤组织的诱导和生长的影响

用红霉素、头孢唑啉钠、头孢拉定、头孢霉素(国产和进口)等5种抗生素对农杆菌LBA4404进行抑菌试验,以头孢霉素的抑菌效果最好.头孢霉素作为抑菌剂用于大豆遗传转化试验时,在下胚轴浓度以300mg/L,在子叶节以500mg/L为宜.大豆品种对卡那霉素的反应在出愈率上表现相似,在褐化率上表现有些不同.大豆不同外植体对卡那霉素的反应存在较大差异,以真叶反应最敏感,下胚轴反应最迟钝.在以卡那霉素作为抗性选择标记时,选择压力真叶和子叶节以50～100mg,L为好,下胚轴以100～200mg/L为宜。 Ahstract:The experiment of inhibiting Agrobacterium LBA4404 was undertaken with 5 antibiotics (the Erythronycin Base,Cefazolin Sodium,Cefradine,2 kinds of Cefotaximes).Among them,Cefotaxime showed the best effect.When Cefotaxime is used in transformation,the ideal concentration is 300mg/L in hypocotyl and 500mg/L in cotyledon node.The response of soybean varieties to Kanamycin is similar in induction of callus rate and is different in brown rate of callus.Differences of the response of soybean explants to Kanamycin were found.The young leaves are sensitive to Kanamycin,but hypoeotyl is not.The ideal selecting pressure of Kanamycin is 50- l00mg/L in young leaf and cotyledon node,andl00-200mg/L in hypocotyl when Kanamycin is used as selection marker.     

抗生素对大豆愈伤组织的诱导和生长的影响

用红霉素、头孢唑唑钠、头孢拉定、头孢霉素（国产和进口）等5种抗生素对农杆菌LBA4404进行抑菌试验,以头孢霉素的抑菌效果最好。头孢霉素作为抑菌剂用大于豆遗传转化试验时,在下胚轴浓度以300mg/L,在子叶节以500mg/L。大豆品种对卡那霉素的反应在出愈率上表现相似,在褐化率上表现有些不同。大豆不同外植体对卡那霉素的反应存在较大差异,以真叶反应最敏感,下胚轴反应最迟钝。在以卡那霉素作为抗性选择标记时,选择压力真叶和子叶节以50－100mg/L为好,下胚轴以100－200mg/L为宜。     

Biosynthesis of Sesquiterpenes from Deuterated Mevalonates in Perilla Callus

Cyclization mechanisms of sesquiterpenes including cuparene, β-bisabolene, and (α-curcumene have been studied by GC/MS analyses of the deuterated sesquiterpenes that were biosynthesized from deuterated mevalonates in callus of Perilla fruterscens Britton var. crispa Decne f. purpurea Makino (akachirimenshiso in Japanese). The labeling patterns of cuparene demonstrate a concurrent mechanism of a 1,4-hydride shift, and a double 1,3-hydride shift to form the cyclopentane ring, and the losses of two H-5 atoms and one H-2 atom of mevalonate to form the aromatic ring. It is postulated that a C = C bond in the cyclohexene ring of β-bisabolene might partly migrate to the neighboring C–C bond with the loss of one H-2 atom in mevalonate in its formation. The labeling patterns of deuterated α-curcumene indicate a 1,2-hydride shift to form the aromatic ring.