The antiviral activity of Thuja orientalis folium against Influenza A virus.

Thuja orientalis Folium (TOF) has been prescribed traditionally as an expectorant for inflammatory airway disease. In this study, we evaluated the anti-influenza A virus (IAV) activity of TOF by detecting GFP expressed by influenza A virus (A/PR/8/34-GFP) infection. The fluorescence microscopy and fluorescence-activated cell sorting analysis showed that TOF potently inhibited IAV infection, dose-dependently. Consistently, immunofluorescence and Q-PCR analysis results confirmed TOF significantly represses IAV protein and RNA expression. TOF inhibited IAV infection at the binding and entry step upon viral infection and interferes with HA protein. Further, TOF exhibited a virucidal effect and inhibited the neuraminidase activity of IAV. Additionally, TOF prevented the cytopathic effect caused by H1N1 and H3N2 IAV infection. Amentoflavone among the constituents in TOF exerted the strongest anti-IAV effect. Myricetin, quercetin, and quercitrin also inhibited IAV infection. However, the potent anti-IAV effect of TOF may be related to the synergistic effect of constituents, not by a single specific compound. Our results suggest TOF exhibits a significant inhibitory effect against IAV infection at multi-stages via the blockage of viral attachment and entry, inhibition of neuraminidase, and induction of virucidal effects.

Apoptotic and autophagic death union by Thuja occidentalis homeopathic drug in cervical cancer cells with thujone as the bioactive principle.

OBJECTIVE: "Multi-targeting" drugs can prove fruitful to combat drug-resistance of multifactorial disease-cervical cancer. This study envisioned to reveal if Thuja homeopathic mother tincture (MT) and its bioactive component could combat human papillomavirus (HPV)-16-infected SiHa cervical cancer cells since it is globally acclaimed for HPV-mediated warts. METHODS: Thuja MT was studied for its antiproliferative and antimigratory properties in SiHa cells followed by microscopic determination of reactive oxygen species (ROS) generation by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining and loss in mitochondrial membrane potential (MtMP) by rhodamine 123 (Rh123) staining. Apoptosis and autophagy inductions were studied by acridine orange/ethidium bromide (AO/EB) staining and immunoblot analyses of marker proteins. The bioactive component of Thuja MT detected by gas chromatography-mass spectrometry was studied for antiproliferative and antimigratory properties along with in silico prediction of its cellular targets by molecular docking and oral drug forming competency. RESULTS: Thuja MT showed significant antiproliferative and antimigratory potential in SiHa cells at a 50% inhibitory concentration (IC50) of 17.3 µL/mL. An increase in DCFDA fluorescence and loss in Rh123 fluorescence prove that Thuja MT acted through the burst of ROS and loss in MtMP respectively. AO/EB-stained cells under the microscope and immunoblot analyses supported Thuja-induced cellular demise via dual pathways-apoptosis and autophagy. Immunoblots showed cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) along with upregulation of Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B)-II, and p62 proteins. Hence, the apoptotic cascade followed a caspase-3-dependent pathway supported by PARP-1 cleavage, while autophagic death was Beclin-1-dependent and mediated by accumulation of LC3BII and p62 proteins. Thujone, detected as the bioactive principle of Thuja MT, showed greater anti-proliferative and anti-migratory potential at an IC50 of 77 µg/mL, along with excellent oral drug competency with the ability for gastrointestinal absorption and blood-brain-barrier permeation with nil toxicity. Molecular docking depicted thujone with the strongest affinity for mammalian target of rapamycin, phosphoinositide 3-kinase, and protein kinase B followed by B-cell lymphoma 2, murine double minute 2 and adenosine monophosphate-activated protein kinase, which might act as upstream triggers of apoptotic-autophagic crosstalk. CONCLUSION: Robust "multi-targeting" anticancer potential of Thuja drug and thujone for HPV-infected cervical cancer ascertained its therapeutic efficacy for HPV infections.

Efflux and assimilation of xylem-transported CO2 in stems and leaves of tree species with different wood anatomy.

Determining the fate of CO2 respired in woody tissues is necessary to understand plant respiratory physiology and to evaluate CO2 recycling mechanisms. An aqueous 13 C-enriched CO2 solution was infused into the stem of 3-4 m tall trees to estimate efflux and assimilation of xylem-transported CO2 via cavity ring-down laser spectroscopy and isotope ratio mass spectrometry, respectively. Different tree locations (lower stem, upper stem and leafy shoots) and tissues (xylem, bark and leaves) were monitored in species with tracheid, diffuse- and ring-porous wood anatomy (cedar, maple and oak, respectively). Radial xylem CO2 diffusivity and xylem [CO2 ] were lower in cedar relative to maple and oak trees, thereby limiting label diffusion. Part of the labeled 13 CO2 was assimilated in cedar (8.7%) and oak (20.6%) trees, mostly in xylem and bark tissues of the stem, while limited solution uptake in maple trees hindered the detection of label assimilation. Little label reached foliar tissues, suggesting substantial label loss along the stem-branch transition following reductions in the radial diffusive pathway. Differences in respiration rates and radial xylem CO2 diffusivity (lower in conifer relative to angiosperm species) might reconcile discrepancies in efflux and assimilation of xylem-transported CO2 so far observed between taxonomic clades.

Defensive functions of volatile organic compounds and essential oils from northern white-cedar in China.

BACKGROUND: Plants are known to emit diverse volatile organic compounds (VOCs), which may function as signaling substances in plant communication with other organisms. Thuja occidentalis, which is widely cultivated throughout China, releases aromatic VOCs into the air in winter and early spring. The relationship of this cultivated plant with its neighboring plants is necessary for the conservation of biodiversity. RESULTS: (-)-α-thujone (60.34 ± 5.58%) was found to be the major component in VOCs from the Shenyang population. The essential oils (EOs) from the Kunming and Shenyang populations included the major components (-)-α-thujone, fenchone, (+)-ß-thujone, and (+)-hibaene, identified using GC-MS analyses. (-)-α-thujone and (+)-hibaene were purified and identified by NMR identification. EOs and (-)-α-thujone exhibited valuable phytotoxic activities against seed germination and seedling growth of the plants Taraxacum mongolicum and Arabidopsis thaliana. Moreover, the EOs displayed potent inhibitory activity against pathogenic fungi of maize, including Fusarium graminearum, Curvularia lunata, and Bipolaris maydis, as well as one human fungal pathogen, Candida albicans. Quantitative analyses revealed high concentrations of (-)-α-thujone in the leaves of T. occidentalis individuals from both the Shenyang and Kunming populations. However, (-)-α-thujone (0.18 ± 0.17 µg/g) was only detected in the rhizosphere soil to a distance of 0.5 m from the plant. CONCLUSIONS: Taken together, our results suggest that the phytotoxic effects and antifungal activities of the EOs and (-)-α-thujone in T. occidentalis certainly increased the adaptability of this plant to the environment. Nevertheless, low concentrations of released (-)-α-thujone indicated that reasonable distance of T. occidentalis with other plant species will impair the effects of allelochemical of T. occidentalis.

Case Reports of Five Cancer Patients with Unusual Course.

INTRODUCTION: The analysis of the periodic table of elements by Jan Scholten opened the way for a new kind of classification and repertorisation of homeopathic remedies. Thereby, group analysis (resorting to series and stages) makes precise prescriptions possible. This approach appears to yield striking results, even in severe cases. Whereas Hahnemann stressed the emotional state ('Gemüthssymptome', Organon § 210) when choosing a remedy, Scholten 200 years later investigated the mental picture that represents a life conflict or even a life theme that may maintain the disease process. The person's environment, emotional traumas or a conflict drives him or her to suppress and dissect painful emotions. Such compensations can become subconscious and so strong that they can no longer be controlled; they then influence the patient with a highly destructive energy. METHODS: We present five case reports, each dealing with an unusual clinical course of severe cancer associated with homeopathic treatment using the Scholten method. RESULTS: By presenting these cases, we consider how the constitution (lifelong signs and symptoms of the patient) and the mental state are interwoven and, as a complex mechanism, might provoke disease. CONCLUSION: The appropriate homeopathic remedy, reflecting the Scholten approach, seemed to have beneficial impact on the disease process of the five individuals presented.

Nitrogen uptake over entire root systems of tree seedlings.

Uptake of nitrogen (N) by sequential root regions in six tree species was measured in roots of 16- to 26-month-old seedlings at 50 and 1500 µM NH4NO3 concentration, at the cell level using oscillating microelectrodes and at the root region level using enriched (15)N application. Our objective was to determine the root regions making the greatest contribution to total N uptake in each species as measured by the two contrasting techniques. White and condensed tannin zones were the regions with the smallest surface area in all species, but these zones often had the highest net flux of NH4(+) and NO3(-). For most species, little variation was found among root regions in N flux calculated using a (15)N mass balance approach, but where significant differences existed, high N flux was observed in white, cork or woody zones. When N fluxes measured by each of the two methods were multiplied by the estimated surface area or biomass of each root region, the effect of root region size had the greatest influence on regional N uptake. Root regions of greatest overall N uptake were the cork and woody zones, on average. Total N uptake may thus be greatest in older regions of tree seedling roots, despite low rates of uptake per unit area.

Genetic diversity of the critically endangered Thuja sutchuenensis revealed by ISSR markers and the implications for conservation.

Thuja sutchuenensis Franch. is a critically endangered plant endemic to the North-East Chongqing, China. Genetic variation was studied to assess the distribution of genetic diversity within and among seven populations from the single remnant locations, using inter-simple sequence repeat (ISSR) markers. A total of 15 primers generated 310 well defined bands, with an average of 20.7 bands per primer. The seven populations revealed a relatively high level of genetic diversity in the species. The percentage of polymorphic bands, Nei's gene diversity and Shannon's information index at the population and species level were 76.1%, 0.155, 0.252 and 100%, 0.165, 0.295, respectively. A low level of genetic differentiation among populations (G(ST) = 0.102), in line with the results of Analyses of Molecular Variance (AMOVA), and a high level of gene flow (N(m) = 4.407) were observed. Both the Unweighted Pair Group Method with Arithmatic Mean (UPGMA) cluster analysis and Principal Coordinates Analysis (PCoA) supported the grouping of all seven populations into two groups. In addition, Mantel test revealed no significant correlation between genetic and geographical distances (r = 0.329, p = 0.100). The low genetic differentiation among populations implies that the conservation efforts should aim to preserve all the extant populations of this endangered species.

Activity, polypeptide and gene identification of thylakoid Ndh complex in trees: potential physiological relevance of fluorescence assays.

Three evergreen (Laurus nobilis, Viburnum tinus and Thuja plicata) and two autumnal abscission deciduous trees (Cydonia oblonga and Prunus domestica) have been investigated for the presence (zymogram and immunodetection) and functionality (post-illumination chlorophyll fluorescence) of the thylakoid Ndh complex. The presence of encoding ndh genes has also been investigated in T. plicata. Western assays allowed tentative identification of zymogram NADH dehydrogenase bands corresponding to the Ndh complex after native electrophoresis of solubilized fractions from L. nobilis, V. tinus, C. oblonga and P. domestica leaves, but not in those of T. plicata. However, Ndh subunits were detected after SDS-PAGE of thylakoid solubilized proteins of T. plicata. The leaves of the five plants showed the post-illumination chlorophyll fluorescence increase dependent on the presence of active Ndh complex. The fluorescence increase was higher in autumn in deciduous, but not in evergreen trees, which suggests that the thylakoid Ndh complex could be involved in autumnal leaf senescence. Two ndhB genes were sequenced from T. plicata that differ at the 350 bp 3' end sequence. Comparison with the mRNA revealed that ndhB genes have a 707-bp type II intron between exons 1 (723 bp) and 2 (729 bp) and that the UCA 259th codon is edited to UUA in mRNA. Phylogenetically, the ndhB genes of T. plicata group close to those of Metasequoia, Cryptomeria, Taxodium, Juniperus and Widdringtonia in the cupresaceae branch and are 5' end shortened by 18 codons with respect to that of angiosperms.

Estimation of canopy average mesophyll conductance using δ(13) C of phloem contents.

Conductance to CO(2) inside leaves, known as mesophyll conductance (g(m)), imposes large limitations on photosynthesis. Because g(m) is difficult to quantify, it is often neglected in calculations of (13)C photosynthetic discrimination. The 'soluble sugar method' estimates g(m) via differences between observed photosynthetic discrimination, calculated from the δ(13)C of soluble sugars, and discrimination when g(m) is infinite. We expand upon this approach and calculate a photosynthesis-weighted average for canopy mesophyll conductance ((c) g(m)) using δ(13)C of stem phloem contents. We measured gas exchange at three canopy positions and collected stem phloem contents in mature trees of three conifer species (Pseudotsuga menziesii, Thuja plicata and Larix occidentalis). We generated species-specific and seasonally variable estimates of (c)g(m) . We found that (c)g(m) was significantly different among species (0.41, 0.22 and 0.09 mol m(-2) s(-1) for Larix, Pseudotsuga and Thuja, respectively), but was similar throughout the season. Ignoring respiratory and photorespiratory fractionations ((c)Δ(ef)) resulted in ≈30% underestimation of (c)g(m) in Larix and Pseudotsuga, but was innocuous in Thuja. Substantial errors (~1-4‰) in photosynthetic discrimination calculations were introduced by neglecting (c)g(m) and (c)Δ(ef) . Our method is easy to apply and cost-effective, captures species variation and would have captured seasonal variation had it existed. The method provides an average canopy value, which makes it suitable for parameterization of canopy-scale models of photosynthesis, even in tall trees.

Relationship of dirigent protein and 18s RNA transcript localization to heartwood formation in western red cedar.

Western red cedar (Thuja plicata) heartwood contains abundant amounts of structurally complex plicatic acid-derived lignans that help confer protective properties and longevity to this tissue type. Although the lignan biochemical entry point is dirigent protein-mediated, the formation of heartwood and its associated lignans in some species remains poorly understood due to technical difficulties of working with the former. To begin to address such questions, this study therefore focused on the anatomical localization of dirigent protein and 18s rRNA (control) gene transcripts within recalcitrant woody tissues, including heartwood. This in situ mRNA hybridization approach enabled detection of dirigent protein transcripts in cork cambia, vascular cambia and ray parenchyma cells of the sapwood, but not the heartwood under the conditions employed. By contrast, the hybridization of the 18s rRNA (control) transcript resulted in its detection in all tissue types, including radial parenchyma cells of apparently preformed heartwood. Application of in situ hybridization to such recalcitrant tissues thus demonstrates the utility of this technique in identifying specific cell types involved in heartwood formation, as well as the relationship of dirigent protein localization to that of heartwood metabolite generation.

Biosorption of a textile dye (Acid Blue 40) by cone biomass of Thuja orientalis: estimation of equilibrium, thermodynamic and kinetic parameters.

Biosorption of Acid Blue 40 (AB40) onto cone biomass of Thuja orientalis was studied with variation in the parameters of pH, contact time, biosorbent and dye concentration and temperature to estimate the equilibrium, thermodynamic and kinetic parameters. The AB40 biosorption was fast and the equilibrium was attained within 50 min. Equilibrium data fitted well to the Langmuir isotherm model in the studied concentration range of AB40 and at various temperatures. Maximum biosorption capacity (q(max)) for AB40 was 2.05 x 10(-4)mol g(-1) or 97.06 mg g(-1) at 20 degrees C. The changes of Gibbs free energy, enthalpy and entropy of biosorption were also evaluated for the biosorption of AB40 onto T. orientalis. The results indicate that the biosorption was spontaneous and exothermic. Kinetics of biosorption of AB40 was analyzed and rate constants were also derived and the results show that the pseudo-second-order kinetic model agrees very well with the experimental data.

Monolignol radical-radical coupling networks in western red cedar and Arabidopsis and their evolutionary implications.

The discovery of a nine-member multigene dirigent family involved in control of monolignol radical-radical coupling in the ancient gymnosperm, western red cedar, suggested that a complex multidimensional network had evolved to regulate such processes in vascular plants. Accordingly, in this study, the corresponding promoter regions for each dirigent multigene member were obtained by genome-walking, with Arabidopsis being subsequently transformed to express each promoter fused to the beta-glucuronidase (GUS) reporter gene. It was found that each component gene of the proposed network is apparently differentially expressed in individual tissues, organs and cells at all stages of plant growth and development. The data so obtained thus further support the hypothesis that a sophisticated monolignol radical-radical coupling network exists in plants which has been highly conserved throughout vascular plant evolution.

The western red cedar (Thuja plicata) 8-8' DIRIGENT family displays diverse expression patterns and conserved monolignol coupling specificity.

The isolation and characterization of a multigene family of the first class of dirigent proteins (namely that mainly involved in 8-8' coupling leading to (+)-pinoresinol in this case) is reported, this comprising of nine western red cedar (Thuja plicata) DIRIGENT genes (DIR1-9) of 72-99.5% identity to each other. Their corresponding cDNA clones had coding regions for 180-183 amino acids with each having a predicted molecular mass of ca. 20 kDa including the signal peptide. Real time-PCR established that the DIRIGENT isovariants were differentially expressed during growth and development of T. plicata (P < 0.05). The phylogenetic relationships and the rates and patterns of nucleotide substitution suggest that the DIRIGENT gene may have evolved via paralogous expansion at an early stage of vascular plant diversification. Thereafter, western red cedar paralogues have maintained an high homogeneity presumably via a concerted evolutionary mode. This, in turn, is assumed to be the driving force for the differential formation of 8-8'-linked pinoresinol derived (poly)lignans in the needles, stems, bark and branches, as well as for massive accumulation of 8-8'-linked plicatic acid-derived (poly)lignans in heartwood.

Linking deer browsing and terpene production among genetic identities in Chamaecyparis nootkatensis and Thuja plicata (Cupressaceae).

To investigate whether differential herbivore browsing reflects genetic variation in plant defense expression, variation in needle terpenes and damage caused by black-tailed deer (Odocoileus hemionus) was analyzed on yellow-cedar (Chamaecyparis nootkatensis) and western redcedar (Thuja plicata). In a 100-genet yellow-cedar population, three genets that were heavily browsed and had extremely low levels of monoterpenes (0-0.36% dry matter), sesquiterpenes, and diterpenes were compared to unbrowsed genets (0.85-3.83% monoterpenes in dry matter). These differences were maintained in individuals protected from browsing, suggesting genetically based variation in constitutive terpene production. In western redcedar, heavily browsed trees had significantly lower total monoterpene concentrations (1.69% dry matter) than lightly browsed trees (3.32% dry matter). One heavily browsed tree expressed no monoterpenes. No differences were found for diterpenes. In both species, the genotypes with extremely low monoterpene concentrations came from the same open-pollinated families.