Chemical Composition, Antitumor Properties, and Mechanism of the Essential Oil from Plagiomnium acutum T. Kop.

Plagiomnium acutum T. Kop. (P. acutum) has been used as a traditional Chinese medicine for thousands of years to treat cancer but lacks evidence. The objective of this work was to reveal the chemical composition of P. acutum essential oil (PEO) and explore its potential antitumor activity and molecular mechanism. PEO was prepared by the simultaneous distillation-extraction method and characterized by gas chromatography/mass spectroscopy. CCK8 assay, flow cytometry, western blot, and immunofluorescence techniques were used to analyze the effects and mechanism of PEO against cancer cells. A total of 74 constituents of PEO were identified, with diterpenes (26.5%), sesquiterpenes (23.89%), and alcohols (21.81%) being the major constituents. Two terpenoids, selina-6-en-4-ol and dolabella-3,7-dien-18-ol, were detected in PEO for the first time. PEO showed significant cell growth inhibitory activity on HepG2 and A549 cells by blocking the G1 phase and inducing apoptosis, which may be attributed to its upregulation of p21Cip1 and p27Kip1 proteins and interference with mitochondrial membrane potential effect. Dolabella-3,7-dien-18-ol accounts for 25.5% of PEO and is one of the main active components of PEO, with IC50 values in HepG2 and A549 cells of (25.820 ± 0.216) µg/mL and (23.597 ± 1.207) µg/mL, respectively. These results confirmed the antitumor medicinal value of P. acutum and showed great application potential in the pharmaceutical industry.

Determination of iron species, including biomineralized jarosite, in the iron-hyperaccumulator moss Scopelophila ligulata by Mössbauer, X-ray diffraction, and elemental analyses.

Scopelophila ligulata is an Fe-hyperaccumulator moss growing in acidic environments, but the mechanism of Fe accumulation remains unknown. To understand the mechanism, we determined Fe species in S. ligulata samples. The moss samples were collected from four sites in Japan. The concentrations of Fe, P, S, Cl, and K in them were measured by induced coupled plasma mass spectrometry. Fe species in some of them were determined by Mössbauer spectroscopy and were confirmed by X-ray diffraction analysis. Fe species in S. ligulata samples were determined to be jarosite, ferritin, high-spin Fe(II) species, and akaganeite. To our knowledge, this is the first report on the biomineralization of jarosite in mosses. This result, combined with the fact that bacteria, a fungus, and a grass mineralize jarosite, suggests that its biomineralization is a common characteristic in a wide variety of living organisms. These findings indicate that the biomineralization of jarosite occurs not only in the region-specific species but in species adapted to a low-pH and metal-contaminated environment in different regions, provide a better understanding of the mechanism of Fe accumulation in the Fe-hyperaccumulator moss S. ligulata, and offer new insights into the biomineralization of jarosite.

Desiccation tolerance in the Antarctic moss Sanionia uncinata

BACKGROUND: One of the most extreme environments on our planet is the Maritime Antarctic territory, due to its low-water availability, which restricts the development of plants. Sanionia uncinata Hedw. (Amblystegiaceae), the main colonizer of the Maritime Antarctic, has effective mechanisms to tolerate this environment. It has been described that the tolerance to desiccation is mediated by the hormone abscisic acid (ABA), antioxidants systems, accumulation of compatible solutes and proteins of the late embryogenesis abundant (LEA). However, to date, these mechanisms have not been described in S. uncinata. Therefore, in this work, we postulate that the tolerance to desiccation in the Antarctic moss S. uncinata is mediated by the accumulation of ABA, the osmolytes proline and glycine betaine, and dehydrins (an LEA class 11 proteins). To demonstrate our hypothesis, S. uncinata was subjected to desiccation for 24 h (loss in 95% of water content), and the effects on its physiological, photosynthetic, antioxidant and biochemical parameters were determined. RESULTS: Our results showed an accumulation of ABA in response to water loss, and the activation of protective responses that involves an increment in levels of proline and glycine betaine, an increment in the activity of antioxidant enzymes such as SOD, CAT, APX and POD, and the accumulation of dehydrins proteins. CONCLUSION: The results showed, suggest that S. uncinata is a desiccation-tolerant moss, property mediated by high cellular plasticity regulated by ABA.

Botrysphones A-C and Botrysphins A-F, Triketides and Diterpenoids from the Fungus Botrysphaeria laricina.

Three new triketides, botrysphones A-C (1-3) and six new isopimarane-type diterpenoids, botrysphins A-F (4-9), together with the known triketides sphaeropsidone (10) and chlorosphaeropsidone (11) and diterpenoids sphaeropsidins A and B (12 and 13), were obtained from culture of the fungus Botrysphaeria laricina associated with the moss Rhodobryum umgiganteum. The structures of the new compounds were established on the basis of extensive spectroscopic techniques including HRMS and 1D and 2D NMR data. Compounds 7 and 12 showed significant quinone reductase inducing activity in Hepa 1c1c7 cells.

Estimation of plant sampling uncertainty: an example based on chemical analysis of moss samples.

In order to estimate the level of uncertainty arising from sampling, 54 samples (primary and duplicate) of the moss species Pleurozium schreberi (Brid.) Mitt. were collected within three forested areas (Wierna Rzeka, Piaski, Poslowice Range) in the Holy Cross Mountains (south-central Poland). During the fieldwork, each primary sample composed of 8 to 10 increments (subsamples) was taken over an area of 10 m2 whereas duplicate samples were collected in the same way at a distance of 1-2 m. Subsequently, all samples were triple rinsed with deionized water, dried, milled, and digested (8 mL HNO3 (1:1) + 1 mL 30 % H2O2) in a closed microwave system Multiwave 3000. The prepared solutions were analyzed twice for Cu, Fe, Mn, and Zn using FAAS and GFAAS techniques. All datasets were checked for normality and for normally distributed elements (Cu from Piaski, Zn from Poslowice, Fe, Zn from Wierna Rzeka). The sampling uncertainty was computed with (i) classical ANOVA, (ii) classical RANOVA, (iii) modified RANOVA, and (iv) range statistics. For the remaining elements, the sampling uncertainty was calculated with traditional and/or modified RANOVA (if the amount of outliers did not exceed 10 %) or classical ANOVA after Box-Cox transformation (if the amount of outliers exceeded 10 %). The highest concentrations of all elements were found in moss samples from Piaski, whereas the sampling uncertainty calculated with different statistical methods ranged from 4.1 to 22 %.

The sources and fate of (210)Po in the urban air: A review.

The origin of (210)Po activity and its fluctuations in the air are discussed in this paper. In the case of atmospheric aerosol samples, a comparison of the (210)Po/(210)Pb and (210)Bi/(210)Pb activity ratios makes it possible not only to determine aerosol residence times but also to appraise the contribution of the unsupported (210)Po coming from other sources than (222)Rn decay, such as human industrial activities, especially coal combustion. A simple mathematical method makes it possible to observe the seasonal fluctuations of the anthropogenic excess of (210)Po in the urban air. The average doses of (210)Po intake with food (including drinking water) and inhalation of urban aerosols are usually lower than those from (210)Po intake by cigarette smokers and negligible in comparison to total natural radiation exposure.

Characterization of the GPI-anchored lipid transfer proteins in the moss Physcomitrella patens.

The non-specific lipid transfer proteins (nsLTPs) are characterized by a compact structure with a central hydrophobic cavity very suitable for binding hydrophobic ligands, such as lipids. The nsLTPs are encoded by large gene families in all land plant lineages, but seem to be absent from green algae. The nsLTPs are classified to different types based on molecular weight, sequence similarity, intron position or spacing between the cysteine residues. The Type G nsLTPs (LTPGs) have a GPI-anchor in the C-terminal region which may attach the protein to the exterior side of the plasma membrane. Here, we present the first characterization of nsLTPs from an early diverged plant, the moss Physcomitrella patens. Moss LTPGs were heterologously produced and purified from Pichia pastoris. The purified moss LTPGs were found to be extremely heat stable and showed a binding preference for unsaturated fatty acids. Structural modeling implied that high alanine content could be important for the heat stability. Lipid profiling revealed that cutin monomers, such as C16 and C18 mono- and di-hydroxylated fatty acids, could be identified in P. patens. Expression of a moss LTPG-YFP fusion revealed localization to the plasma membrane. The expressions of many of the moss LTPGs were found to be upregulated during drought and cold treatments.

Composition and structure of photosystem I in the moss Physcomitrella patens.

Recently, bryophytes, which diverged from the ancestor of seed plants more than 400 million years ago, came into focus in photosynthesis research as they can provide valuable insights into the evolution of photosynthetic complexes during the adaptation to terrestrial life. This study isolated intact photosystem I (PSI) with its associated light-harvesting complex (LHCI) from the moss Physcomitrella patens and characterized its structure, polypeptide composition, and light-harvesting function using electron microscopy, mass spectrometry, biochemical, and physiological methods. It became evident that Physcomitrella possesses a strikingly high number of isoforms for the different PSI core subunits as well as LHCI proteins. It was demonstrated that all these different subunit isoforms are expressed at the protein level and are incorporated into functional PSI-LHCI complexes. Furthermore, in contrast to previous reports, it was demonstrated that Physcomitrella assembles a light-harvesting complex consisting of four light-harvesting proteins forming a higher-plant-like PSI superstructure.

Effect of methanolic and water extract of Leucobryum bowringii Mitt. on growth, migration and invasion of MCF 7 human breast cancer cells in vitro.

Inhibitory effects of methanol and water extract of L. bowringii. on the adhesion, migration, invasion and matrix metalloproteinase (MMP) activities of MCF 7 human breast cancer cell line are reported. Cells were cultured with 10, 25, 50 microg/mL methanolic or water extract of L. bowringii. Culture medium containing 0.1% DMSO was used as a solvent control. Ultra structural analysis by electron microscopy revealed typical features of apoptosis. A remarkable dose-response parallelism was observed between methanolic extract with growth, migration and invasion of breast cancer cells. Fractionation of methanolic extract by RP-HPLC revealed a pool of phenolic acids. Hoechst 33342 staining assay reveals massive chromatin condensation and subsequent cleavage of structural components of nucleus. The results indicate that methanol extracts inhibit the growth of human breast cancer cells partially through the inhibition of metallo proteinases MMP-2 and MMP-9 activities. Methanolic extract has more anti-metastatic effects in cell based assay than water extract. Clinical application of L. bowringii extract as a bioactive chemopreventive compound may be helpful in limiting breast carcinoma invasion and metastasis.

Bacteriocin release protein-mediated secretory expression of recombinant chalcone synthase in Escherichia coli.

Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant, and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at OD(600) induction time of 0.5 was found to be the best condition for chalcone synthase secretion.

[Exploration and identification of Physcomitrella patens moss peptides].

In the current study the isolation and identification of Physcomitrella patens (Hedw.) B.S.G. moss peptides are described. Physcomitrella patens moss is actively used in recent years as a model organism to study the biology of plants. Protoplasts, protonemata and gametophores of the moss are demonstrated for the first time to contain diverse small peptides. From gametophores was isolated and identified 58 peptides that are fragments of 14 proteins, and from protonemata - 49 peptides, fragments of 15 proteins. It was found that the protonemata and gametophores Ph. patens, which are the successive stages of development of this plant, significantly different from each other as a peptide composition and the spectrum of the precursor protein of identified peptides. Isolation of protoplasts of the enzymatic destruction of cell wall protonemata accompanied by massive degradation of intracellular proteins, many of whom are proteins of photosynthesis, which is a characteristic response of plants to stress the impact of environmental factors. A total of moss protoplasts were isolated and identified 323 peptides that are fragments of 79 proteins.

Auxin-binding proteins without KDEL sequence in the moss Funaria hygrometrica.

Whereas the important plant growth regulator auxin has multiple effects in flowering plants, it induces a specific cell differentiation step in the filamentous moss protonema. Here, we analyse the presence of classical auxin-binding protein (ABP1) homologues in the moss Funaria hygrometrica. Microsomal membranes isolated from protonemata of F. hygrometrica have specific indole acetic acid-binding sites, estimated to be about 3-5 pmol/mg protein with an apparent dissociation constant (K (d)) between 3 and 5 microM. Western analyses with anti-ABP1 antiserum detected the canonical endoplasmic reticulum (ER)-localised 22-24 kDa ABP1 in Zea mays, but not in F. hygrometrica. Instead, polypeptides of 31-33 and 46 kDa were labelled in the moss as well as in maize. In F. hygrometrica these proteins were found exclusively in microsomal membrane fractions and were confirmed as ABPs by photo-affinity labelling with 5-azido-[7-(3)H]-indole-3-acetic acid. Unlike the classical corn ABP1, these moss ABPs did not contain the KDEL ER retention sequence. Consistently, the fully sequenced genome of the moss Physcomitrella patens, a close relative of F. hygrometrica, encodes an ABP1-homologue without KDEL sequence. Our study suggests the presence of putative ABPs in F. hygrometrica that share immunological epitopes with ABP1 and bind auxin but are different from the classical corn ABP1.

In silico and biochemical analysis of Physcomitrella patens photosynthetic antenna: identification of subunits which evolved upon land adaptation.

BACKGROUND: In eukaryotes the photosynthetic antenna system is composed of subunits encoded by the light harvesting complex (Lhc) multigene family. These proteins play a key role in photosynthesis and are involved in both light harvesting and photoprotection. The moss Physcomitrella patens is a member of a lineage that diverged from seed plants early after land colonization and therefore by studying this organism, we may gain insight into adaptations to the aerial environment. PRINCIPAL FINDINGS: In this study, we characterized the antenna protein multigene family in Physcomitrella patens, by sequence analysis as well as biochemical and functional investigations. Sequence identification and analysis showed that some antenna polypeptides, such as Lhcb3 and Lhcb6, are present only in land organisms, suggesting they play a role in adaptation to the sub-aerial environment. Our functional analysis which showed that photo-protective mechanisms in Physcomitrella patens are very similar to those in seed plants fits with this hypothesis. In particular, Physcomitrella patens also activates Non Photochemical Quenching upon illumination, consistent with the detection of an ortholog of the PsbS protein. As a further adaptation to terrestrial conditions, the content of Photosystem I low energy absorbing chlorophylls also increased, as demonstrated by differences in Lhca3 and Lhca4 polypeptide sequences, in vitro reconstitution experiments and low temperature fluorescence spectra. CONCLUSIONS: This study highlights the role of Lhc family members in environmental adaptation and allowed proteins associated with mechanisms of stress resistance to be identified within this large family.

Spatial patterns of cadmium and lead deposition on and adjacent to National Park Service lands in the vicinity of Red Dog Mine, Alaska.

Heavy metal escapement associated with ore trucks is known to occur along the DeLong Mountain Regional Transportation System (DMTS) haul road corridor in Cape Krusenstern National Monument, northwest Alaska. Heavy metal concentrations in Hylocomium splendens moss (n = 226) were used in geostatistical models to predict the extent and pattern of atmospheric deposition of Cd and Pb on Monument lands. A stratified grid-based sample design was used with more intensive sampling near mine-related activity areas. Spatial predictions were used to produce maps of concentration patterns, and to estimate the total area in 10 moss concentration categories. Heavy metal levels in moss were highest immediately adjacent to the DMTS haul road (Cd > 24 mg/kg dw; Pb > 900 mg/kg dw). Spatial regression analyses indicated that heavy metal deposition decreased with the log of distance from the DMTS haul road and the DMTS port site. Analysis of subsurface soil suggested that observed patterns of heavy metal deposition reflected in moss were not attributable to subsurface lithology at the sample points. Further, moss Pb concentrations throughout the northern half of the study area were high relative to concentrations previously reported from other Arctic Alaska sites. Collectively, these findings indicate the presence of mine-related heavy metal deposition throughout the northern portion of Cape Krusenstern National Monument. Geospatial analyses suggest that the Pb depositional area extends 25 km north of the haul road to the Kisimilot/Iyikrok hills, and possibly beyond. More study is needed to determine whether higher moss heavy metal concentrations in the northernmost portion of the study area reflect deposition from mining-related activities, weathering from mineralized Pb/Zn outcrops in the broader region, or a combination of the two. South of the DMTS haul road, airborne deposition appears to be constrained by the Tahinichok Mountains. Heavy metal levels continue to diminish south of the mountains, reaching a minimum in the southernmost portion of the study area near the Igichuk Hills (45 km from the haul road). The influence of the mine site was not studied.

Differences in concentration of heavy metals between native and transplanted Plagiothecium denticulatum: a case study of soils contaminated by oil well exudates in South East Poland.

For a period of 60 days, the terrestrial moss Plagiothecium denticulatum was transplanted from a clean control site to a soil contaminated with crude oil exudates (Krosno, South East Poland). Native P. denticulatum growing on this contaminated soil was collected during the same period. Concentrations of the metals Al, Ba, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, V, and Zn as well as the macroelements N, P, K, Ca, Mg, and S were determined in P. denticulatum and in soils from the contaminated and control sites. Contaminated soil was the main contributor of Ba, Co, Cr, Cu, Fe, Mn, Ni, Pb, V, and Zn to native and transplanted P. denticulatum. Other sources of contamination apart from the soil existed for Cd and Cu. Transplanted P. denticulatum accumulated significantly more Al, Cr, Cu, Ni, Pb, and Zn, and native P. denticulatum accumulated significantly more N, P, K, and Ca. Higher levels of N, P, K, and Ca in native P. denticulatum indicates a possible mechanism to prevent the loss of these elements as observed in the transplanted P. denticulatum.

The use of the aquatic moss Fontinalis antipyretica L. ex Hedw. as a bioindicator for heavy metals: 3. Cd2+ accumulation capacities and biochemical stress response of two Fontinalis species.

We studied heavy metal stress responses of two Fontinalis species, F. antipyretica and F. dalecarlica, collected from two habitats in Germany and Canada. The capacities of the two species for extracellular adsorption (biosorption) and intracellular uptake (bioaccumulation) of Cadmium (Cd2+) were investigated in the laboratory. Time-dependent Cd2+ adsorption by cell wall and intracellular uptake differed significantly between the two species. These differences were related to the number of Cd2+ binding sites, resulting from differences in leaflet surface and cell wall composition. Glutathione (GSH) levels in response to Cd2+ exposure were monitored over a 10-day period. GSH synthesis differed significantly between the two species. Both Fontinalis species appear to be suitable for heavy metal biomonitoring in aquatic habitats.

UV-B radiation arising from stratospheric ozone depletion influences the pigmentation of the Antarctic moss Andreaea regularis.

Changes to the radiative environment arising from stratospheric ozone (O(3)) depletion and subsequent associations between these changes and the pigmentation of the moss Andreaea regularis were measured in late austral spring and early summer 1998 at Rothera Point on the western Antarctic Peninsula (67 degrees S, 68 degrees W). A strong relationship between O(3) column depth and the ratio of UV-B to PAR irradiance ( F(uv-b)/ F(par)) was recorded at ground level ( r(2)=92%, P<0.001). Weaker, but significant, associations between O(3) column depth and ground level unweighted and biologically effective UV-B radiation (UV-B(be)) were also found. Regression analyses indicated that F(uv-b)/ F(par) was the best predictor for concentrations of UV-B screening pigments and total carotenoids extracted from plant tissues. Concentrations of these pigments were loosely ( r(2)= ca. 30%) but significantly ( P<0.01) positively associated with F(uv-b)/ F(par). Concentrations of UV-B screening pigments were also positively associated with irradiances and daily doses of unweighted UV-B and UV-B(be) radiation. The concentrations of chlorophylls a and b were apparently unaffected by O(3) depletion. The data derived from this study suggest that changes to the radiative environment associated with stratospheric O(3) depletion influence the pigmentation of A. regularis. As a corollary, flavonoids are shown to be present in tissues of A. regularis.

Origin of lead in eight Central European peat bogs determined from isotope ratios, strengths, and operation times of regional pollution sources.

Lead originating from coal burning, gasoline burning, and ore smelting was identified in 210Pb-dated profiles through eight peat bogs distributed over an area of 60,000 km2. The Sphagnum-dominated bogs were located mainly in mountainous regions of the Czech Republic bordering with Germany, Austria, and Poland. Basal peat 14C-dated at 11,000 years BP had a relatively high 206Pb/207Pb ratio (1.193). Peat deposited around 1800 AD had a lower 206Pb/207Pb ratio of 1.168-1.178, indicating that environmental lead in Central Europe had been largely affected by human activity (smelting) even before the beginning of the Industrial Revolution. Five of the sites exhibited a nearly constant 206Pb/207Pb ratio (1.175) throughout the 19th century, resembling the "anthropogenic baseline" described in Northern Europe (1.17). At all sites, the 206Pb/207Pb ratio of peat decreased at least until 1980; at four sites, a reversal to more radiogenic values (higher 206Pb/207Pb), typical of easing pollution, was observed in the following decade (1980-1990). A time series of annual outputs for 14 different mining districts dispersing lead into the environment has been constructed for the past 200 years. The production of Ag-Pb, coal, and leaded gasoline peaked in 1900, 1980, and 1980, respectively. In contrast to other European countries, no peak in annual Pb accumulation rates was found in 1900, the year of maximum ore smelting. The highest annual Pb accumulation rates in peat were consistent with the highest Pb emission rates from coal-fired power plants and traffic (1980). Although maximum coal and gasoline production coincided in time, their isotope ratios were unique. The mean measured 206Pb/207Pb ratios of local coal, ores, and gasoline were 1.19, 1.16, and 1.11, respectively. A considerable proportion of coal emissions, relative to gasoline emisions, was responsible for the higher 206Pb/207Pb ratios in the recent atmosphere (1.15) compared to Western Europe (1.10). As in West European countries, the gasoline sold in the Czech Republic during the Communist era (1948-1989) contained an admixture of low-radiogenic Precambrian lead from Australia.

Nematocidal activity of natural polyphenols from bryophytes and their derivatives.

The nematocidal in vitro activity of three natural perotetins (phenolic bisbibenzyiethers) and eleven diphenyl ethers used as synthetic precursors has been assayed using two different experimental models, Caenorhabditis elegans and Nippostrongylus brasiliensis. Nine compounds showed some activity against C. elegans and nine against N. brasiliensis. For the former model, three compounds displayed an activity similar to that of the standards, whereas for N. brasiliensis none of the tested compounds was as active as the standards. From the in vitro results, five compounds (3, 4, 8, 9, 13) could be selected as lead compounds to continue the search for improved activity.

Influence of current velocity on cadmium accumulation by an aquatic moss and the consequences for its use as a biomonitor.

Aquatic mosses are widely used as biomonitors of contaminant concentrations in running waters. The results of several previous studies suggest that metal concentrations in mosses are influenced by current velocity and that this variable should be taken into account when mosses are used as metal biomonitors. However, in these studies, the purported influence of water velocity was confounded by other uncontrolled variables. We conducted our study to test the influence of current velocity on Cd accumulation by the riverine moss Fontinalis dalecarlica. We found no difference in Cd accumulation over 2 weeks by moss exposed in the laboratory to a constant Cd concentration over a wide range of current velocities (0.01-0.70 m s-1) that occur in the field. Similarly, the results of a field experiment, in which we exposed F. dalecarlica in a Cd-contaminated creek to four current velocities (0.05-0.50 m s-1), confirmed that in nature Cd accumulation by this moss is not influenced by current velocity. We show that a bioaccumulation model that ignores current velocity describes Cd accumulation by F. dalecarlica very well. Our results suggest that current velocity does not have to be considered when using aquatic mosses as metal biomonitors.