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Phytoestrogen from Pinus ponderosa assayed by competitive binding with 17beta-estradiol to mouse uterine cytosol.

Ponderosa pine needle extracts contained a phytoestrogen that competed with 17beta-estradiol for specific binding to mouse uterine cytosol. Phytoestrogen was separated from the initial aqueous extract by acetone fractionation and eluted from a polyvinylpyrrolidone column with 90% methanol. Forty microg/ml was determined as the concentration of most purified phytoestrogen displacing 50% of 17beta-estradiol (15 pg/ml) from the estrogen specific binding sites of the mouse uterine cytosol. Intraperitoneal administration of 200 microg (0.3 g needle equivalents) of the phytoestrogen was equal in activity to 10 ng of 17beta-estradiol in a 24 hour uterine growth assay in immature mice. The phytoestrogen was retained in dialysis tubing with a 14,000 molecular weight cutoff and displayed an ultraviolet absorbance maximum at 208 nm, void of any phenolic chromophores. Phytoestrogen was being evaluated as a contributory factor in Ponderosa pine needle-induced abortion observed in range cattle.

Journal Article↗

Evidence for trans-cuticular uptake of HNO3 vapor by foliage of Eastern white pine (Pinus strobus L.).

Forest declines in Europe and the northeastern United States are widely believed to be associated with deposition of air pollutants, perhaps including nitric acid vapor. The experiments presented here, which were conducted in indoor chambers, involved measurement of steady-state rates of nitric acid deposition on foliage of seedlings of eastern white pine. Nitric acid concentrations ranged from 37 to 1260 ppb, but were mostly in the 130-180 ppb range. Between 130 and 180 ppb, much of the deposited nitric acid could be removed by washing leaf surfaces with water. Amounts of nitrate removed by washing increased with exposure duration to a maximum of 7.6 nmol cm(-2) after about 8 h. Although nitrate concentrations in the washings remained constant after 8 h, nitric acid deposition nonetheless continued at a steady-state rate. We have termed this steady-state deposition 'trans-cuticular' on the assumption that the nitric acid diffuses through the cuticle. Resistance to trans-cuticular uptake (69 m(2) s mol(-1)) far exceeded resistance to diffusion across the unstirred layer at the surface of the leaf (5 m(2) s mol(-1)). High concentrations used in these experiments preclude quantitative extrapolation to ambient conditions; however, we have demonstrated that the white pine cuticle is both a major storage pool and a major barrier to uptake of deposited nitric acid.

Journal Article↗

Fatty acids and ultrastructure of ozone-exposed Aleppo pine (Pinus halepensis Mill.) needles.

Fifteen or 18-month-old Aleppo pine seedlings were fumigated with different concentrations and doses of ozone over a period of 2-16 days in controlled-environmental growth chambers. The total fatty acid content and ultrastructure of the current year needles were subsequently analysed. In acute, high concentration exposures, significant reductions in the levels of linolenic acid were detected. Increases in myristic or palmitic acid were common in needles exposed to lower concentrations of ozone. Ultrastructural studies revealed reductions in chloroplast size and a darkening of stroma at low ozone exposures while at high concentrations disruption of the chloroplast membranes was also identified.

Journal Article↗

Effects of simulated acid rain and ozone on foliar chemistry of field-grown Pinus ponderosa seedlings and mature trees.

We investigated the additive and interactive effects of simulated acid rain and elevated ozone on C and N contents, and the C:N ratio of one-year-old and current-year foliage of field-grown mature trees and their half-sib seedlings of a stress tolerant genotype of ponderosa pine. Acid rain levels (pH 5.1 and 3.0) were applied weekly to foliage only (no soil acidification or N addition), from January to April, 1992. Plants were exposed to two ozone levels (ambient and twice-ambient) during the day from September 1991 to November 1992. The sequential application of acid rain and elevated ozone mimicked the natural conditions. Twice-ambient ozone significantly decreased foliar N content (by 12-14%) and increased the C:N ratio of both one-year-old and current-year foliage of seedlings. Although similar ozone effects were also observed on one-year-old foliage of mature trees, the only statistically significant effect was an increased C:N ratio when twice-ambient ozone combined with pH 3.0 rain (acid rain by ozone interaction). Enhancing the effect of twice-ambient ozone in increasing the C:N ratio of one-year-old foliage of mature trees in June was the only significant effect of acid rain.

Journal Article↗

The effect of simulated acid rain on the biochemical composition of Scots pine (Pinus sylvestris L.) needles.

The effects of prolonged simulated acid rain on the biochemistry of Scots pine needles were studied in Finnish Lapland. Pine trees were exposed by spraying the foliage and soil with either clean water or simulated acid rain (SAR; both sulphuric and nitric acids) over the period 1985-1991. The concentrations of carbohydrates (starch, glucose, fructose, sucrose) in one-year-old pine needles were not affected by SAR-treatments. The SAR-treatments did not have significant effects on protein bound amino acids, which was true also for most of the free amino acids. However, the citrulline concentration was over three-fold greater in the foliage of pines exposed to SAR of pH 3 compared to irrigated controls. The concentrations of total phenolics, individual low molecular weight phenolics and soluble proanthocyanidins were not affected by the treatments, but insoluble proanthocyanidins had increased in acid-treated trees. Some of the studied biochemical compounds showed significant differences between two sub-areas (similar treatments) only 120 m apart.

Journal Article↗

Ozone exposure-response relationships for biomass and root/shoot ratio of beech (Fagus sylvatica), ash (Fraxinus excelsior), Norway spruce (Picea abies) and Scots pine (Pinus sylvestris).

Current-year seedlings of beech, ash, Norway spruce and Scots pine were exposed during one growing season to different, but moderate, ozone (O(3)) scenarios representative for Switzerland (50, 85, 100% ambient, 50% ambient+30 nl l(-1)) in open-top chambers (OTCs) and to ambient O(3) concentrations in the field. Biomass significantly decreased with increasing O(3) dose in all species except for spruce. Losses of 25.5% (ash), 17.4% (beech), 9.9% (Scots pine) were found per 10 microl l(-1) h accumulated O(3) exposure over a threshold concentration of 40 nl l(-1) during daylight hours (AOT40). Ratios of root/shoot biomass (RSR) also significantly decreased with increasing AOT40 levels in beech and ash, but not in Norway spruce and Scots pine. The data show that the deciduous species beech and ash were more susceptible to O(3) with respect to RSR and biomass than the coniferous species Norway spruce and Scots pine.

Journal Article↗

The effects of multiple metal contamination on ectomycorrhizal Scots pine (Pinus sylvestris) seedlings.

Experiments were conducted to investigate the effects of single and multiple metal contamination (Cd, Pb, Zn, Sb, Cu) on Scots pine seedlings colonised by ectomycorrhizal (ECM) fungi from natural soil inoculum. Seedlings were grown in either contaminated field soil from the site of a chemical accident, soils amended with five metals contaminating the site, or in soil from an uncontaminated control site. Although contaminated and metal-amended soil significantly inhibited root and shoot growth of the Scots pine seedlings, total root tip density was not affected. Of the five metals tested in amended soils, Cd was the most toxic to ECM Scots pine. Field-contaminated soil had a toxic effect on ECM fungi associated with Scots pine seedlings and caused shifts in ECM species composition on ECM seedlings. When compared to soils amended with only one metal, soils amended with a combination of all five metals tested had lower relative toxicity and less accumulation of Pb, Zn and Sb into seedlings. This would indicate that the toxicity of multiple metal contamination cannot be predicted from the individual toxicity of the metals investigated.

Journal Article↗

In vitro inhibition of the activity of phosphorylase kinase, protein kinase C and protein kinase A by caffeic acid and a procyanidin-rich pine bark (Pinus marittima) extract.

Caffeic acid (CA) is a common constituent of human diet while pine bark extract (PBE) is utilized either as nutritional supplement or as phytochemical remedy for different diseases. CA and PBE, are reported as efficient antioxidants and more recently have been described to modulate cellular response to oxidative challenge and to possess many other biological activities, i.e. anti-inflammatory, antimutagenic, antitumoral effects. In order to investigate in depth the mechanism of action of these polyphenols, the effects of CA and PBE on the activity of some protein kinases involved in the regulation of fundamental cellular processes were studied in vitro: phosphorylase kinase (PhK), protein kinase A (PKA), protein kinase C (PKC). PBE at the concentration of 20 microg/ml (corresponding to 69 microM catechin equivalents) inhibited PKA, PhK and PKC by about 90, 59, 57%, respectively, while 100 microM CA inhibited by 37, 52 and 54%, respectively. Considerable inhibitions have been still observed at even lower concentrations of CA and PBE. For PhK and PKA, the inhibition follows a non-competitive mechanism. CA also inhibits PKC activity in a partially purified cellular extract. The results suggest a possible involvement of CA and PBE in modulation of cellular functions.

Caffeic Acids↗

Electron spin resonance study of free radicals formed from a procyanidin-rich pine (Pinus maritima) bark extract, pycnogenol.

The free radical generated from the oxidation of a French maritima pine bark extract Pycnogenol (PYC), by the horseradish peroxidase (HRP)-hydrogen peroxide (H2O2) system at pH 7.4-10.0 was studied using electron spin resonance (ESR) spectrometer. The formation rate of the PYC radical (aH = 0.92 G; g = 2.0055) was dependent on the PYC and HRP concentrations and pH; the lifetime of the radical was up to 90 min. Furthermore, it was found that the PYC radical was mainly composed of the secondary radical formed from procyanidin B3, one of major procyanidins in PYC. The primary radical signal of procyanidin B3 with hyperfine splitting constants aH = 3.67 G (1H), aH = 0.92 G (3H), and g = 2.0055 was transient and disappeared quickly, whereas its secondary radical signal appeared and increased with time. The secondary radical from dimer procyanidin B3 showed quite high stability, differing from the radical from monomer (+)-catechin that could not be observed possibly because of its instability. These results provide evidence to support the idea that the intramolecular hydrogen bond between the O* at the 4' position in one B ring and an OH group in the other B ring of procyanidin B3 is formed during its oxidation in the presence of HRP and H2O2.

Antioxidants↗

Supercritical fluid extraction of polycyclic aromatic hydrocarbons from white pine (Pinus strobus) needles and its implications.

A supercritical fluid extraction (SFE) method was developed for the extraction of polycyclic aromatic hydrocarbons (PAHs) from fresh and fallen pine needles. Toluene-modified CO2 was used as the extracting fluid, and the extracted PAHs were analyzed by GC-MS. Using a two-stage extraction procedure, a static extraction at 180 degrees C and a dynamic extraction at 60 degrees C, and an in-cell silica gel plug plus a post-oven silica gel column, the extraction and fractionation of PAHs can be accomplished in one step. Over a seven month period, a significant variation was observed for PAHs in urban samples, while PAHs in mountain samples were at much lower levels (by a factor of approximately 8) and showed little seasonal change. Although dry fallen needles and fresh needles contained similar amounts of PAHs, in the fallen needles the lower molecular weight PAHs were partially lost while the higher molecular weight PAHs were slightly enriched. Pollution in urban areas was found to be highly localized, and buildings and trees are believed to be important factors in the restriction of atmospheric PAHs.

Air Pollution↗