Rosmarinus officinalis L. essential oil and the related compound 1,8-cineole do not induce direct or cross-protection in Listeria monocytogenes ATCC 7644 cultivated in meat broth

Listeria monocytogenes has the capability of adapting to 1 or more antimicrobial compounds or procedures applied by the food industry to control the growth and survival of microorganisms in foods. In this study, the effects of Rosmarinus officinalis essential oil (EO) and the related compound 1,8-cineole on the inhibition of the growth and survival of L.?monocytogenes ATCC 7644 were determined. The ability of the R.?officinalis EO and 1,8-cineole to induce direct and cross-protection of bacteria against various stresses (lactic acid, pH?5.2; NaCl, 3?g/100?mL; high temperature, 45?°C) was also determined. At all concentrations tested (minimum inhibitory concentration (MIC), ? MIC, and ? MIC), both compounds inhibited the cell viability of L.?monocytogenes over 120?min of exposure. Overnight exposure of L.?monocytogenes to sublethal amounts of either the R.?officinalis EO or 1,8-cineole in meat broth revealed no induction of direct or cross-protection against lactic acid, NaCl, or high temperature. Similarly, cells subjected to 24?h cycles of adaptation with increasing amounts (? MIC to 2× MIC) of the EO and 1,8-cineole showed no increase in direct tolerance, as they were able to survive in growth medium containing up to ? MIC of either substance. These results show the antimicrobial efficacy of R.?officinalis EO and 1,8-cineole for use in systems, particularly as anti-L.?monocytogenes compounds.     

不同株龄薰衣草花生物学性状和精油主要化学成分研究

为了解不同株龄薰衣草花生物学性状、精油主要化学成分及含量差异, 以昆明薰衣草CAS08 （Lavandula angustifolia Mill.×Lavandula latifolia Medik.）为试材, 对不同株龄（一年生 、两年生和三年生）薰衣草的花枝、 精油提取率和精油成分变化进行了研究。 结果显示,不同株龄薰衣草的单支花生物学性状差异显著（P 〈0.05）; 薰衣草花精油的提取率在不同株龄间有差异, 分别为一年生熏衣草花精油提取率3.40%、 两年生2.37%和三年生3.60%; 薰衣草CAS08花精油的主要成分有桉叶油醇、芳樟醇、樟脑和红没药醇, 株龄对精油中各化学成分的相对含量有影响。本研究结果可为云南薰衣草产业化发展及持续开发利用提供科学依据。     

RNAi targeting the gene for 1,8-cineole synthase induces recomposition of leaf essential oil in lavandin (Lavandula × intermedia Emeric)

Recomposition of volatile compounds is an effective way to alter the fragrance of an essential oil. Using RNA interference (RNAi), we attempted to reduce the production of 1,8-cineole, a major monoterpene in essential oils, to alter the composition of the essential oil in lavandin. We obtained 12 transgenic regenerants via inoculation with Agrobacterium tumefaciens, including an RNAi inducing vector targeting five regions of the gene for 1,8-cineole synthase (CINS). Of these, two regenerants, targeting a coding region about 250 nt upstream with 5′-UTR and a coding region about 300 nt downstream of CINS mRNA (CINS I and V), respectively, showed a significant decrease of 1,8-cineole production in leaf essential oil, although the overall composition was barely altered because the production of other compounds decreased concurrently. By contrast, in two other regenerants, targeting a coding region about 1000 nt in the middle and a coding region about 300 nt downstream of CINS mRNA (CINS IV and V), respectively, 1,8-cineole production could be barely observed, without any decrease in production of other compounds. Expression of CINS in these transgenic regenerants was extremely suppressed to 0.02 and 0.08 of that of a nontransgenic regenerant control. The composition of leaf essential oil in the transgenic regenerants was changed by the RNAi. Specifically, the major compounds changed from 1,8-cineole, camphor, and borneol, to linalool, camphor, and borneol. Consequently, the fragrance of essential oils of these plants was perceived as more citrusy than the fragrance of the nontransgenic regenerant. These results suggest that the knockdown strategy was a useful tool for altering the fragrance in lavandin.

     

Comparison of bacteriostatic and bactericidal activity of 13 essential oils against strains with varying sensitivity to antibiotics

Aims:  To compare the bacteriostatic and bactericidal activity of 13 chemotyped essential oils (EO) on 65 bacteria with varying sensitivity to antibiotics.
Methods and Results:  Fifty-five bacterial strains were tested with two methods used for evaluation of antimicrobial activity (CLSI recommendations): the agar dilution method and the time-killing curve method. EO containing aldehydes ( Cinnamomum verum bark and Cymbopogon citratus ), phenols ( Origanum compactum , Trachyspermum ammi , Thymus satureioides , Eugenia caryophyllus and Cinnamomum verum leaf) showed the highest antimicrobial activity with minimum inhibitory concentration (MIC) <2% (v/v) against all strains except Pseudomonas aeruginosa . Alcohol-based EO ( Melaleuca alternifolia , Cymbopogon martinii and Lavandula angustifolia ) exhibited varying degrees of activity depending on Gram status. EO containing 1·8-cineole and hydrocarbons ( Eucalyptus globulus , Melaleuca cajeputii and Citrus sinensis ) had MIC_90% ≥ 10% (v/v). Against P. aeruginosa , only C. verum bark and O. compactum presented MIC ≤2% (v/v). Cinnamomum verum bark, O. compactum , T. satureioides , C. verum leaf and M. alternifolia were bactericidal against Staphylococcus aureus and Escherichia coli at concentrations ranging from to 0·31% to 10% (v/v) after 1 h of contact. Cinnamomum verum bark and O. compactum were bactericidal against P. aeruginosa within 5 min at concentrations <2% (v/v).
Conclusions:  Cinnamomum verum bark had the highest antimicrobial activity, particularly against resistant strains.
Significance and Impact of the Study:  Bacteriostatic and bactericidal activity of EO on nosocomial antibiotic-resistant strains.     

Expressed sequence tags of Chinese cabbage flower bud cDNA.

We randomly selected and partially sequenced cDNA clones from a library of Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower bud cDNAs. Out of 1216 expressed sequence tags (ESTs), 904 cDNA clones were unique or nonredundant. Five hundred eighty-eight clones (48.4%) had sequence homology to functionally defined genes at the peptide level. Only 5 clones encoded known flower-specific proteins. Among the cDNAs with no similarity to known protein sequences (628), 184 clones had significant similarity to nucleotide sequences registered in the databases. Among these 184 clones, 142 exhibited similarities at the nucleotide level only with plant ESTs. Also, sequence similarities were evident between these 142 ESTs and their matching ESTs when compared using the deduced amino acid sequences. Therefore, it is possible that the anonymous ESTs encode plant-specific ubiquitous proteins. Our extensive EST analysis of genes expressed in floral organs not only contributes to the understanding of the dynamics of genome expression patterns in floral organs but also adds data to the repertoire of all genomic genes.     

SlY1, the first active gene cloned from a plant Y chromosome, encodes a WD-repeat protein.

Unlike the majority of flowering plants, which possess hermaphrodite flowers, white campion (Silene latifolia) is dioecious and has flowers of two different sexes. The sex is determined by the combination of heteromorphic sex chromosomes: XX in females and XY in males. The Y chromosome of S.latifolia was microdissected to generate a Y-specific probe which was used to screen a young male flower cDNA library. We identified five genes which represent the first active genes to be cloned from a plant Y chromosome. Here we report a detailed analysis of one of these genes, SlY1 (S.latifolia Y-gene 1). SlY1 is expressed predominantly in male flowers. A closely related gene, SlX1, is predicted to be located on the X chromosome and is strongly expressed in both male and female flowers. SlY1 and SlX1 encode almost identical proteins containing WD repeats. Immunolocalization experiments showed that these proteins are localized in the nucleus, and that they are most abundant in cells that are actively dividing or beginning to differentiate. Interestingly, they do not accumulate in arrested sexual organs and represent potential targets for sex determination genes. These genes will permit investigation of the origin and evolution of sex chromosomes in plants.     

Construction and characterization of normalized cDNA library of maize inbred Mo17 from multiple tissues and developmental stages

Comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10.830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarry was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate gene, and developing microarrays in maize genomics research.     

Synthesis of ‘cineole cassette’ monoterpenes in Nicotiana section Alatae: gene isolation, expression, functional characterization and phylogenetic analysis

The scent bouquets of flowers of Nicotiana species, particularly those of section Alatae, are rich in monoterpenes, including 1,8-cineole, limonene, β-myrcene, α- and β-pinene, sabinene, and α-terpineol. New terpene synthase genes were isolated from flowers of Nicotiana bonariensis, N. forgetiana, N. longiflora, and N. mutabilis. The recombinant enzymes synthesize simultaneously the characteristic 'cineole cassette' monoterpenes with 1,8-cineole as the dominant volatile product. Interestingly, amino acid sequence comparison and phylogenetic tree construction clustered the newly isolated cineole synthases (CIN) of section Alatae together with the catalytically similar CIN of N. suaveolens of section Suaveolentes, thus suggesting a common ancestor. These CIN genes of N. bonariensis, N. forgetiana, N. longiflora, and N. mutabilis are distinct from the terpineol synthases (TERs) of the taxonomically related N. alata and N. langsdorfii (both Alatae), thus indicating gene diversification of monoterpene synthases in section Alatae. Furthermore, the presence of CINs in species of the American section Alatae supports the hypothesis that one parent of the Australian section Suaveolentes was a member of the present section Alatae. Amino acid sequences of the Nicotiana CINs and TERs were compared to identify relevant amino acids of the cyclization reaction from α-terpineol to 1,8-cineole.     

Comparative analysis of sequences expressed during the liquid-cultured mycelia and fruit body stages of Pleurotus ostreatus

To characterize genes involved in fruit body development, two complementary DNA (cDNA) libraries were constructed from RNA isolated from liquid-cultured mycelia and fruit bodies of Pleurotus ostreatus. Using single-pass sequencing of cDNA clones, 952 and 1069 expressed sequence tags (ESTs) were generated from liquid-cultured mycelia and fruit body cDNA library, respectively. A BLASTX search revealed that 390 of the liquid-cultured mycelia ESTs (41%) and 531 of the fruit body ESTs (50%) showed significant similarity to protein sequences described in the nonredudant database (E values < or =1 x 10(-5)). When liquid-cultured mycelia and fruit body ESTs were compared by the SeqMan II program, among the total of 2021 ESTs, 1256 ESTs were unigenes, and 66 unigenes (5.3%) were commonly expressed during both stages. The functional catalogs of the ESTs were made by comparison with functionally identified Saccharomyces cerevisiae genes. Liquid-cultured mycelium ESTs were compared with fruit body ESTs and changes of the expressed genes during fruit body development were analyzed.     

Genetic and clonal diversity of two cattail species, Typha latifolia and T. angustifolia (Typhaceae), from Ukraine

Genetic and clonal diversity vary between two closely related cattail species (Typha angustifolia and T. latifolia) from Ukraine. This diversity was calculated from microsatellite data. Forty-eight percent of the total variation was partitioned between species, which formed distinct clusters in a dendrogram with no indication of hybrid populations. Typha angustifolia had higher heterozygosity at the species (H(es) = 0.66) and population (H(ep) = 0.49) levels than did T. latifolia (H(es) = 0.37 and H(ep) = 0.29, respectively). The higher number of alleles in T. angustifolia may be indicative of larger effective population sizes due to its higher seed production. Clonal diversity of T. angustifolia was lower than that of T. latifolia (N(g)/N(r) = 0.40 and 0.61, Simpson's D = 0.82 and 0.94, respectively). Correlations between clonal and genetic diversity were higher for T. latifolia than T. angustifolia, suggesting that the importance of factors and their interactions affecting this relationship are different for the two species. Latitudinal and longitudinal trends were not observed in either species despite the large sampling area. Population differentiation was relatively high with F(ST) of 0.24 and 0.29 for T. angustifolia and T. latifolia, respectively. Weak isolation by distance was observed for T. latifolia but not for T. angustifolia.     

Inhibitory effect of Ocotea quixos (Lam.) Kosterm. and Piper aduncum L. essential oils from Ecuador on West Nile virus infection

West Nile virus (WNV) is a mosquito-borne flavivirus responsible of neuroinvasive manifestations. Natural products are well-known for their biological activities and pharmaceutical application. In this study, the inhibitory effects of essential oils (EOs) of Ocotea quixos (Lam.) Kosterm. and Piper aduncum L. on WNV replication were investigated. WNV was incubated with EOs before adsorption on Vero cells, viral replication was carried out in the absence or presence of EO. Cells were exposed to EO before the adsorption of untreated-virus. GC-MS and GC-FID were used for chemical characterization of EOs. Cell protection from infection was observed for both EOs. P. aduncum EO was characterized by dillapiole as main compound (48.21%) and O. quixos EO by 1,8-cineole (39.15%). Further investigations, such as the study of molecular and cellular mechanisms of action and in vivo evaluation, should be performed on these essential oils to derive new potential drugs against WNV.     

Chemical Variability of Moroccan Myrtle Oil

Thirty-three oil samples isolated from aerial parts of Myrtus communis L. harvested in seven localities, from Northern to Central Morocco, have been analyzed by combination of chromatographic and spectroscopic techniques. The 33 compositions have been subjected to statistical analysis, hierarchical cluster analysis (HCA) and principal component analysis (PCA). Two groups have been differentiated on the basis of their myrtenyl acetate and α-pinene contents and each one was sub-divided in two sub-groups according to the contents of 1,8-cineole and linalool. The compositions of our 33 myrtle oil samples may be named as follow by their main components: sub-group IA (13/33): α-pinene/1,8-cineole/linalool; sub-group IB (6/33): 1,8-cineole/α-pinene; sub-group IIA (10/33): 1,8-cineole/myrtenyl acetate; sub-group IIB (4/33): myrtenyl acetate.     

Monoterpene variation in two Achillea ageratum chemotypes

Artemisia ketone and artemisia acetate are the main monoterpene components in both the flowers and leaves of A. ageratum growing in central Italy, but are replaced by 1,8-cineole in plants growing in Sardinia (Italy).     

Chemical compositions of Casuarina equisetifolia L., Eucalyptus toreliana L. and Ficus elastica Roxb. ex Hornem cultivated in Nigeria

Essential oils were obtained by separate hydrodistillation of three different plants cultivated in Nigeria and analysed comprehensively for their constituents by means of gas chromatography (GC) and gas chromatography-mass spectrometry (GC–MS). The leaf essential oil of Casuarina equisetifolia L. (Casuarinaceae) comprised mainly of pentadecanal (32.0%) and 1,8-cineole (13.1%), with significant amounts of apiole (7.2%), α-phellandrene (7.0%) and α-terpinene (6.9%), while the fruit oil was dominated by caryophyllene-oxide (11.7%), trans-linalool oxide (11.5%), 1,8-cineole (9.7%), α-terpineol (8.8%) and α-pinene (8.5%). On the other hand, 1,8-cineole (39.4%) and α-terpinyl acetate (10.7%) occurred in large quantities in the essential oils of the leaf of Eucalyptus toreliana L. (Myrtaceae). The oil also features high levels of sabinene (5.9%), caryophyllene-oxide (4.7%) and α-pinene (4.2%). The main compounds identified in the leaf oil of Ficus elastica Roxb. ex Hornem. (Moraceae) were 6,10,14-trimethyl-2-pentadecanone (25.9%), geranyl acetone (9.9%), heneicosene (8.4%) and 1,8-cineole (8.2%).     

Construction and characterization of normalized cDNA library of maize inbred MO17 from multiple tissues and developmental stages

A comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10 830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarray was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using the BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate genes, and developing microarrays in maize genomics research.__________From Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 198–206.Original English Text Copyright © 2005 by Z. Zhang, F. Zhang, Tang, Pi, Zheng.This article was submitted by the authors in English.     

Depth distribution of three Typha species, Typha orientalis Presl, Typha angustifolia L. and Typha latifolia L., in an artificial pond

The depth distribution of the aquatic macrophyte Typha orientalis Presl was examined in comparison with two other Typha species: Typha angustifolia L. and Typha latifolia L. Vegetation surveys mapping the depth distributions were conducted at Ushigafuchi Pond, Tokyo, Japan, in autumn 2004 and 2005. All vegetation had been cleared from this artificial pond in spring 2003. In 2004 T. orientalis was distributed in shallow to deep water habitats between T. latifolia (shallow water regions) and T. angustifolia (shallow to deep water regions). However, by 2005 T. orientalis had almost disappeared from the pond. It had been replaced by Leersia japonica Makino at depths of 0–30 cm, by Schoenoplectus validus (Vahl) at depths of 30–60 cm and by T. angustifolia at depths of 60–100 cm. It appears that T. orientalis is not a strong competitor, particularly with taller species, but rather a pioneer species.     

Metabolism of 1,8-cineole by human cytochrome P450 enzymes: identification of a new hydroxylated metabolite

Human metabolism of the monoterpene cyclic ether 1,8-cineole was investigated in vitro and in vivo. In vitro, the biotransformation of 1,8-cineole was investigated by human liver microsomes and by recombinant cytochrome P450 enzymes coexpressed with human CYP-reductase in Escherichia coli cells. Besides the already described metabolite 2alpha-hydroxy-1,8-cineole we found another metabolite produced at high rates. The structure was identified by a comparison of its mass spectrum and retention time with the reference compounds as 3alpha-hydroxy-1,8-cineole. There was a clear correlation between the concentration of the metabolites, incubation time and enzyme content, respectively. CYP3A4/5 antibody significantly inhibited the 2alpha- and 3alpha-hydroxylation catalyzed by pooled human liver microsomes. Further kinetic analysis revealed that the Michaelis-Menten K(m) and V(max) for oxidation of 1,8-cineole in position three were 19 microM and 64.5 nmol/min/nmol P450 for cytochrome P450 3A4, and 141 microM and 10.9 nmol/min/nmol P450 for cytochrome P450 3A5, respectively. To our knowledge, this is the first time that 3alpha-hydroxy-1,8-cineole is described as a human metabolite of 1,8-cineole. We confirmed these in vitro results by the investigation of human urine after the oral administration of cold medication containing 1,8-cineole. In human urine we found by GC-MS analysis the described metabolites, 2alpha-hydroxy-1,8-cineole and 3alpha-hydroxy-1,8-cineole.