A solid-phase method for the extraction and measurement of anandamide from multiple human biomatrices

N-Arachidonoylethanolamine (AEA, anandamide) was the first endocannabinoid to be identified and has since become associated with the mediation of several physiological functions and disease states. AEA has been isolated from numerous tissues and biofluids, in the low nanomolar range, using lipid extraction techniques with organic solvents. These techniques require the drying down of relatively large volumes of solvents, making them unsuitable for high-throughput analysis. Here we describe a solid-phase extraction (SPE) method for the investigation of AEA concentrations in human plasma, serum, milk, urine, amniotic fluid, peritoneal fluid, saliva, follicular fluid, and fluid from an ovarian cyst. AEA was detected in serum and plasma from blood isolated from 20 adult women (means ± standard deviations: 0.68 ± 0.29 and 0.64 ± 0.28 nM, respectively), from pregnant women at term (1.37 ± 0.42 nM), and from umbilical vein (1.26 ± 0.33 nM) and umbilical artery (1.14 ± 0.35 nM), in milk (0.12 ± 0.05 nM) and from amniotic (0.03 ± 0.02 nM), peritoneal (0.93 ± 0.27 nM), follicular (1.17 ± 0.51 nM), and ovarian cyst (0.32 ± 0.01 nM) fluids. AEA was undetectable in saliva and urine. The 60% AEA extraction efficiency achieved with SPE from plasma was superior to the 19% efficiency achieved using the existing organic solvent extraction method. Limits of quantification and detection for AEA were also improved dramatically using SPE (8 and 4 fmol/ml) compared with organic extraction (25 and 18.75 fmol/ml plasma). These improvements allow the use of smaller plasma samples with SPE. Intra- and interday variability were comparable, and the mean AEA concentration of pooled plasma samples (1.18 nM, n = 15) was identical with the two techniques. Similarly, when 56 plasma samples from laboring and nonlaboring women were analyzed using both techniques, no extraction method-dependent differences were observed. Consequently, we provide evidence for a robust SPE technique for the extraction of AEA from biomatrices to replace the existing liquid extraction methods, with the SPE technique being superior in terms of speed, extraction efficiency, and sample size required.     

Antihypertensive and endothelium-dependent vasodilator effects of aqueous extract of Cistus ladaniferus

Cistus ladaniferus L. (Cistaceae) is a medicinal plant originated from the Mediterranean region which exerts different pharmacological effects. In the present study, our goal was to examine whether the plant possessed antihypertensive properties. Aqueous extract of Cistus leaves (AEC, 500 mg/kg/day) reduced systemic blood pressure (SBP) in two animal models of hypertension, the l-NAME and renovascular two kidney-one clip (2K-1C) hypertensive rats. In the former, AEC prevented the increase in SBP when co-administered with l-NAME during four weeks (164 ± 3 mm Hg in l-NAME vs. 146 ± 1 mm Hg in l-NAME + AEC, p < 0.001). In the latter, AEC reversed the increase in SBP when administered during four weeks after installation of the hypertension (146 ± 5 mm Hg with AEC vs. 179 ± 6 mm Hg without, p < 0.05). AEC treatment also reversed the endothelial dysfunction observed in both animal models of hypertension. A direct effect on cardiac and vascular tissue was also tested by examining the contractile effects of AEC in rat isolated aortic rings and Langendorff perfused hearts. AEC (10 mg/L) had no effect on left ventricular developed pressure and heart rate in isolated perfused heart. However, AEC produced a strong relaxation of pre-contracted rat aortic rings (80 ± 2% relaxation, n = 25). When the rings were denuded from endothelium or were incubated with 1 mM Nω-nitro-l-arginine (l-NNA), the relaxant effect of AEC was lost. We conclude that C. ladaniferus possesses antihypertensive properties which are mainly due to an endothelium-dependent vasodilatory action.     

Effects of the aqueous extract of white tea (Camellia sinensis) in a streptozotocin-induced diabetes model of rats

White tea (WT) is very similar to green tea (GT) but it is exceptionally prepared only from the buds and young tea leaves of Camelia sinensis plant while GT is prepared from the matured tea leaves. The present study was investigated to examine the effects of a 0.5% aqueous extract of WT in a streptozotocin-induced diabetes model of rats. Six-week-old male Sprague-Dawley rats were divided into 3 groups of 6 animals in each group namely: normal control (NC), diabetic control (DBC) and diabetic white tea (DWT). Diabetes was induced by an intraperitoneal injection of streptozotocin (65 mg/kg BW) in DBC and DWT groups except the NC group. After 4 weeks feeding of 0.5% aqueous extracts of WT, the drink intake was significantly (P < 0.05) increased in the DWT group compared to the DBC and NC groups. Blood glucose concentrations were significantly decreased and glucose tolerance ability was significantly improved in the DWT group compared to the DBC group. Liver weight and liver glycogen were significantly increased and serum total cholesterol and LDL-cholesterol were significantly decreased in the DWT group compared to the DBC group. The food intake, body weight gain, serum insulin and fructosamine concentrations were not influenced by the consumption of WT. Data of this study suggest that the 0.5% aqueous extract of WT is effective to reduce most of the diabetes associated abnormalities in a steptozotocin-induced diabetes model of rats.     

The composition of the free fatty acids from Dendrolimus pini exuviae

The pine moth Dendrolimus pini effectively resists many insecticides, but it can be controlled by the use of bioinsecticides such as entomopathogenic fungi. In the use of microbial agents for the biocontrol of D. pini, it is important to identify the cuticular lipids of this pest if we are to understand the factors responsible for the preferential adhesion or selective repulsion of entomopathogenic fungi that are potentially useful in biocontrol. In this work the qualitative and quantitative analyses of free fatty acids in two exuviae extracts (petroleum ether and dichloromethane) and two developmental stages (larval-larval and larval-pupal molts) were studied. The free fatty acid composition of the epicuticular lipids from exuviae of D. pini was characterized chemically using gas chromatography (GC) and gas chromatography-electron impact mass spectrometry (GC-MS). Structural analyses of the dichloromethane extracts from larval-larval exuviae (LLE) and larval-pupal exuviae (LPE) revealed that the carbon numbers for the major acid moieties ranged from C_8:0 to C_34:0. Only C_23:0 was not identified in the LPE extract. The relative contents of fatty acids in the extracts varied from trace amounts to 34%. The fatty acids extracted by dichloromethane were essentially the same as those in the petroleum ether extract. We also identified dehydroabietic acid in the exuviae of D. pini. The respective quantities of dehydroabietic acid obtained from D. pini LLE and LPE were 1763 ± 103 μg/g exuviae and 11521 ± 1198 μg/g of exuviae.     

Separation and purification of echinacoside from Penstemon barbatus (Can.) Roth by recycling high-speed counter-current chromatography

Echinacoside is an important bioactive compound extracted from Cistanche tubulosa which was endangered by overexploitation. It is imperative to find an alternative source. Echinacoside was isolated from Penstemon barbatus (Can.) Roth for the first time. The peak contents of echinacoside are 9.09 ± 0.32 mg/g and 7.25 ± 0.36 mg/g respectively in the leaves and roots annually. The methanolic extracts from 20 g of dried powder of the roots of P. barbatus were pre-purified by AB-8 resin and the fraction containing echinacoside was further purified by conventional high-speed counter-current chromatography (HSCCC) and recycling HSCCC with the solvent system n-butanol–water (1:1, v/v). Totally 42.0 mg echinacoside with a purity of 96.3% was recovered. The recovery rate of echinacoside by recycling HSCCC reached 91.0%. The structure of our echinacoside confirmed by IR, ¹H NMR and ¹³C NMR is identical to the standard sample. This indicates that P. barbatus might be ideal source for preparation of large scale of echinacoside.     

Regulation of growth by the trehalose pathway: Relationship to temperature and sucrose

Carbon signaling can override carbon supply in the regulation of growth. At least some of this regulation is imparted by the sugar signal trehalose 6-phosphate (T6P) through the protein kinase, SnRK1. This signaling pathway regulates biosynthetic processes involved in growth under optimal growing conditions. Recently, using a seedling system we showed that under sub-optimal conditions, such as cold, carbon signaling by T6P/ SnRK1 enables recovery of growth following relief of the stress. The T6P/ SnRK1 mechanism thus could be selected as a means of improving low temperature tolerance. High-throughput automated Fv/Fm measurements provide a potential means to screen for T6P/ SnRK1, and here we confirm through measurements of Fv/Fm in rosettes that T6P promotes low temperature tolerance and recovery during cold to warm transfer. Further, to better understand the coordination between sugars, trehalose pathway, and temperature-dependent growth, we examine the interrelationship between sugars, trehalose phosphate synthase (TPS), and trehalose phosphate phosphatase (TPP) gene expression and T6P content in seedlings. Sucrose, particularly when fed exogenously, correlated well with TPS1 and TPPB gene expression, suggesting that these enzymes are involved in maintaining carbon flux through the pathway in relation to sucrose supply. However, when sucrose accumulated to higher levels under low temperature and low N, TPS1 and TPPB expression were less directly related to sucrose; other factors may also contribute to regulation of TPS1 and TPPB expression under these conditions. TPPA expression was not related to sucrose content and all genes were not well correlated with endogenous glucose. Our work has implications for understanding acclimation to sink-limited growth conditions such as low temperature and for screening cold-tolerant genotypes with altered T6P/ SnRK1 signaling.     

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography–electrospray ionization–tandem mass spectrometry

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) and ³⁴S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC–MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r² > 0.99) over a range of concentrations (SAM 0.01–2.0 μM, SAH 0.002–0.10 μM, Met 0.05–4.0 μM, GSH 0.17–20.0 μM, GSSG 0.07–20.0 μM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1 nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.     

A cytotoxic type-2 ribosome inactivating protein (from leafless mistletoe) lacking sugar binding activity

Articulatin-D, a 66 kDa ribosome inactivating protein (RIP) comprised of 29 kDa A-chain linked to 35 kDa B-chain, is purified from leafless mistletoe (Viscum articulatum) parasitic on Dalbergia sp. from Western Ghats (India). N-terminal sequence and LC-MS/MS analyses of A- and B-chain confirmed that articulatin-D is a type-2 RIP having high homology with other mistletoe lectins. Translation inhibition and diagnostic N-glycosidase activity of articulatin-D illustrate the presence of catalytically active A-chain. Its inability to: (i) bind to acid treated Sepharose CL-6B column, (ii) agglutinate trypsin-treated and untreated RBCs of human (A, B, O, AB), mice, rat, rabbit, buffalo, porcine, pigeon, cock, fish, sheep and goat even with 10 mg/ml of purified articulatin-D, (iii) show change in circular dichroism spectra after addition of sugar to the native protein, (iv) bind to different sugars (galactose, lactose, gal-NAc, rhamnose, arabinose, fucose and mannose) immobilized on Sepharose 4B matrix, and (v) show change in enthalpy during titration with galactose confirm that the B-chain of articulatin-D lacks sugar binding activity. Despite this, articulatin-D is highly toxic as characterized with low IC₅₀ against different cancer cell lines (Jurkat: 0.31 ± 0.02 nM, MOLT-4: 0.51 ± 0.03 nM, U-937: 0.64 ± 0.07 nM, HL-60: 0.79 ± 0.11 nM, Raji: 1.45 ± 0.09 nM). Toxicity of RIPs has been ascribed to the absence/presence of B-chain with sugar binding activity. Identification of articulatin-D, the first cytotoxic RIP with B-chain lacking sugar binding activity opens new vistas in understanding cytotoxic action of RIPs.     

Expression and characterization of the Arabidopsis thaliana 11S globulin family

The 11S globulins are the principal seed storage proteins in a variety of major crop species, including members of the legume and mustard families. They are targets for protein engineering studies attempting to alter the physicochemical properties of seed protein extracts (e.g. soybean) and to improve the nutritional quality of important agricultural crops. A key factor that has limited the success of this approach to date is insufficient accumulation of the engineered protein variants in vivo due to their improper folding and/or reduced stability, compared to the native protein. We have developed the Arabidopsis thaliana 11S proglobulins as a model system to enable studies exploring the factors underlying structural stability in this family of proteins. Yields of 1.5–4 mg/L were achieved for the three A. thaliana 11S proglobulins expressed in the Origami Escherichia coli cell line in super broth media at 20 °C for 16 h and purified via immobilized-metal affinity chromatography. We also demonstrate that differential scanning fluorimetry is an effective and accessible technique to facilitate the screening of variants to enable the successful engineering of 11S seed storage proteins. The relative in vitro stability of the A. thaliana 11S proglobulins (proAtCRU1 > proAtCRU3 > proAtCRU2) is consistent between chemical and thermal denaturation studies.     

Cytokinin profiling in plant tissues using ultra-performance liquid chromatography-electrospray tandem mass spectrometry

We have developed a simple, high-throughput batch immunoextraction (IAE) micropurification procedure for extracting a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) from plant tissues in solutions that are compatible with ultra-performance liquid chromatography (UPLC), thereby facilitating sensitive subsequent analysis. The UPLC system was coupled to a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray interface (ESI). Small (mg) amounts of tissues were purified by solid-phase extraction (SPE) followed by an immunoaffinity clean-up step and two fast chromatographic separations of most cytokinin metabolites (bases, ribosides, and 9-glucosides in the first, O-glucosides and nucleotides in the second). Using UPLC, the runs were up to 4-fold faster than in standard cytokinin analyses, and both retention times and injection volumes were less variable (RSDs, 0.15-0.3% and 1.0-5.5%, respectively). In multiple reaction monitoring (MRM) mode, the detection limit for most of the cytokinins analyzed was close to 1 fmol (5-25 fmol for O-glucosides and nucleotides) and the linear range spanned at least five orders of magnitude. The extraction and purification method was optimized using poplar (Populus × canadensis Moench, cv Robusta) leaf samples, and the analytical accuracy was further validated using IAE-purified 10-day-old Arabidopsis thaliana plants spiked with 1 and 10 pmol of cytokinin derivatives. This approach can be used for rapid, sensitive qualitative and/or quantitative analysis of more than 50 natural cytokinins in minute amounts of plant tissues with high performance, robustness, and accuracy.     

Measurement of glycolysis reactants by high-throughput solid phase extraction with tandem mass spectrometry: Characterization of pyrophosphate-dependent phosphofructokinase as a case study

Glycolysis is a 10-step metabolic pathway involved in producing cellular energy. Many tumors exhibit accelerated glycolytic rates, and enzymes that participate in this pathway are focal points of cancer research. Here, a novel method for the measurement of glycolysis reactants from in vitro samples is presented. Fast and direct measurement is achieved by an automated system that couples on-line solid phase extraction (SPE) with tandem mass spectrometry (MS/MS). The single analytical method enables multiple reactants to be measured concurrently, sustains a cycle time of 8 s, and permits the measurement of up to 10,000 samples per day. Concentration–response curves were conducted using standards for 10 metabolic intermediates, and the results demonstrate that the detection strategy has excellent sensitivity (average limit of detection = 5.4 nM), dynamic range (nanomolar to micromolar), and linear response (average R² = 0.998). To test the analysis method on reactions, pyrophosphate-dependent phosphofructokinase (PPi–PFK) was used as a model system. Data that corroborate the activation and inhibition of PPi–PFK are presented, and the ways in which SPE–MS/MS simplifies experimental design and interpretation are highlighted. In summary, the method for measuring metabolic intermediates described here demonstrates unprecedented speed, performance, and versatility.     

In vitro and in vivo inhibition of plant polyamine oxidase activity by polyamine analogues

Polyamine oxidase from Avena sativa L. cv. Cristal seedlings was purified to homogeneity using a simple four-step purification protocol including an infiltration washing technique. The enzyme had a high affinity for spermidine and spermine (K_m ∼ 5.5 and 1.2 μM, respectively), and also oxidized norspermidine (K_m ∼ 64.0 μM). Natural and synthetic diamines, cyclohexylamine, the putrescine analogue 1-aminooxy-3-aminopropane, and several polyamine analogues had inhibitory effects on polyamine oxidase activity and none were substrates. No inhibitory effect was observed on spermidine oxidation when the reaction product 1,3-diaminopropane was added. By contrast, 1-aminooxy-3-aminopropane showed mixed inhibition kinetics and a K_i value of 0.113 mM. In addition, in vitro enzymatic activity assays showed that the oligoamine [3,8,13,18,23,28,33,38,43,48-deca-aza-(trans-25)-pentacontene], the tetramine 1,14-bis-[ethylamino]-5,10-diazatetradecane, and the pentamine 1,19-bis-[ethylamino]-5,10,15-triazanonadecane, displayed potent competitive inhibitory activities against polyamine oxidase with K_i values of 5.8, 110.0 and 7.6 nM, respectively, where cyclohexylamine was a weak competitive inhibitor with a K_i value of 0.5 mM. These analogues did not inhibit mycelial growth of the fungus Sclerotinia sclerotiorum (Lib.) De Bary and the bacterium Pseudomonas viridiflava (Burkholder) Dowson in vitro. On the contrary, with concentrations similar to those used for polyamine analogues, guazatine (a well-known fungicide and at the same time, a polyamine oxidase inhibitor) inhibited (∼85%) S. sclerotiorum mycelial growth on Czapek-Dox medium.Finally, the analogue 1,19-bis-ethylamino-5,10,15-triazanonadecane inhibited polyamine oxidase activity observed in segments of maize leaves in vivo. The results obtained provide insights into research on the influence of polyamine oxidase activity on plant biotic and abiotic stresses.     

2-Arylimidazo[2,1-b]benzothiazoles: a new family of amyloid binding agents with potential for PET and SPECT imaging of Alzheimer's brain

We designed and synthesized a small series of 2-aryl-imidazo[2,1-b]benzothiazole, representing a combination of motifs from the two most potent amyloid imaging agents, PIB and IMPY. The binding affinity of the new compounds ranged from 6 to 133 nM. Among the best compounds, 3b (K_i = 6 nM) can be labeled with ¹¹CH₃ for PET imaging whereas 3j (K_i = 10.9 nM) can be labeled with ¹²³I for SPECT imaging.     

High-performance liquid chromatographic procedures for the quantitative determination of paclitaxel (Taxol) in human urine

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for the quantitative determination of paclitaxel in human urine. A comparison is made between solid-phase extraction (SPE) and liquid-liquid extraction (LLE) as sample pretreatment. The HPLC system consists of an APEX octyl analytical column and acetonitrile-methanol-0.2 μM ammonium acetate buffer pH 5 (4:1:5, v/v) as the mobile phase. Detection is performed by UV absorbance measurement at 227 nm. The SPE procedure involves extraction on Cyano Bond Elut columns. n-Butylchloride is the organic extraction fluid used for the LLE. The recoveries of paclitaxel in human urine are 79 and 75% for SPE and LLE, respectively. The accuracy for the LLE and SPE sample pretreatment procedures is 100.4 and 104.9%, respectively, at a 5 μg/ml drug concentration. The lower limit of quantitation is 0.01 μg/ml for SPE and 0.25 μg/ml for LLE. Stability data of paclitaxel in human urine are also presented.     

Using a starch-rich mutant of Arabidopsis thaliana as feedstock for fermentative hydrogen production

A mutant plant (Arabidopsis thaliana), sex1-1 (starch excess 1-1), accumulating high starch content in leaves was created to serve as better biomass feedstock for a H₂-producing strain Clostridium butyricum CGS2, which efficiently utilizes starch for H₂ production but cannot assimilate cellulosic materials. The starch content of the mutant plant increased to 10.67 mg/fresh weight, which is four times higher than that of wild type plant. Using sex1-1 mutant plant as feedstock, C. butyricum CGS2 could produce 490.4 ml/l of H₂ with a H₂ production rate of 32.9 ml/h/l. The H₂ production performance appeared to increase with the increase in the concentration of mutant plant from 2.5 to 10 g/l. The highest H₂ to plant biomass yield was nearly 49 ml/g for the mutant plant. This study successfully demonstrated the feasibility of using a starch-rich mutant plant for more effective bioH₂ production with C. butyricum CGS2.     

The physicochemical properties and chemical composition of trehalose lipids produced by Rhodococcus erythropolis 51T7

This study analyzed the chemical and physical properties of a biosurfactant synthesized by Rhodococcus sp. 51T7. The biosurfactant was a trehalose tetraester (THL) consisting of six components: one major and five minor. The hydrophobic moieties ranged in size from 9 to 11 carbons. The critical micelle concentration (CMC) was 0.037 g L⁻¹ and the interfacial tension against hexadecane was 5 mN m⁻¹. At pH 7.4 the glycolipid CMC/critical aggregation concentration (CAC) was 0.05 g L⁻¹ and at pH 4 it was 0.034 g L⁻¹. A phase diagram revealed effective emulsification with water and paraffin or isopropyl myristate. A composition of 11.3-7.5-81.8 (isopropyl myristate-THL-W) was stable for at least 3 months. The HLB was 11 and the phase behaviour of the glycolipid revealed the formation of lamellar and hexagonal liquid-crystalline textures.     

Phytotoxic polyacetylenes from roots of Russian knapweed (Acroptilon repens (L.) DC.)

There are several factors thought to assist invasive weeds in colonization of ecosystems. One of these factors is allelopathy, the negative effect of chemicals produced by one plant on neighboring plants, frequently mediated through root exudates and other plant leachates. Acroptilon repens (Asteraceae) is one of the most invasive and ecologically threatening weed species in western North America. A bioassay-guided fractionation of the root extracts of this plant led to the isolation of five polyacetylenic compounds, of which one [5′-methoxy-1′-(5-prop-1-yn-1-yl-2-thienyl)-hexa-2′,4′-diyin-6′-yl acetate] was hitherto unknown. The structures of these compounds were elucidated on the basis of spectroscopic analysis (IR, ESIMS, ¹H, ¹³C NMR and 2D NMR). All of the compounds obtained, except 1-chloro-4-(5-penta-1,3-diyn-1-yl-2-thienyl)but-3-yn-2-ol, showed phytotoxic activity against Arabidopsis thaliana seedlings. The presence of 4′-chloro-1′-(5-penta-1,3-diyn-1-yl-2-thienyl)-but-2′-yn-3′-ol was detected in the root exudates of aeroponically grown A. repens plants. None of the polyacetylenes isolated in this study were found in Colorado soils collected between September 2006 and July 2007 in an A. repens colonized site. However, polyacetylene 5 in A. repens infested soil from Washington was found in June, 2007. Contrary to our previous report, the compound 7,8-benzoflavone (6) was not detected in root exudates, nor was it encountered in extracts of roots, aerial parts or infested soil. Since we could not repeat this work, the original report has been retracted [Stermitz, F.R., Bais, H.P., Foderaro, T.A., Vivanco, J.M., 2003. 7,8-Benzoflavone: a phytotoxin from root exudates of invasive Russian knapweed [A retraction]. Phytochemistry 64, 493-497.].