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A TLC bioautographic assay for the detection of nitrofurantoin resistance reversal compound.

A simple TLC bioautographic method was developed for detection of antibiotic resistance reversal agents. In this study, the retention factor values of the components of some essential oils not previously shown to have any antibacterial activity were evaluated on nitrofurantoin supplemented agar media. The active component of Artemisia annua, Artemisia dracunculus and Eucalyptus globulus essential oils was piperitone which increased the antibacterial activity of nitrofurantoin against Enterobacter cloacae. Piperitone was not detected in the essential oil of Humulus lupulus and we could not observe any clear areas in this bioautographic method.

Chromatography, Thin Layer↗

Cloning and over-expression of a cDNA encoding a polyketide synthase from Cannabis sativa.

A polyketide synthase has been suggested to play an important role in cannabinoid biosynthesis in Cannabis sativa L. This enzyme catalyzes the biosynthesis of olivetolic acid, one of the precursors for cannabinoid biosynthesis. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) based on the DNA homology of chalcone synthase (EC 2.3.1.156) and valerophenone synthase (EC 2.3.1.156) of hop (Humulus lupulus), a cDNA encoding a polyketide synthase in C. sativa was identified. The coding region of the gene is 1170 bp long encoding a 389 amino acid protein of a predicted 42.7 kDa molecular mass and with a pI of 6.04. The gene shares a high homology with a chalcone synthase gene of H. lupulus, 85% and 94% homology on the level of DNA and protein, respectively. Over-expression of the construct in Escherichia coli M15 resulted in a 45 kDa protein. The protein has chalcone synthase activity as well as valerophenone synthase activity, a chalcone synthase-like activity. Using n-hexanoyl-CoA and malonyl-CoA as substrates did not give olivetol or olivetolic acid as a product.

Acyl Coenzyme A↗

4-Hydroxy-2-pyrone formation by chalcone and stilbene synthase with nonphysiological substrates.

Valerophenone synthase (VPS) is a polyketide synthase that catalyzes the formation of the phloroglucinol derivatives in the synthesis of the bitter acids in hop (Humulus lupulus). The reaction uses isovaleryl-CoA or isobutyryl-CoA, but otherwise it is identical to that of the chalcone synthase in flavonoid biosynthesis. Our study showed that chalcone synthase can perform the function of VPS, but not perfectly, because the majority of the reactions terminated after two condensation reactions (products: 4-hydroxy-2-pyrone derivatives). The same experiments with stilbene synthase yielded exclusively the 4-hydroxy-2-pyrone derivatives, not the products expected from three condensation reactions. The results are discussed in the context of the functional diversity and evolution in the family of CHS-related polyketide synthases.

Acyltransferases↗

Spider mite allergy in apple-cultivating farmers: European red mite (Panonychus ulmi) and two-spotted spider mite (Tetranychus urticae) may be important allergens in the development of work-related asthma and rhinitis symptoms.

BACKGROUND: Recent investigations have suggested that the citrus red mite (Panonychus citri) is the most important allergen affecting citrus-cultivating farmers with asthma, allergic rhinitis, or both. OBJECTIVE: We sought to evaluate type I hypersensitivity to spider mites, particularly the European red mite (Panonychus ulmi) and the two-spotted spider mite (Tetranychus urticae), and to determine the relationship between hypersensitivity to spider mites and respiratory dysfunction. METHODS: We performed a cross-sectional survey. Questionnaires were given, and skin prick tests for 11 inhalant allergens common in Korea and 2 species of spider mites (European red mite and two-spotted spider mite) were performed in 725 apple-cultivating farmers in Korea. RESULTS: Results of skin prick tests in the apple farmers indicated that European red mite (23.2%) was the most common sensitizing allergen, followed by Tyrophagus putrescentiae (21.2%), two-spotted spider mite (16.6%), Dermatophagoides farinae (16.3%), D pteronyssinus (14.4%), cockroach (13.1%), and Hop Japanese (Humulus Japonicus) pollen (12.0%). Positive skin responses (mean wheal size >/=3 mm) to one or more of 13 inhalant allergens were found in 48.2% of farmers tested, whereas 40 subjects (8.6%) had an isolated skin response to the spider mites. Among 119 farmers with work-related asthmatic symptoms, the positive skin response rates to European red mite and two-spotted spider mite were 40.4% and 27.0%, respectively. These figures were significantly higher than those found among farmers without work-related symptoms (19.1% and 14.1%, respectively; P <.01). The prevalence of work-related asthma symptoms was higher in farmers with positive skin responses to spider mites than in those with negative skin responses to spider mites and those with positive skin responses to any allergen tested (31.4% vs 15.0% vs 21.0%, respectively; P <.05). CONCLUSION: Spider mites, particularly European red mite and 2-spotted spider mite, are common sensitizing allergens in apple-cultivating farmers. These spider mites may be important causative allergens in the development of work-related respiratory symptoms in these workers.

Administration, Inhalation↗

A highly polymorphic STR locus in Cannabis sativa.

We report on the first short tandem repeat (STR) locus to be isolated from the plant Cannabis sativa. The STR locus, isolated by a hybrid-capture enrichment procedure, was found to contain a simple sequence repeat motif of 6 bp. This 6 bp repeat motif showed no variation in repeat length but with minor variations in repeat unit sequences. The data show the locus to be highly polymorphic with the number of repeat units ranging from 3 to 40 in 108 screened samples. The observed heterozygosity was approximately 87.04%. The forward and reverse primers (CS1F and CS1R) produced no PCR products in cross-reaction study from 20 species of plants, including highly related species such as Humulus japonicus and Nicotiana tabacum. This hexanucleotide repeat DNA locus could be used to identify cannabis samples and predict their genetic relationship as the test is specific to C. sativa and is highly reproducible.

Alleles↗

Ribosomal DNA analysis as a tool for the identification of Cannabis sativa L. specimens of forensic interest.

A method is described for the identification of Cannabis sativa L., comparing the sequence of the nuclear ribosomal DNA Internal Transcribed Spacer II (ITS2) of an unknown sample with a known predetermined consensus sequence of Cannabis. Hemp ITS2 varied very little among cultivars, but was consistently different from that of hops (Humulus lupulus L.), which belongs to the only other genus of family Cannabinaceae.

Base Sequence↗

A re-consideration of Pseudoperonospora cubensis and P. humuli based on molecular and morphological data.

Phylogenetic analysis of the ITS rDNA region was carried out with two economically important downy mildews, Pseudoperonospora cubensis, which infects species of Cucumis, Cucurbita, and Citrullus belonging to Cucurbitaceae, and P. humuli, which infects plants of the genus Humulus belonging to Cannabaceae. Two closely related species, P. cannabina and P. celtidis, were also included to reveal taxonomic relationships with the first two mildews. All four species formed a well-resolved clade when compared with the ITS sequences of other downy mildew genera, using Bayesian inference and maximum parsimony. The P. cubensis isolates obtained from different hosts and (or) geographical origins in Korea, exhibited no intraspecific variability in the ITS sequences. The phylogenetic analyses of P. cubensis and P. humuli showed that they share a high level of sequence homology; the morphology of the sporangiophores, sporangia, and dehiscence apparatus confirmed the similarity of the two species. We therefore reduce P. humuli to the status of a taxonomic synonym of P. cubensis.

DNA, Fungal↗

Screening method for the discovery of potential cancer chemoprevention agents based on mass spectrometric detection of alkylated Keap1.

Natural products are important sources of drugs such as cancer chemopreventive agents, but most assays for the discovery of compounds in natural product extracts are low throughput and provide little information about lead compounds in these complex mixtures. The induction of enzymes such as quinone reductase, glucuronyl transferases, glutathione S-transferases, and sulfotransferases can protect cells against the toxic and neoplastic effects of carcinogens. An increase in the concentration of Nrf2 in the nucleus of a cell upregulates the antioxidant response element and induces the expression of these chemopreventive enzymes. Based on the hypothesis that ubiquitination and proteosome-mediated degradation of Nrf2 in the cytoplasm decreases upon the covalent modification of 1 or more of the 27 cysteine sulfhydryl groups on Keap1 (a protein that sequesters Nrf2 in the cytoplasm) and results in higher Nrf2 levels both in the cytoplasm and in the nucleus, a high-throughput mass spectrometry-based screening assay was designed to detect alkylation of sulfhydryl groups of human Keap1. As an initial high-throughput screening step, matrix-assisted laser desorption time-of-flight mass spectrometry was used to determine whether incubation of Keap1 with a botanical sample produced adducts of Keap1. Test extracts found to form adducts with Keap1 were then incubated with the alternative biological nucleophile glutathione and characterized using LC-UV-MS-MS. After validation of the assay using two model alkylating agents, fractions of an extract of hops (Humulus lupulus L.) from the brewing industry were screened, and several compounds were detected as potential chemopreventive agents. Two of these electrophilic hops constituents were identified as xanthohumol and xanthohumol D. In a subsequent cell-based assay, xanthohumol and xanthohumol D were confirmed to be potent inducers of quinone reductase, and reaction with Keap1 was also confirmed. Therefore, this new mass spectrometric screening assay was demonstrated to facilitate the discovery of chemoprevention agents in complex natural product mixtures.

Alkylation↗

Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptoms.

Eight botanical preparations that are commonly used for the treatment of menopausal symptoms were tested for estrogenic activity. Methanol extracts of red clover (Trifolium pratense L.), chasteberry (Vitex agnus-castus L.), and hops (Humulus lupulus L.) showed significant competitive binding to estrogen receptors alpha (ER alpha) and beta (ER beta). With cultured Ishikawa (endometrial) cells, red clover and hops exhibited estrogenic activity as indicated by induction of alkaline phosphatase (AP) activity and up-regulation of progesterone receptor (PR) mRNA. Chasteberry also stimulated PR expression, but no induction of AP activity was observed. In S30 breast cancer cells, pS2 (presenelin-2), another estrogen-inducible gene, was up-regulated in the presence of red clover, hops, and chasteberry. Interestingly, extracts of Asian ginseng (Panax ginseng C.A. Meyer) and North American ginseng (Panax quinquefolius L.) induced pS2 mRNA expression in S30 cells, but no significant ER binding affinity, AP induction, or PR expression was noted in Ishikawa cells. Dong quai [Angelica sinensis (Oliv.) Diels] and licorice (Glycyrrhiza glabra L.) showed only weak ER binding and PR and pS2 mRNA induction. Black cohosh [Cimicifuga racemosa (L.) Nutt.] showed no activity in any of the above in vitro assays. Bioassay-guided isolation utilizing ER competitive binding as a monitor and screening using ultrafiltration LC-MS revealed that genistein was the most active component of red clover. Consistent with this observation, genistein was found to be the most effective of four red clover isoflavones tested in the above in vitro assays. Therefore, estrogenic components of plant extracts can be identified using assays for estrogenic activity along with screening and identification of the active components using ultrafiltration LC-MS. These data suggest a potential use for some dietary supplements, ingested by human beings, in the treatment of menopausal symptoms.

Binding, Competitive↗

Fate of xanthohumol and related prenylflavonoids from hops to beer.

The fate of three prenylated flavonoids of the chalcone type, xanthohumol, desmethylxanthohumol, and 3'-geranylchalconaringenin, was monitored with LC/MS-MS from hops (Humulus lupulus L.) to beer in two brewing trials. The three prenylchalcones were largely converted into their isomeric flavanones, isoxanthohumol, prenylnaringenins, and geranylnaringenins, respectively, in the boiling wort. Losses of prenylflavonoids were due to incomplete extraction from the hops into the wort (13-25%), adsorption to insoluble malt proteins (18-26%), and adsorption to yeast cells (11-32%) during fermentation. The overall yield of xanthohumol, after lagering of the beer and largely in the form of isoxanthohumol, amounted to 22-30% of the hops' xanthohumol. About 10% of the hops' desmethylxanthohumol, completely converted into prenylnaringenins, remained in the beers. 3'-Geranylchalconaringenin behaved similarly to desmethylxanthohumol. Solubility experiments indicated that (1) malt carbohydrates form soluble complexes with xanthohumol and isoxanthohumol and (2) solubility does not dictate the isoxanthohumol levels of finished beers.

Beer↗

Qualitative and quantitative variation among volatile profiles induced by Tetranychus urticae feeding on plants from various families.

Many plant species are known to emit herbivore-induced volatiles in response to herbivory. The spider mite Tetranychus urticae Koch is a generalist that can feed on several hundreds of host plant species. Volatiles emitted by T. urticae-infested plants of 11 species were compared: soybean (Glycine max), golden chain (Laburnum anagyroides), black locust (Robinia pseudo-acacia), cowpea (Vigna unguiculata), tobacco (Nicotiana tabacum), eggplant (Solanum melalonga), thorn apple (Datura stramonium), sweet pepper (Capsicum annuum), hop (Humulus lupulus), grapevine (Vitis vinifera), and ginkgo (Ginkgo biloba). The degree to which the plant species produced novel compounds was analyzed when compared to the odors of mechanically damaged leaves. Almost all of the investigated plant species produced novel compounds that dominated the volatile blend, such as methyl salicylate, terpenes, oximes, and nitriles. Only spider mite-infested eggplant and tobacco emitted a blend that was merely quantitatively different from the blend emitted by mechanically damaged or clean leaves. We hypothesized that plant species with a low degree of direct defense would produce more novel compounds. However, although plant species with a low direct defense level do use indirect defense to defend themselves, they do not always emit novel compounds. Plant species with a high level of direct defense seem to invest in the production of novel compounds. When plant species of the Fabaceae were compared to plant species of the Solanaceae, qualitative differences in spider mite-induced volatile blends seemed to be more prominent in the Fabaceae than in the Solanaceae.

Animals↗

Attractive and defensive functions of the ultraviolet pigments of a flower (Hypericum calycinum).

The flower of Hypericum calycinum, which appears uniformly yellow to humans, bears a UV pattern, presumably visible to insects. Two categories of pigments, flavonoids and dearomatized isoprenylated phloroglucinols (DIPs), are responsible for the UV demarcations of this flower. Flavonoids had been shown previously to function as floral UV pigments, but DIPs had not been demonstrated to serve in that capacity. We found the DIPs to be present in high concentration in the anthers and ovarian wall of the flower, suggesting that the compounds also serve in defense. Indeed, feeding tests done with one of the DIPs (hypercalin A) showed the compound to be deterrent and toxic to a caterpillar (Utetheisa ornatrix). The possibility that floral UV pigments fulfill both a visual and a defensive function had not previously been contemplated. DIPs may also serve for protection of female reproductive structures in other plants, for example in hops (Humulus lupulus). The DIPs of hops are put to human use as bitter flavoring agents and preservatives in beer.

Animals↗

A preliminary RAPD-PCR analysis of Cimicifuga species and other botanicals used for women's health.

Traditional taxonomic methods of botanical identification that rely primarily on morphological observations cannot be used efficiently when only powdered plant materials are available. Thus, our objectives were to determine if we could apply a molecular approach to: a) produce unique DNA profiles that are characteristic of the species, and b) determine if the geographical area or time of collection influences these DNA profiles. Towards this end, random amplified polymorphic DNA (RAPD) analyses were performed on a number of botanicals currently used for women's health. The test materials included samples from three species each of the genera Cimicifuga (Actaea) and Trifolium, as well as samples of Vitex agnus-castus L., Glycyrrhiza glabra L., Gingko biloba L., Valeriana officinalis L., Angelica sinensis (Oliv.) Diels, Viburnum prunifolium L., Humulus lupulus L., Vaccinium macrocarpon Ait., Panax ginseng C.A. Mey. Cimicifuga racemosa (L.) Nutt. and Trifolium pratense L. are currently under clinical investigation in our basic research laboratories and medical clinic for the relief of post-menopausal symptoms. Characteristic profiles produced with the OPC-15 primer could distinguish the three Cimicifuga species: C. racemosa, C. americana and C. rubifolia. Similar results were obtained with the three Trifolium species: Trifolium pratense L., Trifolium incarnatum L., and Trifolium repens L. Accessions of cultivated T. pratense collected from the same field at different times, produced identical profiles. Accessions of Cimicifuga species collected from different geographical areas produced similar but not identical DNA profiles; however, species-specific DNA fragments were identified. These results demonstrate that RAPD analysis can be applied to distinguish species when only powdered material is available for testing. This methodology can be applied to identify species of commercial value regardless of collection time or geographic area.

Cimicifuga↗

Fractionation by SFE and microcolumn analysis of the essential oil and the bitter principles of hops.

Supercritical fluid extraction (SFE) is evaluated and optimized for the enrichment and fractionation of the essential oil and the bitter principles of hops (Humulus lupulus), both of which contribute to the flavor of beer. Profiles of the essential oil of different hop varieties are compared. The bitter principles, the humulones and lupulones, are analyzed by miniaturized liquid chromatography (micro-LC) and by micellar electrokinetic chromatography (MEKC).

Chromatography↗

Host-plant diversity of the European corn borer Ostrinia nubilalis: what value for sustainable transgenic insecticidal Bt maize?

The strategies proposed for delaying the development of resistance to the Bacillus thuringiensis toxins produced by transgenic maize require high levels of gene flow between individuals feeding on transgenic and refuge plants. The European corn borer Ostrinia nubilalis (Hübner) may be found on several host plants, which may act as natural refuges. The genetic variability of samples collected on sagebrush (Artemisia sp.), hop (Humulus lupulus L.) and maize (Zea mays L.) was studied by comparing the allozyme frequencies for six polymorphic loci. We found a high level of gene flow within and between samples collected on the same host plant. The level of gene flow between the sagebrush and hop insect samples appeared to be sufficiently high for these populations to be considered a single genetic panmictic unit. Conversely, the samples collected on maize were genetically different from those collected on sagebrush and hop. Three of the six loci considered displayed greater between-host-plant than within-host-plant differentiation in comparisons of the group of samples collected on sagebrush or hop with the group of samples collected on maize. This indicates that either there is genetic isolation of the insects feeding on maize or that there is host-plant divergent selection at these three loci or at linked loci. These results have important implications for the potential sustainability of transgenic insecticidal maize.

Adaptation, Physiological↗

Factors affecting antibacterial activity of hop compounds and their derivatives.

The antibacterial effect of weak acids derived from the hop plant (Humulus lupulus L.) increased with decreasing pH. Analysis of the minimum inhibitory concentration of such compounds against Lactobacillus brevis IFO 3960 over pH 4-7 suggests that undissociated molecules were mainly responsible for inhibition of bacterial growth. The antibacterial activity of trans-isohumulone was ca 20 times greater than that of humulone, 11 times greater than that of colupulone and nine times greater than that of trans-humulinic acid when the degree of ionization was taken into account. Monovalent cations (K+, Na+, NH4+, Rb+, Li+) stimulated antibacterial activity of trans-isohumulone but the effect was smaller than that observed with H+. The response to divalent cations varied: Ca2+ had little effect on antibacterial activity, whereas Mg2+ reduced activity. Lipid materials and beta-cyclodextrin also antagonized the antibacterial action of trans-isohumulone.

Anti-Bacterial Agents↗

Development of a bipartite method for Fusarium identification based on cellobiohydrolase-C: CAPS and Western blot analysis.

The identification of 12 Fusarium strains isolated from diseased hops (Humulus lupulus, L.) was achieved by a strategy based on cellobiohydrolase-C: cleaved amplified polymorphic sequence analysis targeting the gene and the use of an antibody directed against a peptide of the Fusarium graminearum enzyme. This strategy is shown to be rapid and reliable for all the Fusarium of our collection: F. avenaceum, F. graminearum, F. sambucinum, F. sporotrichioides, F. tricinctum and F. venenatum.

Blotting, Western↗

Humulon, a bitter in the hop, inhibits tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin.

Humulon, one of the bitters in the hop, was isolated from the female flowers of Humulus lupulus. This component has inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation. At 1 mg/mouse, humulon inhibited markedly the tumor-promoting effect of TPA (1 microgram/mouse) on skin tumor formation following initiation with 7,12-dimethylbenz[a]anthracene (50 micrograms/mouse). Furthermore, humulon inhibited arachidonic acid-induced inflammatory ear edema in mice.

9,10-Dimethyl-1,2-benzanthracene↗