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Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Relaxation of functional constraint on light-independent protochlorophyllide oxidoreductase in Thuja.

The light-independent protochlorophyllide oxidoreductase (DPOR) plays a key role in the ability of nonflowering plants and algae to synthesize chlorophyll in darkness. This enzyme consists of three subunits encoded by the chlB, chlL, and chlN genes in the plastid genome. Previously, we found a high nonsynonymous substitution rate (dN) of the chlL gene in the lineage of Thuja standishii, a conifer belonging to the Cupressaceae. Here we revealed that the acceleration of dN in the chlL occurred as well in other species of Thuja, Thuja occidentalis and Thuja plicata. In addition, dark-grown seedlings of T. occidentalis were found to exhibit a pale yellowish color, and their chlorophyll concentration was much lower than that of other species of Cupressaceae. The results suggested that the species of Thuja have lost the ability to synthesize chlorophyll in darkness, and the functional constraint on the DPOR would thus be expected to be relaxed in this genus. Therefore, we expected to find that the evolutionary rates of all subunits of DPOR would in this case be accelerated. Sequence analyses of the chlN and chlB (encoding the other subunits of DPOR) in 18 species of Cupressaceae revealed that the dN of the chlN gene was accelerated in Thuja as was the dN of the chlL gene, but the dN of the chlB gene did not appear to differ significantly among the species of Cupressaceae. Sequencing of reverse transcription-polymerase chain reaction (RT-PCR) products of these genes showed that RNA editing was rare and unlikely to have contributed to the acceleration. Moreover, the RT-PCR analysis indicated that all chl genes were still transcriptionally active in T. occidentalis. Based on these results, it appears that species of Thuja still bear the DPOR protein, although the enzyme has lost its activity because of nonsynonymous mutations of some of the chl genes. The lack of acceleration of the dN of the chlB gene might be accounted for by various unknown functions of its gene product.

Evolution, Molecular↗

(S,S)-2,12-, (S,S)-2,13-, and (S,S)-2,14-diacetoxyheptadecanes: sex pheromone components of red cedar cone midge, Mayetiola thujae.

We identified, synthesized, and field-tested the sex pheromone of female red cedar cone midge Mayetiola thujae (Hedlin) (Diptera: Cecidomyiidae), a pest insect in red cedar Thuja plicata seed orchards. Coupled gas chromatographic (GC)-electroantennographic detection analyses of pheromone extract revealed three components (A, B, C) that elicited responses from antennae of males, all of which occurred below the detection threshold of the mass spectrometer and thus had to be identified without spectroscopic data. Taking into account (1) their retention indices (RI) on three GC columns (DB-5, DB-23, and DB-210), (2) intercolumn RI differentials, and (3) the molecular structures of known cecidomyiid pheromones, we synthesized seven candidate pheromone components: 2,10-, 2,11-, 2,12-, 2,13-, 2,14-, 2,15- and 2,16-diacetoxyheptadecanes. Of these, 2,12-, 2,13-, and 2,14-diacetoxyheptadecane had RIs on all columns consistent with those of A, B, and C and elicited strong antennal responses when tested at picogram levels. In field experiments with the twelve stereoselectively synthesized stereoisomers, only the SS-stereoisomers of 2,12-, 2,13-, and 2,14-diacetoxyheptadecane attracted male M. thujae. The three-component SS-stereoisomer blend was more attractive than the 12-component blend of all stereoisomers, suggesting that one or several nonnatural stereoisomers are inhibitory. One-, two-, and three-component lures of the SS-stereoisomers were equally effective in attracting male M. thujae, indicating redundancy in the pheromone. Identification of the M. thujae sex pheromone will allow development of pheromone-based monitoring, and possibly control, of M. thujae populations in T. plicata seed orchards.

Animals↗

Allergic rhinitis to thuja pollen.

Allergy to pollen of Cupressaceae has been linked to pollens of Cupressus, Juniper and Cryptomeria. The authors report 2 cases of rhinitis and conjunctivitis induced by thuja, another member of the Cupressaceae family. Monosensitization to thuja pollen has been identified as the causal agent: (1) of a long-standing springtime rhinitis in 1 patient with negative skin tests and specific IgE titers to the main inhalants (specificity of the prick test and nasal provocation with the thuja extract was confirmed by a positive RAST) and (2) in a 2nd patient without former history of allergy, who consulted for conjunctivitis following acquisition of a dog; the standard battery of skin tests, and Phadiotop were all negative. After controls, only the thuja extract gave significant skin test response. The level of total IgE was low, and RAST was negative. Sensitization and pollen provocation were produced by the intermediary of the dog, carrying thuja pollens on its fur. The immunoprint and the crossed radioimmunoelectrophoresis revealed common antigenicity between cypress and thuja extracts.

Adult↗

GC analysis of essential oils in the rumen fluid after incubation of Thuja orientalis twigs in the Rusitec system.

Methods for the chemical analysis of toxic plant substances in the rumen of ruminants are of importance for the diagnosis of intoxications with poisonous plants. The present work establishes a method to estimate monoterpene components of the essential oil of thuja (Thuja orientalis, Cupressaceae) in these types of samples. Alpha-thujone, which is regarded as the toxic principle, is present at a concentration of 50-60% in the essential oil. The rumen simulation technique (Rusitec) was used to simulate natural digestion. Chopped twigs of thuja were subjected to rumen content in a closed container with an overflow device. The flow of saliva was simulated by the continuous addition of a buffer solution. Samples for analysis were taken from the overflow at 24 and 48 h. A further sample was taken from the remaining liquid fraction of the rumen content in the container at 48 h. The essential oils were extracted with hexane and concentrated. A quantitative determination was done by capillary gas chromatography. Together in the three fractions analysed this resulted in total mean recoveries of 6.8% for alpha-thujone, 5.3% for beta-thujone, 18.9% for fenchone and 27.8% for camphor. The observation that the thujones were recovered to a lesser extent than other oil components is evidence of their fast decomposition in the rumen medium. Under these circumstances the calculated detection limit is 100-200 g thuja twigs in cows with rumen volumes of 60-100 litres. The main essential oil degradation products found in the rumen fluid of all three fractions in the Rusitec system were discovered to be iso-3-thujanol, neo-3-thujanol, carvomenthol and carvomenthone.

Animals↗

Thuja occidentalis (Arbor vitae): A Review of its Pharmaceutical, Pharmacological and Clinical Properties.

Arbor vitae (Thuja occidentalis L.) is a native European tree widely used in homeopathy and evidence-based phytotherapy. Many reviews and monographs have been published on the herbal substance's description, mode of action and clinical use. However, no comprehensive evidence-based review is available. Therefore, our aim was to search MEDLINE databases and survey manufacturers for further details or unpublished data. This review presents the botany, ethnobotany and phytochemistry, especially the different contents of essential oil (Thujone) in relation to different extraction procedures of this medicinal plant. Thuja's antiviral action and immunopharmacological potential, such as stimulatory and co-stimulatory effects on cytokine and antibody production and activation of macrophages and other immunocompetent cells, have been evaluated in numerous in vitro and in vivo investigations. Although no controlled trials have been conducted on Thuja occ alone, many clinical studies have been performed with a herbal medicinal product containing a special extract of Thuja occ and other immunostimulants, demonstrating its therapeutic efficacy and safety in respiratory tract infections.

Journal Article↗

Contact allergy from Frullania and respiratory allergy from Thuja.

Occupational allergic contact dermatitis in 52 forest-workers was caused by sesquiterpene lactones from liverworts (Frullania) and by usnic acid from lichens which grow on various trees including cedar (Thuja). Occupational asthma and rhinitis in 35 wood-workers was caused by wood dust of western red cedar (Thuja plicata). Characteristically, the respiratory symptoms occurred in the evening and at night and not during working hours; inhalation challenge with plicatic acid from the wood provoked immediate, late or dual (combined immediate and late) asthmatic reactions. Another class of compounds, tropolones, derived from Thuja plicata wood, was responsible for dermatitis in a wood-worker. These distinct industrial hazards in two groups of workers at the tree-felling and wood-working levels in the forest-products industry can be identified by clinical history and examination supplemented by specific cutaneous or respiratory clinical investigation.

Alcohols↗

Branch and foliage morphological plasticity in old-growth Thuja plicata.

At the Wind River Canopy Crane Facility in southeastern Washington State, USA, we examined phenotypic variation between upper- and lower-canopy branches of old-growth Thuja plicata J. Donn ex D. Don (western red cedar). Lower-canopy branches were longer, sprouted fewer daughter branches per unit stem length and were more horizontal than upper-canopy branches. Thuja plicata holds its foliage in fronds, and these had less projected area per unit mass, measured by specific frond area, and less overlap, measured by silhouette to projected area ratio (SPARmax), in the lower canopy than in the upper canopy. The value of SPARmax, used as an indicator of sun and shade foliage in needle-bearing species, did not differ greatly between upper- and lower-canopy branches. We suggest that branching patterns, as well as frond structure, are important components of morphological plasticity in T. plicata. Our results imply that branches of old-growth T. plicata trees have a guerilla growth pattern, responding to changes in solar irradiance in a localized manner.

Light↗

Protective effect of Thuja occidentalis against radiation-induced toxicity in mice.

The effect of Thuja occidentalis against damage induced by gamma radiation was studied. Whole-body exposure of Swiss albino mice to gamma-rays (6 Gy) reduced the total white blood cell count to 1900 cells/mm(3) on the third day, which was elevated to 2050 cells/mm(3) by the administration of alcoholic extract ofT occidentalis (5 mg/dose/animal, intraperitoneally). Six animals from each group were killed after 2, 7, and 11 days of irradiation to detect the bone marrow cellularity and radiation-induced toxicity. The number of bone marrow cells and alpha-esterase positive cells in control animals after 11 days was reduced to 12.2 x 10(6) cells/femur and 693.5/4000 cells, respectively. In T occidentalis-treated animals, bone marrow cellularity was increased to 16.9 x 10(6) cells/femur and alpha-esterase positive cells were 940/4000 cells, a nearly normal level. Alcoholic extract of T occidentalis reduced the elevated levels of GPT and alkaline phosphatase in liver and serum after irradiation. The lipid peroxidation levels were also lowered in the irradiated animals treated with the Thuja extract.

Animals↗

Occupational asthma caused by eastern white cedar (Thuja occidentalis) with demonstration that plicatic acid is present in this wood dust and is the causal agent.

A worker developed symptoms of work-related asthma a few weeks after starting to work in a sawmill where eastern white cedar (Thuja occidentalis) was transformed into shingles. The diagnosis of occupational asthma was confirmed by monitoring of peak expiratory flow rates and bronchial responsiveness to histamine off work and at work, and specific inhalation challenges in the laboratory that demonstrated an isolated late asthmatic reaction after exposure for 4 hours to the wood dust. Specific inhalation challenges with western red cedar (thuja plicata) for 2 hours and plicatic acid (PA) for 7 minutes also caused an isolated late asthmatic reaction. Elevated specific IgE levels to PA were present. Antiserum was produced in rabbits that were immunized with PA conjugated to human serum albumin. With this antiserum in inhibition experiments, cross-reactivity between western red cedar and eastern white cedar, both belonging to the family of arborvitae, was found. It is estimated that eastern white cedar contains approximately half the amount of PA present in western red cedar.

Adult↗

Water Relations and Hydraulic Architecture of a Tropical Tree (Schefflera morototoni) : Data, Models, and a Comparison with Two Temperate Species (Acer saccharum and Thuja occidentalis).

The water relations and hydraulic architecture of a tropical tree (Schefflera morototoni) and of two temperate species (Acer saccharum and Thuja occidentalis) are reported. Among the water relations parameters measured were leaf and stem water storage capacity, leaf water potential, transpiration, and vulnerability of stems to cavitation and loss of hydraulic conductivity by embolisms. Among the hydraulic architecture parameters measured were hydraulic conductivity per unit pressure gradient, specific conductivity, leaf-specific conductivity, and Huber value. In terms of vulnerability of stems to cavitation, stem and leaf capacitances, and leaf-specific conductivity, all three species followed the same sequence: Schefflera > Acer > Thuja. It is argued here that the high stem capacitance and high leaf-specific conductivity of Schefflera are necessary to compensate for its high vulnerability to cavitation. Extractable water storage per unit leaf area in Schefflera stems is >100 times that of Acer and may permit the species to survive unusually long, dry seasons in Panama. Although Schefflera frequently grows >20 meters, the biggest resistance to water flow in the shoots resides in the leaves.

Journal Article↗

Thuja plicata exclusion in ectomycorrhiza-dominated forests: testing the role of inoculum potential of arbuscular mycorrhizal fungi.

The ability of trees dependent on arbuscular mycorrhizal (AM) fungi to establish in ectomycorrhizal forests is unknown. On northern Vancouver Island, Canada, there are sharp boundaries between mixed red cedar (Thuja plicata)-hemlock (Tsuga heterophylla) (CH) stands, and stands of hemlock and amabilis fir (Abies amabilis) (HA). We tested differences in AM colonization of red cedar between ectomycorrhiza-dominated (HA) stands and stands containing red cedar (CH), across a range of light levels. We used a soil bioassay approach to determine whether there was sufficient AM fungal inoculum in the HA tree stands to colonize red cedar seedlings. Seeds of hemlock and red cedar were sown in forest floor samples collected from the two types of forests, and shade treatments ranging from < 1 to 53% of full sunlight were imposed. After 6 months, seedling survival and root and shoot biomass were quantified, and red cedar seedlings were sampled for AM fungal colonization. Hemlock survival and growth did not differ between soil types, suggesting there was no substrate-associated limitation to its establishment in either forest type. Red cedar colonization by AM fungi was significantly correlated with light levels in CH soils but arbuscular mycorrhizas were absent in roots of red cedar seedlings grown in HA soil. Red cedar survival and relative growth rate were significantly greater in the CH than in HA soil; higher growth was due primarily to greater shoot growth in CH soils at high light levels. The low soil inoculum potential for red cedar in ectomycorrhiza-dominated stands may account for the virtual exclusion of red cedar seedlings from these forests.

Abies↗

Ni(II) removal from aqueous solutions using cone biomass of Thuja orientalis.

The biomass of terrestrial-plant materials has high removal capacities for a number of heavy metal ions. The Ni(II) biosorption capacity of the cone biomass of Thuja orientalis was studied in the batch mode. The biosorption equilibrium level was determined as a function of contact time, pH, temperature, agitation speed at several initial metal ion and adsorbent concentrations. The removal of Ni(II) from aqueous solutions increased with adsorbent concentration, temperature and agitation speed of the solution were increased. The biosorption process was very fast; 90% of biosorption occurred within 3 min and equilibrium was reached at around 7 min. It is found that the biosorption of Ni(II) on the cone biomass was correlated well (R2 > 0.99) with the Langmuir equation as compared to Freundlich, BET Temkin and D-R isotherm equation under the concentration range studied. According to Langmuir isotherm, the monolayer saturation capacity (Q(o)) is 12.42 mg g(-1). The pseudo-first-order, pseudo-second-order and intraparticle diffusion kinetic models were applied to test the experimental data for initial Ni(II) and cone biomass concentrations. The pseudo-second-order kinetic model provided the best correlation of the used experimental data compared to the pseudo-first-order and intraparticle diffusion kinetic models. The activation energy of biosorption (E(a)) was determined as 36.85 kJ mol(-1) using the Arrhenius equation. This study indicated that the cone biomass of T. orientalis can be used as an effective and environmentally friendly adsorbent for the treatment of Ni(II) containing aqueous solutions.

Adsorption↗

A randomized, double-blind, placebo-controlled, clinical dose-response trial of an extract of Baptisia, Echinacea and Thuja for the treatment of patients with common cold.

The aim of this study was to verify the efficacy and safety of an herbal medication containing an extract of a mixture of Baptisiae tinctoriae radix, Echinaceae pallidae/purpureae radix and Thujae, occidentalis herba (SB-TOX) in the treatment of upper respiratory tract infections (URIs), and to test whether SB-TOX's clinical efficacy is dose dependent. A total of 91 adults (mean age 42.1 +/- 13.0 years) were randomised to receive 19.2 mg of SB-TOX (n=31), 9.6 mg SB-TOX (n=29) or placebo (n=31) three times daily for 3-12 days. Since a "running nose" is the main symptom of a common cold, the total number of facial tissues used throughout the clinical duration of their cold was the primary efficacy parameter. In the intention-to-treat analysis, this total number of tissues decreased with increasing extract dose. The slope across groups according to the Jonckheere test was significant (p = 0.0259). In the high-dose group, the standardised effect size delta/SD was 0.46 compared with placebo. Time to relevant improvement in cold symptoms (measured as the time until less than 30 tissues per day were used) was 1.1 days (95% CI 0.52; 1.67), 0.76 days (95% CI 0.28; 1.24) and 0.52 days (95% CI 0.22; 0.82) in the placebo, low-dose and high-dose groups, respectively (p(LogRank) = 0.0175). No adverse events were reported. This study demonstrates the efficacy and safety of SB-TOX in the treatment of URIs, and that its efficacy is dose dependent.

Adult↗

The extract of Thujae occidentalis semen inhibited 5alpha-reductase and androchronogenetic alopecia of B6CBAF1/j hybrid mouse.

BACKGROUND: The conversion of testosterone to dihydrotestosterone; 5alpha-androstan-17beta-ol-3-one by 5alpha-reductase plays a crucial role in hair baldness and prostatomegaly. Recent approach showed specific inhibitors for 5alpha-reductase type 2 such as finasteride promoted hair growth in male pattern alopecia. OBJECTIVE: In order to search for effective medicinal plant extracts applied topically for androgenetic alopecia, we screened natural plant extracts having inhibitory activities of 5alpha-reductase type 2 and demonstrated its biological function in androgen-related animal models. METHODS: We evaluated the inhibition activities of numerous plant extracts by contact cell based metabolic method using a stable HEK 293 cell line expressing human 5alpha-reductase (type 2). To elucidate the biological activity in vivo, the Thujae occidentalis semen (TOS) extract was topically applied to fuzzy rat and androchronogenetic alopecia (AGA) mouse, respectively. The secreted sebum and the size of sebaceous glands of fuzzy rat were measured after 6 weeks. Also, after the topical treatment with TOS extract and androgen receptor antagonist (cyproterone acetate) simultaneously with subcutaneous injection of testosterone (1 mg/mice/day), hair loss patterns of female B6CBAF1/j hybrid mouse were observed. RESULTS: TOS extract showed higher inhibition activity of 5alpha-reductase type 2(IC(50) value=2.6 microg/ml) than that of gamma-linolenic acid, but lower than that of finasteride. When applied to fuzzy rat, the amount of sebum and sebaceous gland size decreased remarkably. In AGA model, alopecia degrees of two groups, treated with TOS extract (P<0.015) or cyproterone acetate (P<0.01), were lower than that of vehicle (propylene glycol:ethanol=7:3) and there was no difference between above two groups. CONCLUSION: We have demonstrated the inhibitory activity of TOS extract for 5alpha-reductase type 2 and its biological action in two animal models, suggesting that TOS extract would be used as an effective agent for male pattern baldness by modifying androgen conversion.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

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.

Amino Acid Sequence↗

Aromatase inhibitory activities of standishinal and the diterpenoids from the bark of Thuja standishii.

The absolute stereostructure of a novel skeletal diterpene, standishinal (1), from the bark of Thuja standishii was confirmed by X-ray crystallographic analyses of 1 and its p-bromobenzoate derivative. Aromatase inhibitory activities of standishinal, eight known diterpenes, totarol, 12-methoxyabieta-8,11,13-trien-11-ol, 12-hydroxy-6,7-seco-abieta-8,11,13-triene-6,7-dial, trans-communic acid, labda-8(17),13-dien-12 R,15-olid-19-oic acid, 12 S-hydroxylabda-8(17),13(16),14-trien-19-oic acid, 13-oxo-15,16-dinorlabda-8(17),11 E-dien-19-oic acid and 14-oxo-15-norlabda-8 (17),12 E-dien-19-oic acid from the plant, and four synthetic analogs were evaluated using a recombinant human aromatase. Among them, standishinal and its diacetate derivative had significant inhibitory activities.

Abietanes↗