Detoxification of naturally occurring chromenes in larvae of the generalist herbivore Spodoptera littoralis (Noctuidae)

Five naturally occurring chromenes from the Asteraceae including the insecticidal compounds precocene II (1) and encecalin (2) were administered to last instar larvae of Spodoptera littoralis via the food or by topical contact. Metabolites formed and excreted via the frass were analysed by GC-MS and by direct comparison with reference compounds obtained by partial synthesis. In total 28 different metabolites were identified, many of them reported here for the first time. All metabolites detected originated from phase I reactions (most probably catalysed by Cytochrom P-450-dependent monooxygenases) by hydroxylation, demethylation or reduction of the parent chromenes. The resulting metabolites can be regarded as detoxification products based on previous structure-activity studies. The increased polarity of the metabolites will furthermore facilitate their excretion by the larvae compared to the more apolar parent chromenes. The largest number of metabolites (eight for each compound) was detected following oral treatment with precocene II and encecalin respectively. 3-Monool as well as 3,4-trans-diol derivatives predominated in the frass of larvae treated with the latter compounds whereas the 6-hydroxyethyl derivatives were the major metabolites of the other chromenes investigated. The patterns of metabolites originating from precocene II or encecalin were the same following oral application or topical treatment.     

Style proteins of a wild tomato (Lycopersicon peruvianum) associated with expression of self-incompatibility

The identification, isolation and aminoterminal sequencing of two S-genotype-associated proteins from style extracts of Lycopersicon peruvianum Mill. is reported. There is a high level of homology between these two sequences and with the amino-terminal sequences of other S-allele-associated glycoproteins isolated from Nicotiana alata Link et Otto. These sequences were obtained by a new high-sensitivity method of selected twodimensional gel analysis followed by electroelution and purification of proteins by inverse-gradient high-performance liquid chromatography before sequencing.Abbreviations HPLC high-performance liquid chromatography - Mr relative molecular mass - PTH phenylthiohydrantoin - SDS sodium dodecyl sulphate     

Deterrent and insecticidal chromenes and benzofurans from Encelia (asteraceae)

Four analogous chromenes and benzofurans that are among the major natural products of the genus Encelia were bioassayed for deterrency and toxicity against pest insects. The chromene encecalin has both antifeedant and insecticidal properties towards larvae of the noctuid pests Peridroma saucia and Plusia gamma. In addition to reducing larval growth and decreasing survivorship of neonate larvae fed artificial diets containing encecalin, this chromene was directly insecticidal to neonate larvae after 4 hr using a residue contact bioassay. Topical application of encecalin to third instar larvae of Peridroma led to a reduction in dietary utilization, indicative of a metabolic cost of handling the compound. The demethyl analogue of encecalin is considerably less active, and the benzofurans euparin and methyleuparin, all common constituents of Encelia, were essentially inactive or only weakly active in all bioassays against the insects.     

Chemical variation and defense of Encelia farinosa

Chemical analysis of leaves from 12 different localities of Encelia farinosa (including var. phenicodonta and var. radians) collected on the peninsula of Baja California (Mexico) revealed the presence of various chemotypes that differed with regard to the concentrations of chromenes and sesquiterpene lactones. Localities of E. farinosa collected in the northern part of Baja California were characterized by high concentrations of the chromene encecalin (up to 252 μmol g⁻¹ dry wt.), whereas the sesquiterpene lactone farinosin was not detected. Localities of E. farinosa collected at the southern tip of the peninsula lacked encecalin, but were shown to accumulate farinosin (up to 85 μmol g⁻¹ dry wt.) instead. On the mainland of Mexico, as well as in Arizona (U.S.A.), farinosin concentrations varied from 18 to 44 μmol g⁻¹ dry wt. for 10 different localities analyzed. Chromenes were not detected or present only in minor amounts (up to 13 μmol g⁻¹ dry wt.), when compared to the samples from northern Baja California. Chemical variation within localities was small when compared to variation between different localities. Accumulation of encecalin and aridity seem to coincide at least on the peninsula of Baja California, as localities of E. farinosa that receive the least amount of rainfall contained the largest amounts of encecalin in their leaves. Leaves of E. farinosa that contained sufficiently large amounts of either encecalin or farinosin were both detrimental to neonate larvae of the polyphagous pest insect Spodoptera littoralis as shown by addition of the respective crude leaf extracts to artificial diet. Possible advantages of the observed intraspecific chemical variability of E. farinosa with regard to adaptation by generalist insect herbivores are discussed.     

Occurrence of menaquinone as the sole isoprenoid quinone in the photosynthetic bacterium Heliobacterium chlorum

Thin-layer chromatography, ultraviolet spectrophotometry, high-performance liquid chromatography, and mass spectroscopy revealed that Heliobacterium chlorum contained menaquinone as the sole quinone with an average amount of 0.35 mol per g dry weight of cells. A menaquinone homologue with nine isoprene units occurred as the major component.Non-standard abbreviations MK-n menaquinone with n isoprene units - TLC thin-layer chromatography - HPLC high-performance liquid chromatography     

Microbial metabolism of d- and l-phenylglycine by Pseudomonas putida LW-4

A strain of Pseudomonas putida capable of utilizing both stereoisomers of phenylglycine as the sole carbon and energy source was isolated from soil. No phenylglycine racemase was detected in cells grown on either stereoisomer. In an initial reaction each steroisomer of phenylglycine was transaminated yielding phenylglyoxylate which was further metabolized via benzaldehyde to benzoate. Subsequently, benzoate was further degraded via an ortho-cleavage of catechol.Abbreviation HPLC high-performance liquid chromatography     

A plant lipid and the platelet-activating factor stimulate ATP-dependent H+ transport in isolated plant membrane vesicles

A plant lipid was isolated from zucchini (Cucurbita pepo L.) membranes and from soybean (Glycine max [L.] Merr) phospholipids by thinlayer chromatography and further purified by high-performance liquid chromatography. This plant lipid was chromatographically very similar to the platelet-activating factor, an ether phospho-lipid with hormone-like properties found in mammals. Both the plant lipid and the platelet-activating factor stimulated ATP-dependent H⁺ transport in isolated membrane vesicles from zucchini hypocotyls.Abbreviations HPLC high-performance liquid chromatography - PAF platelet-activating factor     

Quantitative variation of the sesquiterpene lactones and chromenes of encelia farinosa

The components of the two major classes of natural products of the composite Encelia farinosa, sesquiterpene lactones and benzopyrans/benzofurans (chromenes), were resolved and quantified by high performance liquid chromatography. Geographic variation was examined over a range of mesic and xeric populations from California and Arizona (USA). The concentration of farinosin, a sesquiterpene lactone, varies significantly between individuals and seasonally within individuals. Ontogenetic comparisons revealed interpopulation variations within sesquiterpene lactones and chromenes. A geographic survey suggests the presence of two putative chemical races which vary in the amounts of sesquiterpene lactones and chromenes present. The adaptive significance of this quantitative variation is discussed.     

Immunochemical detection of ochratoxin A in black Aspergillus strains

One hundred and fifty-seven strains belonging to Aspergillus section Nigri were tested for ochratoxin A production using three different methods: a relatively new immunochemical method based on an enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The monoclonal antibody-based ELISA technique was successfully used to screen for low levels of ochratoxin A in the black Aspergilli without concentrating the culture filtrates. The results were confirmed by TLC and HPLC analysis and chemical derivatization. These latter methods required concentrated filtrates. Ochratoxin A was detected in the culture filtrates of five of the 12 A. carbonarius strains, none of the 45 A.japonicus strains and three of the 100 isolates in the A. niger aggregate (A. foetidus, A. awamori and A. niger).Abbreviations ELISA enzyme-linked immunosorbent assay - HPLC high-performance liquid chromatography - OA ochratoxin A - TLC thin-layer chromatography     

Determination of trans-resveratrol in human plasma by high-performance liquid chromatography

The first method using high-performance liquid chromatography (HPLC) has been developed for the determination of trans-resveratrol in human plasma. The method involves a liquid–liquid extraction followed by reversed-phase HPLC with UV detection. The detection limit of trans-resveratrol in human plasma was 5.0 ng/ml. Standard curves are linear over the concentration range of 5.0–5000.0 ng/ml. Intra-assay variability ranged from 1.9 to 3.7% and inter-assay variability ranged from 2.5 to 4.0% at the concentration range of 15.0–4000.0 ng/ml.     

Enantioselective analysis of disopyramide and mono-N-dealkyldisopyramide in plasma and urine by high-performance liquid chromatography on an amylose-derived chiral stationary phase

An enantioselective high-performance liquid chromatography method was developed for the simultaneous determination of disopyramide (DP) and mono-N-dealkyldisopyramide (MND) enantiomers in plasma and urine. The drugs were extracted from plasma samples by liquid–liquid extraction with dichloromethane after protein precipitation with trichloroacetic acid; the urine samples were processed by liquid–liquid extraction with dichloromethane. The enantiomers were resolved on a Chiralpak AD column using hexane–ethanol (91:9, v/v) plus 0.1% diethylamine as the mobile phase and monitored at 270 nm. Under these conditions the enantiomeric fractions of the drug and of its metabolite were analyzed within 20 min. The extraction procedure was efficient in removing endogenous interferents and low values for the relative standard deviations were demonstrated for both within-day and between-day assays. The method described in this paper allows the determination of DP and MND enantiomers at plasma levels as low as 12.5 ng/ml and can be used in clinical pharmacokinetic studies.     

Seasonal age-specific measurements of the sesquiterpene lactones and chromenes of Encelia farinosa

Age-specific concentrations of farinosin, a sesquiterpene lactone, and encecalin and euparin, both chromenes, were measured in two semi-desert populations of Encelia farinosa in California over a three month period. All three natural products differed significantly in concentration. The average concentration of encecalin is 10 times that of euparin and eight times that of farinosin. Farinosin concentrations did not differ between the two populations, but both euparin and encecalin were in higher amounts in the UC Riverside population than in the Home Gardens population. Seasonal changes in natural products differed at the 0.05 level for all populations and compounds except euparin in the UC Riverside population. Percent nitrogen content of dried leaves was similar in both populations, but differed between collection dates. Water content did not vary significantly with leaf age on one collection date. The significance of these results is discussed in the light of herbivore history.     

Measurement of serum levels of natalizumab, an immunoglobulin G4 therapeutic monoclonal antibody

Chlorophyllide a is a metabolite late in the biosynthesis of chlorophylls and bacteriochlorophylls. Isolation procedures for chlorophyllide a from Rhodobacter capsulatus CB1200 and barley (Hordeum vulgare L.) are described and compared. R. capsulatus CB1200 is a double mutant in the bacteriochlorophyllide a biosynthetic pathway, and chlorophyllide a is excreted by the cells when grown in Tween 80-containing liquid medium. It was purified by liquid or solid phase extraction, yielding 7 mg of chlorophyllide a from 1 L of culture. In a second approach, intrinsic chlorophyllase activity was used to dephytylate chlorophyll in an acetonic preparation of leaves of wild-type or chlorophyll b-deficient barley. Purification was achieved by liquid phase extraction, yielding 14 μg of chlorophyllide a per gram of barley leaves. Chlorophyllide a was identified by thin layer chromatography, absorption spectroscopy, and mass spectrometry.     

Metabolism of [14C]indole-3-acetic acid by the cortical and stelar tissues of Zea mays L. roots

Reverse-phase high-performance liquid chromatography was used to analyse ¹⁴C-labelled metabolites of indole-3-acetic acid (IAA) formed in the cortical and stelar tissues of Zea mays roots. After a 2-h incubation in [¹⁴C]IAA, stelar segments had metabolised between 1–6% of the methanol-extractable radioactivity compared with 91–92% by the cortical segments. The pattern of metabolites produced by cortical segments was similar to that produced by intact segments bathed in aqueous solutions of [¹⁴C]IAA. In contrast, when IAA was supplied in agar blocks to stelar tissue protruding from the basal ends of segments, negligible metabolism was evident. On the basis of its retention characteristics both before and after methylation, the major metabolite of [¹⁴C]IAA in Zea mays root segments was tentatively identified by high-performance liquid chromatography as oxindole-3-acetic acid.Abbreviations HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid     

Metallo-proteinase from the seedlings of kale (Brassica oleracea L. var. sabellica):

Metallo-proteinase from 8-d-old seedlings of kale was isolated. The enzyme was extracted with 1% NaCl, concentrated by ammonium sulfate and finally purified by high-performance liquid chromatography. The isolated enzyme had a molecular weight of 22.4 kDa and showed a maximum activity at pH 9.0 using casein as a substrate. Proteolytic activity of proteinase was inhibited by chelators. The inhibition by ethylenediaminetetraacetate (EDTA) was abolished by some divalent metals ions, especially by Zn²⁺. The enzyme showed activity against the synthetic peptides Suc-Ala-Ala-Pro-Leu-pNA and Suc-Ala-Ala-Pro-Phe-pNA, and hydrolized the following peptide bonds in the oxidized insulin B-chain: Leu6-Cya7, Leu15-Tyr16, Leu17—Val18 and Phe25-Tyr26.Abbreviations EDTA ethylenediaminotetraacetic acid - HPLC high-performance liquid chromatography - NEM N-ethylmaleimide - PCMB p-mecuribenzoic acid - PMSF phenylmethylsulfonyl fluoride This work was supported by the University Science Programme, Ministry of National Education, and Polish Academy of Science, Warsaw, Poland.     

Identification of the major chlorosomal bacteriochlorophylls of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides; their function in lateral energy transfer

The chlorosomal bacteriochlorophyll (BChl) composition of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides was investigated by means of normal-phase high-performance liquid chromatography. From both species a number of homologues was isolated, which were identified by absorption and ²⁵²Cf-plasma desorption mass spectroscopy. Besides BChl d, C. vibrioforme contained a significant amount of BChl c, which may provide an explanation for the previous observation of at least two spectrally different pools of BChl in the chlorosomes of green sulfur bacteria (Otte et al. 1991). C. phaeovibrioides contained various homologues of BChl e only. Absorption spectra in acetone of BChl c, d and e, as well as bacteriopheophytin e are presented. No systematic differences were found for the various homologues of each pigment. In addition to farnesol, the mass spectra revealed the presence of various minor esterifying alcohols in both species, including phytol, oleol, cetol and 4-undecyl-2-furanmethanol, as well as an alcohol of low molecular mass, which is tentatively assumed to be decenol.Abbreviations BChl bacteriochlorophyll - BPh bacteriopheophytin (used as a general name for the Mg-free compound, irrespective of the esterifying alcohol) - HPLC high-performance liquid chromatography     

Heterologous expression of the kanamycin biosynthetic gene cluster (pSKC2) in <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis> YJ003

The pSKC2 cosmid, which has 32 kb and 28 open-reading frames, was isolated from Streptomyces kanamyceticus ATCC12853 as the gene cluster of kanamycin. This gene cluster includes the minimal biosynthetic genes of kanamycin with the resistance and regulatory genes. It was heterologously expressed in Streptomyces venezuelae YJ003, which has the advantage of fast growth, good efficiency of the transformation host, and rapid production of the aminoglycosides antibiotic. The isolated compound was analyzed by electrospray ionization–mass spectrometry, liquid chromatography–mass spectrometry, high-performance liquid chromatography, and tandem mass spectrometry and shows a molecular weight of 485 as kanamycin A.     

Cytokinins in transfer RNA of normal and crown-gall tissue of Vinca rosea

cis-Zeatin riboside was identified in transfer-RNA hydrolysates from both normal and crown-gall tissue of Vinca rosea L. The trans-isomer was associated exclusively with the crowngall transfer-RNA. The importance of these observations is discussed in relation to biosynthesis of free cytokinins.Abbreviations GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - TMSi trimethylsilyl - tRNA transfer RNA - ZR zeatin riboside     

<Emphasis Type="Italic">Bacillus subtilis</Emphasis> as a bioindicator for estimating pentachlorophenol toxicity and concentration

Pentachlorophenol (PCP) and its sodium salt (Na-PCP) are extremely toxic chemicals responsible for important soil and groundwater pollution, mainly caused by wastes from wood-treatment plants, because chlorinated phenols are widely used as wood preservatives. The methods most commonly used for routine analysis of pesticides such as PCP and Na-PCP are high-performance liquid chromatography (HPLC) and gas chromatography–mass spectroscopy (GC–MS). A variety of rapid biological screening tests using marine organisms, bioluminescent bacteria, and enzymes have also been reported. In this study, rapid biological screening analysis using Bacillus subtilis was developed, to assess the biodegradation of PCP and its by-products in liquid samples. An empirical model is proposed for spectrophotometric analysis of Na-PCP concentration after growth of Bacillus subtilis.     

Purification and cloning of a novel antimicrobial peptide from salivary glands of the hard tick, Ixodes sinensis

A novel antimicrobial peptide named as ixosin-B was isolated from the salivary glands of the hard tick, Ixodes sinensis, by gel filtration, ion exchange chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY by Edman degradation. The cDNA encoding ixosin-B was cloned by cDNA library screening. The predicted protein from the cDNA sequence composed of 89 amino acids including mature ixosin-B. Purified ixosin-B exerted its antimicrobial activities against bacteria and fungi. No similarity was found by BLAST search to any database entries and, thus, our findings describe a novel antimicrobial peptide. It is also the fourth family of antimicrobial peptide from hard ticks.