Antimicrobial activity of extracts of chemical races of the lichen Pseudevernia furfuracea and their physodic acid, chloroatranorin, atranorin, and olivetoric acid constituents

The antimicrobial activity and the MIC values of the ethanol, chloroform, diethyl ether, and acetone extracts of the chemical races of Pseudevernia furfuracea (var. furfuracea and var. ceratea) and their physodic acid, chloroatranorin, atranorin, and olivetoric acid constituents have been investigated against some microorganisms. Nearly all extracts of both chemical races showed antimicrobial activity against Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, Proteus vulgaris, Staphylococcus aureus, Streptococcus faecalis, Yersinia enterocolitica, Candida albicans, Candida glabrata, Alternaria alternata, Ascochyta rabiei, Aspergillus niger, Fusarium culmorum, Fusarium moniliforme, Fusarium oxysporum, Fusarium solani, and Penicillium notatum. There was no antimicrobial activity of the extracts against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas syringae, Salmonella typhimurium, Alternaria citri, Alternaria tenuissima, and Gaeumannomyces graminis. Chloroatranorin and olivetoric acid were active against the same microorganisms with few exceptions. Physodic acid was active against about the same bacteria and yeasts and inactive against all of the filamentous fungi tested. Also no activity of atranorin against the filamentous fungi was observed.     

袋衣属二新种

本文报道袋衣属中两个新种,即亚霜袋衣(Hypogymnia subpruino Chen)和丽江袋衣(Hypogymnia lijiangensis Chen),并从形态和化学相结合比较了新种与其近似种的异同。     

Characterization of two chemotypes of <Emphasis Type="Italic">Pinus pinaster</Emphasis> by their terpene and acid patterns in needles

The existence of two chemotypes of Pinus pinaster, on the basis of the chemical composition of the resin acids in their needles, is known. An investigation was performed on 54 samples of needles of Spanish Pinus pinaster to study the differences between these chemotypes on the basis of monoterpene, sesquiterpene, neutral diterpene, fatty acid, and resin acid composition. One-hundred and twelve compounds were identified by GC–FID and GC–MS. Statistical analysis of the results established the existence of two groups or chemotypes, in the ratio of 5:1. In one chemotype, total acid compounds were more abundant than neutral compounds, whereas in the other the concentrations of both neutral and acid compounds were similar. Distinction of the chemotypes was based on the presence/absence of a sesquiterpene (germacrene d-4-ol acetate), neutral diterpenes (8(14),13(15)-abietadiene, anticopalol, an isomer of anticopalol, and pimarol), fatty acids (10-octadecenoic, 14-hydroxy-10-octadecenoic, and 13-hydroxy-9-octadenoic acids and an unidentified fatty acid), and resin acids (levopimaric + palustric, eperuic, and anticopalic acids, and three isomers of anticopalic acid); and on the different relative percentages of other compounds of these types. This study gives a wide view of the composition of the needles of Pinus pinaster, improving the differentiation of chemotypes on the basis of terpene and acid composition.     

Production of phenolics by immobilized cells of the lichen Pseudevernia furfuracea: the role of epiphytic bacteria

Immobilized lichen cells from the thalli of the lichen Pseudevernia furfuracea, supplied with acetate as the only source of carbon, continuously produced phenolic substances, atranorin and physodic acid, over 23 days. Epiphytic bacteria associated with the lichen thallus grew actively, probably using both acetate and reduced compounds supplied by lichen cells, since their active growth was avoided by including 10 microM 3,3'-dichlorophenyl-1,1'dimethylurea in the bath solution. Penicillin largely impeded the growth of epiphytic bacteria and decreased phenolic production, which was recovered only at the end of the experimental period, just when the bacteria started a slow, but active growth. We suggest the cooperation of epiphytic bacteria in the biosynthesis of both atranotrin and physodic acid.     

Chemical profiling of Ocimum americanum using external flavonoids

A HPLC survey was undertaken of the external flavonoids in 111 herbarium specimens of Ocimum americanum L. (O. canum Sims), which were largely collected from their natural habitats throughout Africa and Asia. The purpose of this study was to establish the flavonoid profiles of this species over the full range of its geographic distribution in order to use these for authentication purposes. Six different external flavonoid chemotypes were found. The major chemotype, present in circa 80% of the specimens of both var. americanum and var. pilosum collected throughout the distribution area of the species, was characterised by very high levels of nevadensin, slightly lower levels of salvigenin and much lower levels of up to 15 other external flavones. Of the remaining five chemotypes, two were found in var. americanum and three in var. pilosum. All specimens belonging to these chemotypes were collected in South or East Africa and represented by only a few specimens. These samples contained much smaller levels of flavones than present in the major chemotype of O. americanum and all lacked nevadensin. Xanthomicrol, a compound absent from the main chemotype, was the dominant flavone in two of the minor chemotypes. The external flavonoid profiles found in the six chemotypes of O. americanum were compared with those of O. x citriodorum (11 herbarium specimens studied) and seven other closely related species of Ocimum. The main nevadensin/salvigenin pattern present in O. americanum was also found in O. x citriodorum, O. basilicum and some specimens of O. minimum, but there were strong quantitative differences in external flavonoids among these taxa. The other chemotypes of O. americanum showed some similarities in their external flavone profiles to those found in the closely related East African species O. fischeri, O. forskolei, O. kenyense and O. kilimandscharicum, which occur in the same geographic areas. This suggests that the uncommon chemotypes of O. americanum may have originated by an exchange of genes with other Ocimum species, e.g. by introgressive hybridisation. Despite some similarities in profiles, chemical differences were also found among the species, so that it should be possible to authenticate a large proportion of leaf samples of O. americanum on the basis of external flavonoid profiles.     

Possible implications of chytrid parasitism for population subdivision in freshwater cyanobacteria of the genus Planktothrix

Populations of the cyanobacterium Planktothrix comprise multiple coexisting oligopeptide chemotypes that can behave differently in nature. We tested whether this population subdivision can, in principle, be driven by parasitic chytrid fungi, which are almost neglected agents of Planktothrix mortality. Two chytrid strains, Chy-Lys2009 and Chy-Kol2008, were isolated from Planktothrix-dominated lakes in Norway. The two strains shared 98.2% and 86.2% of their 28S and internal transcribe spacer rRNA gene sequences, respectively. A phylogenetic analysis placed them in the order Rhizophydiales family Angulomycetaceae. Chy-Lys2009 and Chy-Kol2008 could completely lyse Planktothrix cultures within days, while they failed to infect other filamentous cyanobacteria. The effect on Planktothrix was chemotype dependent, and both chytrid strains showed distinct chemotype preferences. These findings identify chytrid fungi infecting Planktothrix as highly potent and specialized parasites which may exert strong selective pressure on their hosts. According to established hypotheses on host-parasite coevolution, parasitism with the above properties may result in subdivision of Planktothrix populations into coexisting chemotypes and periodic shifts in the relative Planktothrix chemotype composition. These predictions are in agreement with field observations. Moreover, a genetic analysis verified the co-occurrence of Chy-Lys2009 and Chy-Kol2008 or related chytrid strains along with distinct Planktothrix chemotypes in at least one water body. Our findings are consistent with a scenario where chytrid parasitism is one driving force of Planktothrix population subdivision, which in turn leads to polymorphism in parasitic chytrid fungi. Future studies should test the validity of this scenario under field conditions.     

DISTRIBUTION OF OLIGOPEPTIDE CHEMOTYPES OF THE CYANOBACTERIUM PLANKTOTHRIX AND THEIR PERSISTENCE IN SELECTED LAKES IN FENNOSCANDIA1

Eighty‐seven Planktothrix (Anagnostidis and Komàrek 1988) strains isolated from 13 lakes in Scandinavia and Finland between 1964 and 2007 were screened for oligopeptides. Forty‐six individual compounds were detected in total, belonging to the structural classes anabaenopeptins (six variants), aeruginosins (six variants), cyanopeptolins (21 variants), microcystins (five variants), microginins (two variants), and microviridins (two variants). Oscillatorin was also found. Three additional compounds could not be assigned to known oligopeptide classes. Thirty oligopeptides have not been described in previous studies. Of these new compounds, five were aeruginosins and 20 cyanopeptolins. The number of oligopeptides per strain ranged from one to 13. No oligopeptide‐free strains were found, suggesting that oligopeptide production is vital for Planktothrix. On the basis of their oligopeptide patterns, the Planktothrix strains of the present study were assigned to 17 chemotypes. Three major chemotypes occurred in up to six lakes. One chemotype occurred in lakes around the city of Oslo (Norway), on the Finnish island Fasta Åland, which is situated in the Baltic Sea, and on the Finnish mainland. This wide distribution suggests that chemotypes can be subjects of recurrent dispersal and/or strong directional selection. Lake size, maximum depth, and nutrient availability appeared to be of minor importance for the ability of some chemotypes to colonize a water body successfully as long as the general requirements of Planktothrix were met. Four chemotypes were reisolated from the Oslo lake district over a period of 33–40 years, suggesting that they have been members of local Planktothrix populations for decades.     

A Chemotaxonomic Study of Vibrio fluvialis Based on the Sugar Composition of the Polysaccharide Portion of the Lipopolysaccharides

A chemotaxonomic study was carried out with a new serotyping scheme comprising 35 O-antigen groups of Vibrio fluvialis on the basis of the sugar composition of the polysaccharide portion of their lipopolysaccharide (LPS). A previously developed rapid method of preparing samples for compositional sugar analysis was employed. The 35 O-antigen groups were divided into 21 chemotypes. It is noted that a rarely occurring component sugar of gram-negative bacterial LPS, D -glycero-D -manno-heptose, and two kinds of uronic acids, i.e., galacturonic acid of a weakly bound type and glucuronic acid of a strongly bound type, were found in common in all the 21 chemotypes. A characteristic sugar component of gram-negative bacterial LPS, 2-keto-3-deoxyoctonate (KDO), was not detectable in any of the 21 chemotypes. Instead, three kinds of “KDO-like substances” were found, one in each of three chemotypes (III, XI and XVII). They were strongly positive in Weissbach's periodate-thiobarbituric acid test for KDO, but definitely not identical to it in high-voltage paper electrophoresis (HVPE); the substance present in chemotype XI was indicated by HVPE to be 3-deoxy-D -threo-hexulosonic acid which is a sugar constituent of Vibrio parahaemolyticus O7 and O12 LPS.     

Geographical and environment-related variations of essential oils in isolated populations of Thymus richardii Pers. in the Mediterranean basin

Composition of essential oils of different populations of Thymus richardii grex of six localities from Bosnia-Herzegovina (Konjic, Borci), Spain (Majorca, Ibiza, Valencia) and Italy (Marettimo, Sicily) were determined by GC/FID and GC/MS. The main constituents in most of the samples were aromatic monoterpenes corresponding to non-phenolic cyclic compounds (p-cymene, γ-terpinene). The highest monoterpene concentrations were found in the Bosnian samples (70%), and the lowest in samples from the Balearic Islands (<30%; Ibiza and Majorca). Sesquiterpenes were the major component (average > 50%) in samples from Majorca with β-bisabolene (>40%) being the principal constituent. Discriminant analysis (LDA) shows the differentiation of two chemotypes: A (phenol chemotype), with p-cymene and γ-terpinene as characteristic compounds and B, with β-bisabolene and carvacrol, as major and significative compounds. The occurrence of the chemotypes was related to summer positive precipitation and to deep of soils.     

A study of the genetic variability of Rhizoplaca chrysoleuca using DNA sequences and secondary metabolic substances

Specimens of Rhizoplaca chrysoleuca from Mount Wuling can be divided into two distinct groups based on obvious differences in morphological characters. Here we investigated 26 specimens of R. chrysoleuca from Mount Wuling, 10 specimens of this species from other areas and seven specimens of other Rhizoplaca species by analyzing morphology, chemistry and genetics. Nine chemotypes were detected among the specimens of R. chrysoleuca from Mount Wuling, and five of them were reported for the first time. Based on the ITS phylogenetic analysis, the chemotypes and the insertion distribution patterns in SSU rDNA, the samples of R. chrysoleuca from Mount Wuling were grouped in two distinct clades corresponding to two phenotypic groups and no gene flow was detected between these two groups. Our results establish all individuals of Rhizoplaca chrysoleuca are conspecific although some populations have been isolated on Mount Wuling, indicating that they are in the process of speciation. Our study also reveals that the relationships between genotypes and chemotypes are complicated and should be avoided, and we instead recommend using single individuals or few individuals from the same site to represent the population or whole species in systematics study. The results also indicate that Rhizoplaca chrysoleuca might provide a good model for studying the speciation of saxicolous lichenized fungi.     

Quantification of 22 plasma amino acids combining derivatization and ion-pair LC-MS/MS

Time efficient and comprehensive quantification of amino acids continues to be a challenge. We developed a sensitive and precise method for quantitative analysis of amino acids from very small plasma and serum volumes. Ion-pair chromatography of amino acid butyl esters proved to provide an optimal combination of selectivity, sensitivity and robustness. 10 μL of plasma or serum are added to precipitation reagent containing stable isotope standards. After protein precipitation, the supernatants is dried and incubated with 3N butanolic HCl for improving chromatographic separation and ionization efficiency. Amino acid butyl esters are separated using ion-pair (heptafluorobutyric acid) reversed-phase chromatography coupled to triple quadrupole mass spectrometry. The established method enables quantitative analysis of 22 amino acids, all 20 proteinogenic amino acids, ornithine and citrulline. Cysteine is measured as cystine. The combination of precipitation, derivatization and chromatographic separation effectively avoids ion suppression and coelution. Simultaneous with quantification, analyte identity is verified in each sample using qualifier ions. The micro-method is very sensitive and accurate. The intra-assay precision for the analysis of plasma was 2.6-10.1%. Absolute accuracy as determined by comparison of external reference samples was 82-117.7%. Excellent linearity of detection response was demonstrated for all compounds in the range representative for clinical samples from infants and adults. Lower limits of quantification were in the range of 1 μmol/L for all analytes. In conclusion, the method is ideally suited for cost-effective high-throughput analysis of large numbers of samples in clinical studies and metabolomics research.     

Integrated removal of nucleic acids and recovery of LDH from homogenate of beef heart by affinity precipitation

Lactate dehydrogenase (LDH) was purified from beef heart homogenate by affinity precipitation. The protein purification was integrated with nucleic acid removal and was done by precipitation of nucleic acids by addition of poly(ethylene imine) PEI onto which a ligand, Cibacron blue, had been coupled. The yield of LDH after elution from the precipitate was 63%, the purification factor 6.9 and the nucleic acid content was reduced by 98%. The capacity of the affinity polymer Cibacron blue-PEI is dependent on the nucleic acid concentration in the homogenate. The beef heart homogenate had an unfavourable ratio of nucleic acids to LDH. Precipitation with recirculated Cibacron blue-PEI, already complexed with some nucleic acids, improved the yield of the enzyme to 74%. The loss of Cibacron blue-PEI, when recirculated, was less than 1% after each cycle. This revised version was published online in July 2006 with corrections to the Cover Date.     

Quantitative trait loci regulating the fatty acid profile of acylsugars in tomato

Acylsugars are secondary metabolites with proven insect resistance properties that are produced by many Solanaceous species including Solanum pennellii, a wild relative of tomato. The acylsugar chemotypes of S. pennellii varies greatly within its natural range and might be the product of plant/insect coevolution. The S. pennellii accession LA716 was used to transfer increased levels of acylsugar production into the cultivated tomato, resulting in the acylsugar tomato breeding line CU071026. S. pennellii accession LA716 produces high levels of acylsugars with chemotypes that differ greatly from those produced by CU071026 or the trace acylsugars of cultivated tomato. Understanding the genetic regulation of acylsugar chemistry will aid efforts to breed acylsugar production into cultivated tomato, allowing for alteration of both acylsugar base moieties and fatty acid profiles. This study uses a BC1F1 population produced from the cross of S. pennellii LA716 and CU071026 with CU071026 as the recurrent parent to identify QTL that change the fatty acid profile of acylsugars. Multiple QTL and epistatic interactions between QTL were detected including three QTL on chromosomes 2, 5, and 7, which significantly alter the percentage of extended iso-odd branched fatty acids and straight chain fatty acids on the acylsugars. We also report the introgression of one of these QTL, FA 2, into CU071026, resulting in a new tomato line with significantly increased i11:0 as a percentage of the fatty acids in its acylsugars. Candidate genes for these QTL and the impacts of altering acylsugar fatty acid are discussed.     

Disparate proteome reactivity profiles of carbon electrophiles

Insights into the proteome reactivity of electrophiles are crucial for designing activity-based probes for enzymes lacking cognate affinity labels. Here, we show that different classes of carbon electrophiles exhibit markedly distinct amino acid labeling profiles in proteomes, ranging from selective reactivity with cysteine to adducts with several amino acids. These data specify electrophilic chemotypes with restricted and permissive reactivity profiles to guide the design of next-generation functional proteomics probes.     

The effect of previously infested spruce needles on the growth of the green spruce aphid, Elatobium abietinum, and the effect of the aphid on the amino acid balance of the host plant

The effect of chlorosis induced in needles of Sitka and Norway spruce by the green spruce aphid on growth of the aphid is investigated, and the effect of infestation of the aphid on amino acid levels in Sitka spruce foliage is reported. On both Sitka and Norway spruce green spruce aphids were heavier when reared on chlorotic (previously infested) needles than when reared on green (previously uninfested) needles. The effect was more pronounced on Sitka than on Norway spruce. Infestation of the aphid altered the amino acid balance of Sitka spruce foliage but not the concentration of total amino acids. Possible causes of chlorosis, the influence of individual amino acids on aphid growth, the potential effect of chlorosis on outbreaks of the aphid and the differences in susceptibility of Sitka and Norway spruce to damage by the aphid are discussed.     

中国砖孢发属地衣研究

简短回顾了砖孢发属地衣研究概况。在已知的8种亚洲砖孢发属地衣中,中国有七种,其中两种为台湾特有。根据150余份标本的形态与化学特征,承认中国大陆砖孢发属地衣5种,指出了个别异名,发现亚洲砖孢发(Okpogon asiaticus)存在两个化学型,提供了中国5种砖孢发地衣分布的新资料。研究结果表明,5种砖孢发地衣集中分布于我国云南、四川和西藏东部。作者认为中国西南地区是砖孢发属地衣在亚洲的一个分布中心。     

The antimicrobial activity of substances derived from the lichens Physcia aipolia, Umbilicaria polyphylla, Parmelia caperata and Hypogymnia physodes

In this study, in vitro antimicrobial activity of the physodic acid, usnic acid, atranorin and gyrophoric acid isolated from the lichens Hypogymnia physodes, Parmelia caperata, Physcia aipolia and Umbilicaria polyphylla, has been investigated. An antibiotic assessment was done against six bacteria (three Gram-positive and three Gram-negative) and eight fungi by determining the minimal inhibitory concentration (MIC) by the broth tube dilution method. The tested lichen substances inhibited growth of all the tested microorganisms. The bacteria showed a higher sensitivity against the tested fungi. The highest antimicrobial activity was found in the usnic acid of the Parmelia caperata lichen, where the lowest MIC was 0.0037 mg/ml against the Klebsiella pneumoniae (even lower than the one given by the streptomycin standard). The weakest antimicrobial activity was found in the physodic acid, which inhibited most of the microorganisms in the concentration of 1 mg/ml. Generally, all the components had relatively strong antimicrobial activity against the tested microorganisms, among which were human and animal pathogens. This could be of significance for their use for pharmaceutical purposes.     

Chemical variation in the lichen genus <Emphasis Type="Italic">Letrouitia</Emphasis> (Ascomycota,Letrouitiaceae)

Secondary metabolites from 193 specimens belonging to 15 species of Letrouitia were analyzed by HPLC. Significant quantities of the anthraquinones parietin and fragilin were found in most species and occasionally minor quantities of emodin, 5-chloroemodin, 7-chloroemodin, 7-chloroteloschistin, 7-chlorofallacinal and 7-chloroparietinic acid were present. Eight previously unknown lichen substances were identified. A chemotype containing seven new dibenzofurans (8-chlorodioxocondidymic acid, 8-chlorodioxodidymic acid, 8-chloroxodidymic acid, dioxocondidymic acid, dioxodidymic acid, letrouitic acid and oxodidymic acid) was found in 3 species. In addition, a chemotype containing four unknowns (possibly chlorodepsidones) occurred in L. subvulpina. Eight different chemotypes were identified in the genus. The secondary chemistry was important for the precise identification of some species in Letrouitia. The similarity in secondary chemistry between Letrouitiaceae and Teloschistaceae is not particularly strong, as the shared compounds are also known to occur in several other lichen families.     

Rapid small-scale preparation method of cell surface polysaccharides

A rapid small-scale method of extraction of lipopolysaccharide (LPS) and capsular polysaccharides was developed for the purpose of identification of chemotypes of LPS and serotypes of capsular antigens. Cell surface polysaccharides were prepared within less than 2 hr from 1.5 ml of broth or suspension of colonies cultured overnight. The preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for LPS, and by double diffusion gel precipitation (Ouchterlony) test and blotting to nitrocellulose membrane for capsular polysaccharide. The analyses with the preparations obtained by the method could provide adequate results capable of identifying chemotypes of LPS and serotypes of capsular antigens.