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Rapid in situ detection of alkaloids in plant tissue under ambient conditions using desorption electrospray ionization.

Desorption electrospray ionization (DESI) mass spectrometry is applied to the in situ detection of alkaloids in the tissue of poison hemlock (Conium maculatum), jimsonweed (Datura stramonium) and deadly nightshade (Atropa belladonna). The experiment is carried out by electrospraying micro-droplets of solvent onto native or freshly-cut plant tissue surfaces. No sample preparation is required and the mass spectra are recorded under ambient conditions, in times of a few seconds. The impact of the sprayed droplets on the surface produces gaseous ions from organic compounds originally present in the plant tissue. The effects of operating parameters, including the electrospray high voltage, heated capillary temperature, the solvent infusion rate and the carrier gas pressure on analytical performance are evaluated and optimized. Different types of plant material are analyzed including seeds, stems, leaves, roots and flowers. All the previously reported alkaloids have been detected in C. maculatum, while fifteen out of nineteen known alkaloids for D. stramonium and the principal alkaloids of A. belladonna were also identified. All identifications were confirmed by tandem mass spectrometry. Results obtained show similar mass spectra, number of alkaloids, and signal intensities to those obtained when extraction and separation processes are performed prior to mass spectrometric analysis. Evidence is provided that DESI ionization occurs by both a gas-phase ionization process and by a droplet pick-up mechanism. Quantitative precision of DESI is compared with conventional electrospray ionization mass spectrometry (after sample workup) and the RSD values for the same set of 25 dicotyledonous C. maculatum seeds (one half of each seed analyzed by ESI and the other by DESI) are 9.8% and 5.2%, respectively.

Alkaloids↗

Mitochondrial DNA sequence variation among populations and host races of Lambdina fiscellaria (Gn.) (Lepidoptera: Geometridae).

The hemlock looper, Lambdina fiscellaria (Gn.), is a recurring major forest pest that is widely distributed in North America. Three subspecies (L. f. fiscellaria, L. f. lugubrosa (Hulst) and L. f. somniaria (Hulst)) have been recognized based on larval host or adult pheromone differences, but no consistent morphological differences have been reported. To clarify their taxonomic status, we surveyed mitochondrial DNA (mtDNA) sequence and restriction site variation in two protein coding genes, cytochrome oxidase I and II (COI and COII), in populations across the range of L. fiscellaria. In addition to variation in COI and COII, we found an intergenic spacer region of 20-23 bp located between the tRNA tyrosine gene and the start of COI. Of the 141 specimens of L. fiscellaria assayed, 137 were grouped into two distinct mtDNA lineages, one of which was disproportionately associated with eastern populations and one with western populations. However, single specimens and two populations in eastern Canada had mtDNA resembling that of western populations. Three divergent and rare haplotypes had basal affinities to the two common lineages. The two major lineages of L. fiscellaria were diverged by approximately 2% from each other, as well as from the mtDNA of two outgroup species, L. athasaria (Walker) and L. pellucidaria(G. & R.). The two outgroup species had essentially the same mtDNA and may be conspecific. We interpret the pattern of mtDNA variation within L. fiscellaria as indicating genetic polymorphism within a single species without clear subspecific divisions, rather than evidence of multiple cryptic species.

Animals↗

Phenylethylamine and piperidine alkaloids in aloe species.

Of the approximately 300 species of ALOE (Liliaceae) native to Africa and Arabia, leaf extracts of 224 species have been examined chromatographically for alkaloids using ninhydrin, Dragendorff's reagent, nitroprusside, and iodoplatinate as revealing agents. From these, 48 (21%) species contained compounds giving a strong colorimetric reaction with at least one of these reagents. Tyramine derivatives were identified in 18 species and piperidine derivatives in a further 6 species. The other coloured zones remain unidentified but often appeared to represent common amino acids. Some taxonomic correlations are suggested. The presence of the toxic hemlock alkaloids in aloes readily available for potential medicinal use by local inhabitants sounds a note of caution against the unthinking use of these otherwise useful plants.

Journal Article↗

Seasonal biochemical changes in coniferous forest canopies and their response to fertilization.

Seasonal changes in concentrations of total nitrogen, free amino acids, chlorophyll, starch and sugar were measured in foliage from fertilized and unfertilized conifer forests in New Mexico and Oregon. In the New Mexico Douglas-fir (Pseudotsuga menziesii var glauca (Beissn.) Franco) forest, fertilization resulted in elevated foliar nitrogen concentrations on all dates, from an average of 9 mg g(-1) in unfertilized trees to 14 mg g(-1) in fertilized trees. In the Oregon western hemlock (Tsuga heterophylla (Raf.) Sarg.) forest, fertilization increased total N by only 15%, from 13 mg g(-1) in unfertilized trees to 15 mg g(-1) in fertilized trees. Foliar nitrogen concentrations on a weight basis were lowest in winter and spring, but did not vary seasonally when expressed on a leaf area basis. Chlorophyll concentrations increased with fertilization and had greater seasonal variation than did total nitrogen concentrations. Chlorophyll concentrations were significantly higher during the growing season than in the winter and spring months. Fertilization did not result in major changes in the proportion of total nitrogen in chlorophyll at either the Oregon or the New Mexico site. Concentrations of free amino acids varied with date and fertilization treatment; in New Mexico, amino acids were highest in the winter sample, whereas in Oregon, they were lowest in winter and spring. At both sites, amino acid concentrations were significantly higher in fertilized trees than in control trees on most dates and the ratios of amino acid-N to total N were also significantly higher in fertilized trees. For both sites, starch concentrations were nearly zero for most of the year, but increased sharply just before bud break and initiation of new growth in the spring. Although fertilization resulted in increased nitrogen concentrations in foliage at both sites, the response in New Mexico was much greater than in Oregon. These results are in agreement with forest productivity data that suggest that growth in the New Mexico site is limited by nitrogen, whereas in the Oregon site it is not.

Journal Article↗

Carbon and oxygen isotope ratios of ecosystem respiration along an Oregon conifer transect: preliminary observations based on small-flask sampling.

Isotope ratio analyses of atmospheric CO(2) at natural abundance have significant potential for contributing to our understanding of photosynthetic and respiration processes in forest ecosystems. Recent advances in isotope ratio mass spectrometry allow for rapid, on-line analysis of small volumes of CO(2) in air, and open new research opportunities at the ecophysiological, whole-organism, and atmospheric levels. Among the immediate applications are the carbon and oxygen isotope ratio analyses of carbon dioxide in atmospheric air. Routine analysis of carbon dioxide in air volumes of approximately 50-300 &mgr;l is accomplished by linking a commercially available, trace gas condenser and gas chromatograph to an isotope ratio mass spectrometer operated in continuous-flow mode. Samples collected in the field are stored in either gas-tight syringes or 100-ml flasks. The small sample volume required makes it possible to subsample the air in flasks for CO(2) and then to sample the remaining air volume for the analysis of the isotopic composition of either methane or nitrous oxide. Reliable delta(13)C and delta(18)O values can be obtained from samples collected and stored for 1-3 days. Longer-term storage, on the order of weeks, is possible for delta(13)C measurements without drift in the isotope ratio signal, and should also be possible for delta(18)O measurements. When linked with an infrared gas analyzer, pump and flask sampling system, it is feasible to sample CO(2) extensively in remote forest locations. The air-sampling system was used to measure the isotope ratios of atmospheric CO(2) and to conduct a regression analysis of the relationship between these two parameters. From the regression, we calculated the delta(13)C of ecosystem respiration of four coniferous ecosystems along a precipitation gradient in central Oregon. The ecosystems along the coast-to-interior Oregon (OTTER) gradient are dominated by spruce-hemlock forests at the wet, coastal sites (> 200 cm precipitation annually) to juniper woodlands (20 cm precipitation) at the interior, dry end of the transect. The delta(13)C values of ecosystem respiration along this transect differed by only 1.3 per thousand (range of -25.2 to -23.9 per thousand ) during August at the peak of the summer drought. Following autumn rains in September, the delta(13)C of ecosystem respiration in the four stands decreased; overall the difference in the carbon isotope ratio of ecosystem respiration among sites increased to 3.9 per thousand (-26.8 to -22.9 per thousand ).

Journal Article↗

Stomatal behavior of four woody species in relation to leaf-specific hydraulic conductance and threshold water potential.

Midday stomatal closure is mediated by the availability of water in the soil, leaf and atmosphere, but the response to these environmental and internal variables is highly species specific. We tested the hypothesis that species differences in stomatal response to humidity and soil water availability can be explained by two parameters: leaf-specific hydraulic conductance (K(L)) and a threshold leaf water potential (Psi(threshold)). We used a combination of original and published data to estimate characteristic values of K(L) and Psi(threshold) for four common tree species that have distinctly different stomatal behaviors: black cottonwood (Populus trichocarpa Torr. & Gray.), Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), red alder (Alnus rubra Bong.) and western hemlock (Tsuga heterophylla (Raf.) Sarg.). We used the values to parameterize a simple, nonelastic model that predicts stomatal conductance by linking hydraulic flux to transpirational flux and maintaining Psi(leaf) above Psi(threshold). The model successfully predicted fundamental features of stomatal behavior that have been reported in the literature for these species. We conclude that much of the variation among the species in stomatal response to soil and atmospheric water deficits can be explained by K(L) and Psi(threshold). The relationship between Psi(threshold) and xylem vulnerability to cavitation differed among these species.

Journal Article↗

Winter water relations of New England conifers and factors influencing their upper elevational limits. II. Modeling.

We used the water relations model, WINWAT, to model winter water relations of three conifer species-eastern hemlock (Tsuga canadensis (L.) Carr.), eastern white pine (Pinus strobus L.) and red pine (P. resinosa Ait.)-growing at their upper elevational limits on Mt. Ascutney, Vermont, USA, in the winters of 1997 and 1999. Modeled relative water contents remained above 60% in the two youngest foliar age classes of all three species during both winters, indicating that desiccation stress in winter is not responsible for setting the upper elevational limits of these species at this site under present climatic conditions. WINWAT indicated that winter water relations of these low-elevational species were sensitive to low relative humidity, which increased transpiration rates, and low temperatures, which inhibited recharge, but are much less sensitive to summer climate than in the case of subalpine conifers in Colorado. Our results indicate that summer and winter temperatures and relative humidities (or precipitation/potential evapotranspiration ratios) should be incorporated into climate change models designed to simulate future tree distributions.

Altitude↗

Biophysical controls of carbon flows in three successional Douglas-fir stands based on eddy-covariance measurements.

We measured net carbon flux (F(CO2)) and net H2O flux (F(H2O)) by the eddy-covariance method at three Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco)-western hemlock (Tsuga heterophylla (Raf.) Sarg.) sites located in the Wind River Valley of southern Washington State, USA. Stands were approximately 20, 40 and 450 years old and measurements were made between June 15 and October 15 of 1998 in the 40- and 450-year-old stands, and of 1999 in the 20- and 450-year-old stands. Our objectives were to determine if there were differences among the stands in: (1) patterns of daytime F(CO2) during summer and early autumn; (2) empirically modeled relationships between local climatic factors (e.g., light, vapor pressure deficit (VPD), soil water content, temperature and net radiation) and daytime F(CO2); and (3) water-use efficiency (WUE). We used the Landsberg equation, a logarithmic power function and linear regression to model relationships between F(CO2) and physical variables. Overall, given the same irradiance, F(CO2) was 1.0-3.9 mol m-2 s-1 higher (P < 0.0001 for both seasons) at the two young stands than at the old-growth stand. During summer and early autumn, F(CO2) averaged 4.2 and 6.1 mol m-2 s-1 at the 20- and 40-year-old stand, respectively. In contrast, the 450-year-old forest averaged 2.2 and 3.2 mol m-2 s-1 in 1998 and 1999, respectively. Increases in VPD were associated with reduced F(CO2) at all three stands, with the greatest apparent constraints occurring at the old-growth stand. Correlations between F(CO2) and all other environmental variables differed among ecosystems, with soil temperature showing a negative correlation and net radiation showing a positive correlation. In the old-growth stand, WUE was significantly greater (P < 0.0001) in the drier summer of 1998 (2.7 mg g-1) than in 1999 (1.0 mg g-1). Although we did not use replicates in our study, the results indicate that there are large differences in F(CO2) among Douglas-fir stands of different ages growing in the same general area, and that variations in age structure and site conditions need to be considered when scaling flux measurements from individual points to the landscape level.

Carbon Dioxide↗

Structural and compositional controls on transpiration in 40- and 450-year-old riparian forests in western Oregon, USA.

Large areas of forests in the Pacific Northwest are being transformed to younger forests, yet little is known about the impact this may have on hydrological cycles. Previous work suggests that old trees use less water per unit leaf area or sapwood area than young mature trees of the same species in similar environments. Do old forests, therefore, use less water than young mature forests in similar environments, or are there other structural or compositional components in the forests that compensate for tree-level differences? We investigated the impacts of tree age, species composition and sapwood basal area on stand-level transpiration in adjacent watersheds at the H.J. Andrews Forest in the western Cascades of Oregon, one containing a young, mature (about 40 years since disturbance) conifer forest and the other an old growth (about 450 years since disturbance) forest. Sap flow measurements were used to evaluate the degree to which differences in age and species composition affect water use. Stand sapwood basal area was evaluated based on a vegetation survey for species, basal area and sapwood basal area in the riparian area of two watersheds. A simple scaling exercise derived from estimated differences in water use as a result of differences in age, species composition and stand sapwood area was used to estimate transpiration from late June through October within the entire riparian area of these watersheds. Transpiration was higher in the young stand because of greater sap flux density (sap flow per unit sapwood area) by age class and species, and greater total stand sapwood area. During the measurement period, mean daily sap flux density was 2.30 times higher in young compared with old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) trees. Sap flux density was 1.41 times higher in young red alder (Alnus rubra Bong.) compared with young P. menziesii trees, and was 1.45 times higher in old P. menziesii compared with old western hemlock (Tsuga heterophylla (Raf.) Sarg.) trees. Overall, sapwood basal area was 21% higher in the young stand than in the old stand. In the old forest, T. heterophylla is an important co-dominant, accounting for 58% of total sapwood basal area, whereas P. menziesii is the only dominant conifer in the young stand. Angiosperms accounted for 36% of total sapwood basal area in the young stand, but only 7% in the old stand. For all factors combined, we estimated 3.27 times more water use by vegetation in the riparian area of the young stand over the measurement period. Tree age had the greatest effect on stand differences in water use, followed by differences in sapwood basal area, and finally species composition. The large differences in transpiration provide further evidence that forest management alters site water balance via elevated transpiration in vigorous young stands.

Alnus↗

Element accumulation patterns of deciduous and evergreen tree seedlings on acid soils: implications for sensitivity to manganese toxicity.

Foliar nutrient imbalances, including the hyperaccumulation of manganese (Mn), are correlated with symptoms of declining health in sensitive tree species growing on acidic forest soils. The objectives of this study were to: (1) compare foliar nutrient accumulation patterns of six deciduous (sugar maple (Acer saccharum Marsh.), red maple (Acer rubrum L.), red oak (Quercus rubra L.), white oak (Quercus alba L.), black cherry (Prunus serotina Ehrh.) and white ash (Fraxinus americana L.)) and three evergreen (eastern hemlock (Tsuga canadensis L.), white pine (Pinus strobus L.) and white spruce (Picea glauca (Moench) Voss.)) tree species growing on acidic forest soils; and (2) examine how leaf phenology and other traits that distinguish evergreen and deciduous tree species influence foliar Mn accumulation rates and sensitivity to excess Mn. For the first objective, leaf samples of seedlings from five acidic, non-glaciated field sites on Pennsylvania's Allegheny Plateau were collected and analyzed for leaf element concentrations. In a second study, we examined growth and photosynthetic responses of seedlings exposed to excess Mn in sand culture. In field samples, Mn in deciduous foliage hyperaccumulated to concentrations more than twice as high as those found in evergreen needles. Among species, sugar maple was the most sensitive to excess Mn based on growth and photosynthetic measurements. Photosynthesis in red maple and red oak was also sensitive to excess Mn, whereas white oak, black cherry, white ash and the three evergreen species were tolerant of excess Mn. Among the nine species, relative rates of photosynthesis were negatively correlated with foliar Mn concentrations, suggesting that photosynthetic sensitivity to Mn is a function of its rate of accumulation in seedling foliage.

Acer↗

Asphyxial suicides using plastic bags.

Fifty-three suicides using plastic bags were identified in a review of cases within the jurisdiction of the King County Medical Examiner's Office, Seattle, Washington from 1984 to 1993. We found that this method was used at a greater frequency by individuals older than 50 in comparison with other methods. The most commonly identified stressor leading to the suicide in this population was failing health. The use of this method as a means of "self deliverance," as advocated by the Hemlock Society, could be inferred in only a small minority of cases where terminal illnesses were identified. This method may be preferred by those older than 50 years because of the ready availability of plastic bags and the relative nonviolence of the death. Analysis of the autopsy findings showed no specific features for this method of suicide. In particular, petechiae, which are often considered a marker of asphyxia, were present in only a small minority of cases (3%). Furthermore, the scene investigation rarely revealed specific features, other than the plastic bag in place. Thus, if the plastic bag were removed after death, the cause and manner of death would be obscure.

Adolescent↗

Freezing of xylem sap without cavitation.

Freezing of stem sections and entire twigs of hemlock (Tsuga canadensis) has been demonstrated to occur without increasing the resistance to the movement of water through the frozen part after rewarming. This was interpreted to mean that freezing did not produce cavitation in the xylem sap even though A) the sap was unquestionably frozen; B) it contained dissolved gases; and C) it was under tension before freezing and after. Freezing stem sections of some other evergreen gymnosperms during the summer again produced no evidence for cavitation of the xylem sap. On the other hand, freezing stem sections of some angiosperms invariably increased the resistance to sap flow leading to wilting and death in a few hours when the sap tension was at normal daytime values at the time of freezing. These results were interpreted to mean that the bordered pits on the tracheids of gymnosperms function to isolate the freezing sap in each tracheid so that the expansion of water upon freezing not only eliminates any existing tension but also develops positive pressure in the sap. Dissolved gases frozen out of solution may then be redissolved under this positive pressure as melting occurs. As the bubbles are reduced in size by this ice pressure developed in an isolated tracheid, further pressure is applied by the surface tension of the water against air. If the bubbles are redissolved or are reduced to sufficient small size by the time the tension returns to the sap as the last ice crystals melt, then the internal pressure from surface tension in any existing small bubbles may exceed the hydrostatic tension of the melted sap and the bubbles cannot expand and will continue to dissolve.

Journal Article↗

Effects of Pleistocene glaciations on population structure of North American chestnut-backed chickadees.

The postglacial recolonization of northern North America was heavily influenced by the Pleistocene glaciation. In the Pacific Northwest, there are two disjunct regions of mesic temperate forest, one coastal and the other interior. The chestnut-backed chickadee is one of the species associated with this distinctive ecosystem. Using seven microsatellite markers we found evidence of population structure among nine populations of chestnut-backed chickadees. High levels of allelic variation were found in each of the populations. Northern British Columbia and central Alaska populations contained a large number of private alleles compared to other populations, including those from unglaciated regions. The disjunct population in the interior was genetically distinct from the coastal population. Genetic and historical records indicate that the interior population originated from postglacial inland dispersal. Population structuring was found within the continuous coastal population, among which the peripheral populations, specifically those on the Queen Charlotte Islands and the central Alaska mainland, were genetically distinct. The pattern of population structure among contemporary chickadee populations is consistent with a pioneer model of recolonization. The persistence of genetic structure in western North American chestnut-backed chickadees may be aided by their sedentary behaviour, linear distribution, and dependence on cedar-hemlock forests.

Alleles↗

A streptomycete antagonist to Phellinus weirii, Fomes annosus, and Phytophthora cinnamomi.

An actinomycete isolated from the rhizoplane of nitrogen-fixing nodules of Ceanothus velutinus was identified as a variety of Streptomyces griseoloalbus. Streptomyces griseoloalbus is a strong antagonist to three destructive root pathogens, Phellinus weirii, Fomes annosus, and Phytophthora cinnamomi, inhibiting all three on several culture media and preventing establishment of F. annosus on hemlock wood disks. The stability and longevity of the antimicrobial substance produced by it, its consistent effect on the pathogens on all substrates, its ability to colonize wood, and its ability to grow at 10 degrees C suggest biological control possibilities for this organism in the Pacific Northwest.

Anti-Bacterial Agents↗

Dr John Andree, MD (Rheims) LRCP, founding physician of the London Hospital.

The contributions of the Huguenot physician John Andree to the founding of the London Hospital in 1740, and to the development of the new voluntary hospital in its early years, are assessed in the context of his own special interests. These included his promotion of the merits of Tilbury water, his observations on epilepsy and his criticism of the use of hemlock for the treatment of cancer.

England↗

Euthanasia and physician assisted suicide; a social work update.

Considering the recent actions of Dr. Jack Kevorkian, Dr. Timothy Quill and the Hemlock Society's initiatives for legalizing physician assisted suicide for special cases in the state of Washington, Oregon, and California, arguments for and against euthanasia and physician assisted suicide have grown into a substantial and impassioned debate. This paper outlines and analyzes salient issues within the controversy of legalizing physician assisted suicide for terminally ill patients. It highlights significant issues for state and federal policy formation and further suggests unique roles which medical social workers might play in developing and implementing a compassionate and reliable system for caring for the terminally ill.

Euthanasia↗

Temperature stress tolerance of conifer seedlings after exposure to UV-B radiation.

Ground-level UV-B radiation has increased globally due to a thinning stratospheric ozone layer. We estimated the effects of increased UV-B on 10 conifer species grown in chambers in greenhouses with supplemental UV-B. Species were selected from a wide range of geographic locations. Plant material of two ages (germinants, first growing season; seedlings, second season) were exposed to three levels of UV-B from ambient (at Victoria, B.C., Canada) to three times ambient (12 kJ m(-2) d(-1)) for up to four months. Frost hardiness and heat tolerance of shoots were estimated from changes in chlorophyll fluorescence after exposure to test temperatures. There were no significant differences among seed sources from different elevations in their response to temperature stresses. When UV-B increased above the ambient level, three species (interior Douglas-fir, Engelmann spruce, and interior lodgepole pine) increased in frost hardiness and four (grand fir, interior spruce, yellow-cedar, and western redcedar) decreased. Two species (western redcedar and western hemlock) increased in heat tolerance when UV-B increased to the 12 kJ level. The main differences in stress tolerance were between the triple ambient and the other two treatments, not between ambient and double ambient, suggesting that any changes in UV-B would have to be large to elicit physiological changes in conifer seedlings.

Freezing↗

Species richness and stand stability in conifer forests of the Sierra Nevada.

Theoretical and empirical studies have long suggested that stability and complexity are intimately related, but evidence from long-lived systems at large scales is lacking. Stability can either be driven by complex species interactions, or it can be driven by the presence/absence and abundance of a species best able to perform a specific ecosystem function. We use 64 years of stand productivity measures in forest systems composed of four dominant conifer tree species to contrast the effect of species richness and abundance on three stability measures. To perform this contrast, we measured the annual growth increments of > 900 trees in mixed and pure forest stands to test three hypotheses: increased species richness will (1) decrease stand variance, (2) increase stand resistance to drought events, and (3) increase stand resilience to drought events. In each case, the alternate hypothesis was that species richness had no effect, but that species composition and abundance within a stand drove variance, resistance, and resilience. In pure stands, the four species demonstrated significant differences in productivity, and in their resistance and resilience to drought events. The two pine species were the most drought resistant and resilient, whereas mountain hemlock was the least resistant and resilient, and red fir was intermediate. For community measures we found a moderately significant (P = 0.08) increase in the community coefficient of variation and a significant (P = 0.03) increase in resilience with increased species richness, but no significant relationship between species richness and community resistance, though the variance in community resistance to drought decreased with species richness. Community resistance to drought was significantly (P = 0.001) correlated to the relative abundance of lodgepole pine, the most resistant species. We propose that resistance is driven by competition for a single limiting resource, with negative diversity effects. In contrast resilience measures the capacity of communities to partition resources in the absence of a single limiting resource, demonstrating positive diversity effects.

Analysis of Variance↗