One-Step Microbial Conversion of a Racemic Mixture of Pantoyl Lactone to Optically Active d-(-)-Pantoyl Lactone

Washed cells of Rhodococcus erythropolis IFO 12540 were found to convert only the l-(+)-isomer of pantoyl lactone to the d-(-)-isomer in a racemic mixture of pantoyl lactone. Under suitable reaction conditions, the amount of d-(-)-pantoyl lactone synthesized was 18.2 mg/ml (94.4% enantiomer excess; molar yield, 90.5%). This conversion was suggested to proceed through the following successive reactions: first, the enzymatic oxidation of l-(+)-pantoyl lactone to ketopantoyl lactone; second, the rapid and spontaneous hydrolysis of the ketopantoyl lactone to ketopantoic acid; and then, the enzymatic reduction of the ketopantoic acid to d-(-)-pantoic acid. After the reaction d-(-)-pantoic acid could be lactonized by means of acid treatment. During the conversion, the d-(-)-isomer, which was initially present in the reaction mixture, did not undergo any modification.     

去氢木香内酯干预人乳腺癌MCF-7细胞机制的探究

目的:探讨去氢木香内酯对乳腺癌MCF-7细胞凋亡、线粒体跨膜电位及代谢物的影响,为研究去氢木香内酯诱导MCF-7细胞凋亡的作用机制提供新的视角。方法:采用流式细胞仪测定不同浓度去氢木香内酯(0、2、4、8μg/m L)对MCF-7细胞凋亡及线粒体跨膜电位的影响;GC-TOFMS测定去氢木香内酯作用前后,MCF-7细胞内具有显著性变化的代谢差异物。结果:研究结果表明,去氢木香内酯能诱导MCF-7细胞的凋亡、促进线粒体跨膜电位的降低;正交偏最小二乘法判别分析(OPLS-DA)多维统计方法对代谢组学数据分析得到柠檬酸、D-核糖、脯氨酸、苯丙氨酸、赖氨酸等16种代谢差异物。结论:推测去氢木香内酯通过引起线粒体跨膜电位降低而破坏了线粒体的结构,进一步阻碍了线粒体的功能,导致了细胞内代谢物的紊乱,最终诱导了细胞的凋亡。     

Purification and Characterization of Oxopantoyl Lactone Reductase from Higher Plants: Role in Pantothenate Synthesis

Oxopantoyl lactone reductase has been purified to homogeneity from a crude extract of spinach leaves (Spinacia oleracea L.) using affinity chromatography on Red-Agarose and several subsequent ion exchange steps. The enzyme is monomeric with a relative molecular mass between 33,000 to 36,000. Affinity-purified antibodies directed against the homogenous enzyme have been used to determine the amount of oxopantoyl lactone reductase in the crude leaf extract as well as the chloroplast stroma. The overall purification factor has been determined to be 22,000. The subcellular location of the enzyme is chloroplastic. The final specific activity (strictly NADPH-dependent) is 4.5 μmole . min^?1 . mg^?1. The enzyme is also able to reduce isatin, bornanedione and acenaphthenequinone. The enzyme activity is strongly and uncompetitively inhibited by 2-keto-4-hydroxybutyrolactone and substituted 4,5-dioxopyrrolidines. An oxopantoate reductase associated with acetohydroxy acid isomeroreductase could be detected in the plant extract. Using a specific inhibitor of this latter enzyme or oxopyrrolidines, complementation studies with branched chain amino-acids and pantothenate have shown that oxopantoyl lactone reductase is likely to be involved in pantothenate biosynthesis. Furthermore, pantoyl lactone, the putative product of the reaction, together with β-alanine and ATP, has been shown to be the substrate of pantothenate synthase using a novel assay for pantothenate.     

Excretion of Glycolate, Mesotartrate and Isocitrate Lactone by Synchronized Cultures of Ankistrodesmus braunii

Fixation of ¹⁴CO₂ by synchronized cultures of Ankistrodesmus braunii was highest for young growing cells, low for mature cells, and lowest for dividing cells. The amount of ¹⁴C excreted during photosynthesis followed the same trend. Cells at the end of the growing phase, after 10 hours of a 16-hour light phase, excreted nearly 35% of the total ¹⁴C fixed as one product, glycolate. Dividing cells from the dark phase, when tested in the light, excreted only 4% as much glycolate-¹⁴C as the young growing cells. Dividing cells also excreted as much mesotartrate as glycolate and also some isocitrate lactone and an unidentified acid. None of these excreted acids were found inside the cells in significant amounts. Methods for isolation and identification of the excreted acids are present. With ¹⁴C-labeled algae, it was shown that the excretion of glycolate was light-dependent and inhibited by 1,1-dimethyl-3-(p-chlorophenyl) urea. The excretion of labeled mesotartrate, isocitrate lactone, and an unknown acid, but not glycolate, also occurred in the dark. The excreted mesotartrate was predominantly carboxyl-labeled even after long periods of ¹⁴CO₂ fixation. Since glycolate is known to be uniformly labeled, glycolate could not be the precursor of the carboxyl-labeled mesotartrate. The reason for the specific excretion of glycolate, mesotartrate, and isocitrate lactone is not known, but the metabolism of all three acids by the algae may be limited and each can form dilactides or lactones by dehydration. In this context isocitrate lactone was excreted rather than the free acid.     

Lactone diterpenes from the aquatic plant Potamogeton natans

Four lactone diterpenes and two related glucosides with a labdane skeleton have been isolated from the aquatic plant Potamogeton natans. The structures of three new compounds were determined as 19-acetoxy-20-oxo-8(17),13-ent-labdadien-15-->16 lactone, 8(17), 13-ent-labdadien-15-->16,19-->20 dilactone and 6'-acetyl-19-glucopyranosyloxy-8(17),13-ent-labdadien-15-->16 lactone, respectively, by means of spectral analysis. Antialgal assays showed inhibitory activity for some compounds.     

Methyl Jasmonate and Lactones Including Jasmine Lactone in Ceylon Tea

The ester and lactone fraction possessing the most attractive aroma was separated from the aroma concentrate of Ceylon flavory tea by silica-gel column chromatography and analyzed by GC-MS.

Methyl 2-(cis-2′-pentenyl)-cyclopentanone-3-acetate(methyl jasmonate), 5-(cis-2′-pentenyl)-5-pentanolide (jasmine lactone), 2,3-dimethyl-2-nonen-4-olide, 4-octanolide, 4-nonanolide and 5-decanolide were newly identified as the constituents of tes aroma. Former two compounds seemed to carry a major share of aroma character of Ceylon flavory tea.     

朝鲜蓟叶中一个新的倍半萜内酯

从朝鲜蓟（Cynarascolyrnus）叶中分离得到2个倍半萜内酯,其中一个是新化合物,通过波谱学方法确定其结构为3β,8α,11α,13-四羟基-10（14）-愈创木烯-1α,4β,5α,6β氢-6α,12-内酯（1）。     

Multiple N-Acyl Homoserine Lactone Signals of Rhizobium leguminosarum Are Synthesized in a Distinct Temporal Pattern

A common form of bacterial quorum sensing involves the production and release of acyl homoserine lactone (AHL) signal metabolites. The nitrogen-fixing symbiont Rhizobium leguminosarum reportedly produces at least six different AHLs, but little is known about the regulation of biosynthesis of these molecules. We used a radiolabeling protocol to quantify the relative amounts of AHLs synthesized over time by R. leguminosarum cells with and without the symbiosis plasmid pRL1JI. Cells containing pRL1JI were found to produce three predominant signals. In decreasing order of abundance, these were N-(3-oxo)octanoyl homoserine lactone [(3-O)C(8)HSL], N-octanoyl homoserine lactone, and N-hexanoyl homoserine lactone. Cells without pRL1JI produced only two major signals, N-(3-hydroxy-7-cis)tetradecanoyl homoserine lactone [(3-OH)C(14:1)HSL] and (3-O)C(8)HSL. Each AHL exhibited a distinct temporal pattern of synthesis, suggesting that each AHL is subject to unique regulatory mechanisms. While (3-O)C(8)HSL was produced in both cultures, the patterns of synthesis were different in cells with and without pRL1JI, possibly as a result of redundant gene functions that are present on both the chromosome and the symbiosis plasmid. None of the AHLs appeared to regulate its own biosynthesis, although exogenous (3-OH)C(14:1)HSL did activate synthesis of the three AHLs made by cells containing pRL1JI. These results indicate that the synthesis of multiple AHLs in R. leguminosarum is regulated by complex mechanisms that operate independently of quorum sensing itself but that (3-OH)C(14:1)HSL can supersede these controls in pRL1JI-containing cells. This work provides an important global perspective for AHL regulation that both complements and contrasts with the results of previous studies performed with isolated gene systems.     

The Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxododecanoyl)Homoserine Lactone Contributes to Virulence and Induces Inflammation In Vivo

Pseudomonas aeruginosa has two well-characterized quorum-sensing systems, Las and Rhl. These systems are composed of LuxR-type proteins, LasR and RhlR, and two acyl homoserine lactone (AHL) synthases, LasI and RhlI. LasI catalyzes the synthesis of N-(3-oxododecanoyl)homoserine lactone (3O-C12-HSL), whereas RhlI catalyzes the synthesis of N-butyryl-homoserine lactone. There is little known about the importance of AHLs in vivo and what effects these molecules have on eukaryotic cells. In order to understand the role of AHLs in vivo, we first tested the effects that deletions of the synthase genes in P. aeruginosa had on colonization of the lung. We demonstrate that in an adult mouse acute-pneumonia model, deletion of the lasI gene or both the lasI and rhlI genes greatly diminished the ability of P. aeruginosa to colonize the lung. To determine whether AHLs have a direct effect on the host, we examined the effects of 3O-C12-HSL injected into the skin of mice. In this model, 3O-C(12)-HSL stimulated a significant induction of mRNAs for the cytokines interleukin-1alpha (IL-1alpha) and IL-6 and the chemokines macrophage inflammatory protein 2 (MIP-2), monocyte chemotactic protein 1, MIP-1beta, inducible protein 10, and T-cell activation gene 3. Additionally, dermal injections of 3O-C12-HSL also induced cyclooxygenase 2 (Cox-2) expression. The Cox-2 enzyme is important for the conversion of arachidonic acid to prostaglandins and is associated with edema, inflammatory infiltrate, fever, and pain. We also demonstrate that 3O-C12-HSL activates T cells to produce the inflammatory cytokine gamma interferon and therefore potentially promotes a Th1 environment. Induction of these inflammatory mediators in vivo is potentially responsible for the significant influx of white blood cells and subsequent tissue destruction associated with 3O-C12-HSL dermal injections. Therefore, the quorum-sensing systems of P. aeruginosa contribute to its pathogenesis both by regulating expression of virulence factors (exoenzymes and toxins) and by inducing inflammation.     

Identification of Acyl-Homoserine Lactone Signal Molecules Produced by Nitrosomonas europaea Strain Schmidt

Nitrosomonas europaea strain Schmidt produces at least three acyl homoserine lactone (AHL) signal molecules: C₆-homoserine lactone (HSL), C₈-HSL, and C₁₀-HSL. These compounds were identified in extracts of chemostat culture effluent by three independent methods. The concentrations of AHL in effluent were low (0.4 to 2.2 nM) but within the range known to induce AHL-responsive systems. The absence of LuxI and LuxM homologs from the genome of N. europaea strain Schmidt suggested that AHL synthesis occurs by an alternate pathway, possibly mediated by an HdtS homolog. To the best of our knowledge, the present report is the first to document the types and levels of AHLs produced by N. europaea.     

Costunolide and Dehydrocostus Lactone as Inhibitors of Killing Function of Cytotoxic T Lymphocytes

Costunolide and dehydrocostus lactone were isolated from an extract of mokko (Saussurea lappa Clarke) as inhibitors of killing activity of cytotoxic T lymphocytes (CTL). Mokko lactone was also isolated as an inactive compound from the extract. The structure-activity relationship indicated that α-methylenel- γ-butyrolactone is required for the inhibitory effect. Costunolide markedly inhibited the granule exocytosis and the production of inositol phosphates in response to anti-CD3 monoclonal antibody (rnAb) stimulation at a concentration that did not affect the binding of anti-CD3 mAb. Tyrosine phosphorylation induced by crosslinking of CD3 molecules was significantly inhibited by costunolide in a dose-dependent manner. These results suggest that costunolide inhibits the killing activity of CTL through preventing the increase in tyrosine phosphorylation in response to the crosslinking of T-cell receptors.     

Reaction of Acylated Homoserine Lactone Bacterial Signaling Molecules with Oxidized Halogen Antimicrobials

Oxidized halogen antimicrobials, such as hypochlorous and hypobromous acids, have been used extensively for microbial control in industrial systems. Recent discoveries have shown that acylated homoserine lactone cell-to-cell signaling molecules are important for biofilm formation in Pseudomonas aeruginosa, suggesting that biofouling can be controlled by interfering with bacterial cell-to-cell communication. This study was conducted to investigate the potential for oxidized halogens to react with acylated homoserine lactone-based signaling molecules. Acylated homoserine lactones containing a 3-oxo group were found to rapidly react with oxidized halogens, while acylated homoserine lactones lacking the 3-oxo functionality did not react. The Chromobacterium violaceum CV026 bioassay was used to determine the effects of such reactions on acylated homoserine lactone activity. The results demonstrated that 3-oxo acyl homoserine lactone activity was rapidly lost upon exposure to oxidized halogens; however, acylated homoserine lactones lacking the 3-oxo group retained activity. Experiments with the marine alga Laminaria digitata demonstrated that natural haloperoxidase systems are capable of mediating the deactivation of acylated homoserine lactones. This may illustrate a natural defense mechanism to prevent biofouling on the surface of this marine alga. The Chromobacterium violaceum activity assay illustrates that reactions between 3-oxo acylated homoserine lactone molecules and oxidized halogens do occur despite the presence of biofilm components at much greater concentrations. This work suggests that oxidized halogens may control biofilm not only via a cidal mechanism, but also by possibly interfering with 3-oxo acylated homoserine lactone-based cell signaling.     

Quantitation of cerivastatin and its seven acid and lactone biotransformation products in human serum by liquid chromatography–electrospray tandem mass spectrometry

A method for the simultaneous quantitation of cerivastatin (acid) and its biotransformation products, cerivastatin lactone, M-1 (acid), M-1 lactone, M-23 (acid), M-23 lactone, M-24 (acid) and M-24 lactone, in human serum by high-performance liquid chromatography (LC) with positive ion electrospray tandem mass spectrometry (MS–MS) was developed and validated. The method involves extraction of cerivastatin and its biotransformation products from acidified human serum (0.5 ml) using methyl tert.-butyl ether. The standard curve ranges in human serum were from 0.0100 to 10.0 ng/ml for cerivastatin and cerivastatin lactone, 0.0500 to 10.0 ng/ml for M-1 (acid) and M-1 lactone, 0.100 to 10.0 ng/ml for M-23 (acid) and M-23 lactone, and 0.500 to 10.0 ng/ml for M-24 (acid) and M-24 lactone. The lactone compounds in human serum at room temperature underwent considerable conversion to the corresponding acid compounds after only 4 h. Lowering the serum pH with a pH 5.0 buffer stabilized the lactone compounds for up to 24 h at room temperature. The degree of lactonization of the acid compounds was ≤3.5% and the degree of hydrolysis of the lactone compounds was ≤6.0% during the entire assay procedure. All the eight analytes eluted within 2.0 min and the total run time was only 3.5 min.     

Lactone ring of pectenotoxins: a key factor for their activity on cytoskeletal dynamics.

BACKGROUND: Pectenotoxins are a group of natural products from marine origin that can accumulate in shellfish and intoxicate humans. Recently, novel homologues such as pectenotoxin-11 (PTX-11) and pectenotoxin-2 seco acid (PTX-2SA) have been identified. Their toxic potential towards experimental animals has been evaluated however their interaction with cellular systems is almost unknown. This is the first report showing (i) the biological activity of PTX-11 and PTX-2SA on actin cytoskeleton and morphology of living cells and (ii) the structure- activity relationship for this family of toxic compounds. METHODS: Fluorescent phalloidin was utilized to quantify and visualize any modification in polymerized actin. Fluorescence values were obtained with laser-scanning cytometer and cells were imaged through confocal microscopy. For structure-activity evaluations, pectenotoxin-1 (PTX-1) and pectenotoxin-2 (PTX-2) was also analyzed. RESULTS: Data showed that PTX-11 triggered a remarkable depolymerizing effect on actin cytoskeleton and also modifications in the shape of cells. In contrast, PTX-2SA did not evidence the same effects. CONCLUSION: Our findings point out that (i) the actin cytoskeleton is a common target for PTX-11, PTX-2 and PTX-1, but not for PTX-2SA, and (ii) this difference in activity is related to the presence or absence of an intact lactone ring in their structures.     

Synthesis of 3′-C-Phosphono Nucleosides from α,β-Unsaturated Lactone

Abstract

The reaction of 5-protected α,β-unsaturated γ-lactone 4 with trialkylphosphite gave 3′-C-dialkylphosphono-erythro lactone 5 in high yields. The lactone 5 was reduced with DIBAL to the corresponding lactol, which was converted to the acetate 6 by treatment with acetic anhydride in pyridine. The acetate 6 was coupled with silylated thymine in the presence of TMS-triflate and the resulting anomeric mixture of nucleotides could be separated chromatographically and after desilylation using TBAF in THF the 3′-C-dialkylphosphono nucleosides 7 and 8 were obtained.     

Inhibition of Quorum Sensing in Serratia marcescens AS-1 by Synthetic Analogs of N-Acylhomoserine Lactone

Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C₆-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C₉-CPA), had a strong inhibitory effect on prodigiosin production. C₉-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C₉-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C₆-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C₉-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.     

Quantitative gas chromatographic determination of two oxidized metabolites of the diuretic mefruside in human urine,plasma and red blood cells

A gas chromatographic method is reported for the quantitative analysis of two metabolites of mefruside, viz., 5-oxo-mefruside (mefruside lactone) and its hydroxycarboxylic acid analogue in human body fluids. Use was made of extractive methylation as the derivatization technique, and quantitation was achieved, with a suitable internal standard, by means of a nitrogen-sensitive detector.Because the two metabolites are linked chemically through a lactone—open acid equilibrium, interconversion prior to their separation had to be avoided. A pH partitioning study was performed to find optimal separation conditions. The lactone could be extracted quantitatively at pH 7.4, without any trace of co-extracted hydroxy acid. The latter was extracted either at pH 2 directly (in the case of plasma and urine), or after conversion to the lactone at pH 7.4 (in the case of red cells or whole blood). Concentrations down to 25 ng per sample of both compounds could be analysed with a standard deviation of 5%.The two metabolites of mefruside equilibrated instantaneously between red cells and plasma in vitro. At 37°, the red cell/plasma concentration ratio was 20 for the lactone, but only 0.1 for the open acid compound. 5-Oxo-mefruside was able to displace mefruside from its red blood cell binding sites in vitro.     

马桑内酯对粘虫体内蛋白质和消化酶的影响

用马桑内酯分别对粘虫采用注射和饲喂处理,48 h后测定粘虫不同部位消化酶活性及蛋白质含量,以探讨马桑有效成分的作用及其杀虫机理.结果显示:(1)注射羟基马桑毒素处理后粘虫组织中蛋白质含量(与丙酮处理比较)均降低,皮组织降低幅度最大为29.47%,饲喂处理后各组织中蛋白质含量均升高,其中皮组织的升高幅度最大为56.87%;但该毒素对粘虫体内蛋白酶和羧酸酯酶的影响不明显.(2)注射与饲喂马桑亭、马桑宁,粘虫体内蛋白质含量均明显比对照处理升高,蛋白酶活性增强,其中饲喂马桑亭处理的粘虫血淋巴中蛋白质含量升高最大为511.49%,蛋白酶活性增强幅度最大为4 640.26%.马桑亭和马桑宁均可使粘虫组织中羧酸酯酶活性降低,其中饲喂马桑宁处理降低幅度最大为82.94%.结果表明:羟基马桑毒素对粘虫体内蛋白质代谢的影响与用药方式有关,马桑亭和马桑宁处理后的粘虫体内蛋白质含量增高、蛋白酶活性增强及酯酶活性降低均达到显著水平.     

Validation of an HPLC method for analysis of DB-67 and its water soluble prodrug in mouse plasma

A method for the quantitation of DB-67 ((20S)-10-hydroxy-7-tert-butyldimethylsilylcamptothecin) lactone and carboxylate in mouse plasma has been developed, validated, and applied in pharmacokinetic studies. The analytes were separated by reversed-phase chromatography with fluorescence detection. Validation demonstrated the selectivity and specificity for the carboxylate and lactone, with linearity between 1-300ng/mL and 2.5-300ng/mL for the carboxylate and lactone, respectively (accuracy 90-110% of theory and coefficient of variation < or =5.7%). Carboxylate to lactone conversion was <4% using this method. The assay was found to be suitable for the analysis of DB-67 lactone and carboxylate in pharmacokinetic studies following intravenous administration of DB-67 or its delta-aminobutyric acid ester derivative.