New Aspects in the Free-Radical Chemistry of Pyrimidine Nucleobases

The contribution will cover three aspects:

i) It has been known for some time that OH radicals and H atoms react with the pyrimidines by adding to the C(5)-C(6) double bond, but only the u.v.-spectra of the sum of these radicals have been reported so far. It will be shown how to arrive at the individual spectra of the C(5) and the C(6) adduct radicals.

ii) α-Hydroxyalkyl radicals are known to inactivate biologically active DNA. In contrast to the electrophilic radicals H and OH they are nucleophilic and the hydroxymethyl radicals add exclusively at the C(6) position of 1,3-dimethyluracil (k ～ 10⁴dm³ mol^?1 s^?1). In the corresponding thymine derivative this reaction also occurs, but one third of the hydroxymethyl radicals abstract an H-atom from the C(5)-methyl group thereby forming an allylic radical. In the course of these reactions pyrimidines with an exocyclic double bond are formed. These products react much more rapidly with hydroxymethyl radicals than the starting material leading to highly hydroxymethylated material at very low doses.

iii) The direct effect of ionizing radiation which would produce a pyrimidine base radical cation can be mimicked by reacting the pyrimidine with SO₄^?, a very good electron acceptor. In water, the radical cation of 1,3-dimethyluracil is rapidly (t_1/2 2μs) converted into the C(5) OH adduct radical. In the presence of peroxodisulphate a chain reaction sets in which leads to the cis-glycol.

The relevance of these findings to radiobiological aspects of nucleic acid research will be discussed.     

In situ oxygen microelectrode measurements of bottom-ice algal production in McMurdo Sound, Antarctica

In-situ estimates of fast-ice algal productivity at Cape Evans, McMurdo Sound, in 1999 were lower than at the same site in previous years. Under-ice irradiance was between 0 and 8 µmol photons m^-2 s^-1; the ice was between 1.9 and 2.0 m thick and the algal biomass averaged 150 mg chl a m^-2, although values as high as 378 mg chl a m^-2 were recorded. Production on 11 and 12 November was between 0.053 and 1.474 mg C m^-2 h^-1. When the data from 11 November were fitted to a hyperbolic tangent function, a multilinear regression gave estimates for P_max of 0.571 nmol O₂ cm^-2 s^-1, an ! of 0.167 nmol O₂ cm^-2 s^-1 µmol^-1 photons m^-2 s^-1 and an E_k of 3.419 µmol photons m^-2 s^-1. A P_max of 2.674 nmol O₂ cm^-2 s^-1, an ! of 0.275 nmol O₂ cm^-2 s^-1 µmol^-1 photons m^-2 s^-1, r of 0.305 nmol O₂ cm^-2 s^-1 and an E_k of 9.724 µmol^-1 photons m^-2 s^-1 were estimated from the 12 November data. The sea-ice algal community was principally comprised of Nitzschia stellata, Entomoneis kjellmanii and Berkeleya adeliensis. Other taxa present included N. lecointei, Fragilariopsis spp., Navicula glaciei, Pleurosigma spp. and Amphora spp. Variations in the method for estimating the thickness of the diffusive boundary layer were not found to significantly affect the measurements of oxygen flux. However, the inability to accurately measure fine-scale variations in biomass is thought to contribute to the scatter of the P versus E data.     

Drugs with susceptible sites for free radical induced oxidative transformations: the case of a penicillin

Penicillins, as bactericidal antibiotics, have been widely used to treat infections for several decades. Their structure contains both aromatic and thioether moieties susceptible to free radical oxidation. The ^?OH induced oxidation mechanism of amoxicillin was investigated by pulse radiolysis techniques and by final product analysis performed after steady-state γ-irradiation. The predominant sites of the ^?OH attack are suggested to be the thioether group, initially yielding an ^?OH adduct to the sulfur, and the aromatic ring. This adduct to the sulfur converts to sulfur radical cation, which has three competitive reaction paths: (1) by deprotonation at the adjacent carbon α-(alkylthio)alkyl radicals form, which undergo disproportionation leading presumably to sulfoxide as main product; (2) via the pseudo-Kolbe mechanism it may transform to α-aminoalkyl radicals; (3) the radical cation can be stabilized through intramolecular S.˙.O bond formation. The reaction mechanism suggests the presence of a short-living and a stabilized (via hydrogen bonding) long-living ^?OH adduct to the sulfur. The three-electron bonded dimers of amoxicillin were not formed owing to steric hindrance. Thiyl radicals were also present in equilibrium with α-aminoalkyl radicals. In the presence of dissolved oxygen, aromatic ring hydroxylation occurred along with complex reactions resulting in e.g. oxidation of the methyl groups. The formation of the sulfoxide is especially effective in the presence of dissolved oxygen, under anaerobic condition, however, it is also generated owing to H₂O₂ and α-(alkylthio)alkyl radicals. The thioether moiety appears to be more sensitive to oxidation compared to the aromatic ring in case of amoxicillin.     

Free Radicals in the Atmosphere

Like the oxidation in a flame, the oxidation in the atmosphere is mediated by free radicals. Unlike a flame, however, atmospheric oxidation needs an external source of energy: the sun light. In fact the most important radical acting in the lower atmosphere, the hydroxyl radical, OH, is produced following the UV-photolysis of ozone, O,which yields an excited oxygen atom, O'D:

OH reacts with most atmospheric trace gases, in many cases as the first and rate determining step in the reaction chain leading to oxidation. In this way a host of various other radicals (e.g. peroxy radicals), most of them very short lived, are generated. Usually these oxidation reactions form chains which regenerate OH, thus maintaining OH at a relatively high concentration level on the order of 10⁶cm～³ during the day. The reactions which control the OH concentration will be discussed in detail. During the night radical formation is greatly diminished. It proceeds, for example, through the reaction of defines with O, and. in dry air, through reaction of defines and aldehydes with the nitrate radical, NO,.     

Reactions of reactive oxygen species (ROS) with curcumin analogues: Structure-activity relationship

Three curcumin analogues viz., bisdemethoxy curcumin, monodemethoxy curcumin, and dimethoxycurcumin that differ at the phenolic substitution were synthesized. These compounds have been subjected for free radical reactions with DPPH radicals, superoxide radicals (O(2)(?-)), singlet oxygen ((1)O(2)) and peroxyl radicals (CCl(3)O(2)(?)) and the bimolecular rate constants were determined. The DPPH radical reactions were followed by stopped-flow spectrometer, (1)O(2) reactions by transient luminescence spectrometer, and CCl(3)O(2)(?) reactions using pulse radiolysis technique. The rate constants indicate that the presence of o-methoxy phenolic OH increases its reactivity with DPPH and CCl(3)O(2)(?), while for molecules lacking phenolic OH, this reaction is very sluggish. Reaction of O(2)(?-) and (1)O(2) with curcumin analogues takes place preferably at β-diketone moiety. The studies thus suggested that both phenolic OH and the β-diketone moiety of curcumin are involved in neutralizing the free radicals and their relative scavenging ability depends on the nature of the free radicals.     

Carnivorous feeding and respiration of the Arctic under-ice amphipod Gammarus wilkitzkii

. The dominant Arctic under-ice amphipod Gammarus wilkitzkii consumes a wide range of food items. The carnivorous feeding activity and energy budget of this large species were studied using three different approaches. Maximum potential ingestion rates I_max estimated from an allometric function taken from the literature and based on body mass were 2.1ǂ.4% of body carbon day^-1. Based on respiration measurements, the specific ingestion rates required to meet metabolic demands were lower (1.4ǂ.4% of body carbon day^-1). Feeding experiments, in which co-occurring pelagic calanoid (Calanus hyperboreus) or sympagic harpacticoid (Halectinosoma sp.) copepods were offered as prey, yielded actual ingestion rates of 8.0LJ.6% of body carbon day^-1 and 0.1ǂ.1% of body carbon day^-1, respectively. These results indicate that predatory feeding on pelagic copepods may constitute an important food source for G. wilkitzkii. Abundances of G. wilkitzkii at the ice underside (median: 1.6 ind. m^-2), Calanus spp. in the upper metre below the ice (2.6 ind. m^-3), and Halectinosoma sp. in the lowermost 2-3 cm of the ice (393.5 ind. m^-2) were determined from several multi-year pack-ice floes in the northern Greenland Sea and Fram Strait. Potential predation impact of G. wilkitzkii was estimated by combining information on ingestion rates with population densities. It was very high on Calanus spp. in the under-ice water layer (61.5% of the under-ice standing stock day^-1), but comparatively low on Halectinosoma sp. in the bottom of the ice (3.8% of standing stock day^-1). The observation of G. wilkitzkii preying on pelagic copepods in the under-ice water layer represents a hitherto unknown but obviously significant process and a new direction in the cryo-pelagic coupling in the Arctic marine ecosystem.     

μ-H-Bridged Bicyclo[3.3.3]undecyl Cations. Theoretical Calculations of Physical and Chemical Properties

7-H-Bridged carbocations 1 and 2, which are still unknown experimentally, are structures with fascinating possibilities as intermediates for the synthesis of very strained in-bicyclic and tricyclic alkanes and alkenes. They are also expected to possess record high pK_a values. In conjunction with our experimental program to try to prepare 1 and 2, we have carried out ab initio calculations on these structures and various reference compounds, with the aim of assessing just how stable these cations might be, and what physical and chemical properties they might possess. The results of these studies confirm that 1 and 2 should be viable species; they have energies similar to those of the conventional out-cations, and the 7-H bond distances are not very different from those calculated for known 7-H cations. The calculated ¹H NMR chemical shift for the mu-H of 1 is -12 - 0.5 and -9.5 - 0.5 for 2, both values considerably more negative than in known 7-H cations. The possible effect of large amplitude motions of the 7-H on the ¹H NMR chemical shift was investigated and not found to be significant. The pK_as for 1 and 2 are estimated to be 17-18 with respect to an alkene conjugate base, a virtually unimaginable size for a formally alkyl cation. The paper also discusses the possibility of the 7-H being involved in the acid-conjugate base chemistry, since this is shown to be a hugely exothermic reaction. Finally, the alkenes and alkanes associated with cations 1 and 2 have been calculated, the in-alkene 7 from cation 1 is shown, for example, to possess a large steric strain, yet this structure should be accessible via deprotonation of 1 with a strong base.     

On the mechanisms of the reaction of dodecatungstophosphate with alkyl radicals in aqueous solutions

The reduced lacunary polyoxotungstate, [PW₁₁O₃₉]⁸⁻, reacts with the ^.CH₂CH(OH)CH₃ and ^.CH₂C(CH₃)₂OH radicals via a mechanism involving β-hydroxide elimination to yield propene and 2-methyl propene respectively, and [PW₁₁O₃₉]⁷⁻. [PW₁₁O₃₉]⁸⁻ is also oxidized by methyl radicals in a reaction which yields methane as the major product. It is proposed that the reactions proceed via the formation of short lived transients with W-C σ bonds.     

Regulation of xylem sap flow in an evergreen, a semi-deciduous, and a deciduous Meliaceae species from the Amazon

The significance of phenological characteristics, stomatal conductance of the leaves, and stem water storage fluctuations for the regulation of xylem sap flow in an evergreen (Carapa guianensis Aubl.), in a semi-deciduous (Swietenia macrophylla King), and in a deciduous (Cedrela odorata L.) Meliaceae species was studied in a 7-year-old plantation near Manaus, Brazil. The study responds to the increasing demand for knowledge on the water relations of highly exploited timber trees of the Amazon. Xylem sap flow measurements indicated that the daily sap flow of Carapa (3.8 l day^-1 tree^-1 to 16.4 l day^-1 tree^-1) exceeded the daily sap flow of Swietenia (2.4 l day^-1 tree^-1 to 7.0 l day^-1 tree^-1) and Cedrela (1.6 l day^-1 tree^-1 to 11.6 l day^-1 tree^-1) during the entire year, although the highest flux densities were measured in Cedrela. The decrease in xylem sap flow observed in periods with low soil water potentials and high atmospheric vapor saturation deficits was more pronounced in the deciduous (Cedrela) and semi-deciduous species (Swietenia) than the evergreen species (Carapa). Carapa, which has the highest daily sap flow, had the highest biomass and sapwood portion. The high flux densities measured in Cedrela most likely result from the large earlywood vessels in this species. The seasonal variation of xylem sap flow of the three species was correlated with the stomatal conductance of the leaves measured by infiltration experiments. Stem water storage fluctuations in Carapa and Swietenia were predominantly due to transpiration; in Cedrela it was predominantly due to evaporative water loss on the stem surface during dry periods.     

Water availability and carbon isotope discrimination in conifers

The stable C isotope composition ('¹³C) of leaf and wood tissue has been used as an index of water availability at both the species and landscape level. However, the generality of this relationship across species has received little attention. We compiled literature data for a range of conifers and examined relationships among landscape and environmental variables (altitude, precipitation, evaporation) and '¹³C. A significant component of the variation in '¹³C was related to altitude (discrimination decreased with altitude in stemwood, 2.53‰ km^-1 altitude, r²=0.49, and in foliage, 1.91‰ km^-1, r²=0.42), as has been noted previously. The decrease in discrimination with altitude was such that the gradient in CO₂ partial pressure into the leaf (P_a-P_i) and altitude were generally unrelated. The ratio of precipitation to evaporation (P/E) explained significant variation in P_a-P_i of stemwood (r²=0.45) and foliage (r²=0.27), but only at low (<0.8) P/E. At greater P/E there was little or no relationship, and other influences on '¹³C probably dominated the effect of water availability. We also examined the relationship between plant drought stress (O) and '¹³C within annual rings of stemwood from Pinus radiata and Pinus pinaster in south-western Australia. Differential thinning and fertiliser application produced large differences in the availability of water, nutrients and light to individual trees. At a density of 750 stems ha^-1, O and '¹³C were less (more negative) than at 250 stems ha^-1 indicating greater drought stress and less efficient water use, contrary to what was expected in light of the general relationship between discrimination and P/E. The greater '¹³C of trees from heavily thinned plots may well be related to an increased interception of radiation by individual trees and greater concentrations of nutrients in foliage - attributes that increase rates of photosynthesis, reduce P_i and increase '¹³C. '¹³C was thus modified to a greater extent by interception of radiation and by nutrient concentrations than by water availability and the '¹³C-O relationship varied between thinning treatments. Within treatments, the relationship between '¹³C and O was strong (0.38<r²<0.58). We conclude that '¹³C may well be a useful indicator of water availability or drought stress, but only in seasonally dry climates (P/E<1) and where variation in other environmental factors can be accounted for.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated production of transgenic plants of<Emphasis Type="Italic"> Muscari armeniacum</Emphasis> Leichtl. ex Bak.

A system for the production of transgenic plants has been developed for the Liliaceous ornamental plant Muscari armeniacum Leichtl. ex Bak via Agrobacterium-mediated transformation of embryogenic cultures. Leaf-derived embryogenic cultures were co-cultivated with each of three A. tumefaciens strains, all of which harbored the binary vector carrying the neomycin phosphotransferase II (nptII), hygromycin phosphotransferase (hpt) and intron-containing #-glucuronidase (gus-intron) genes in the T-DNA region. Following co-cultivation, the embryogenic cultures were cultured on a medium containing 500 mg l^-1 cefotaxime for 1 week followed by a medium containing 75 mg l^-1 hygromycin in addition to cefotaxime. After 4-5 weeks, several hygromycin-resistant (Hyg^r) cell clusters were produced from the co-cultivated embryogenic cultures. The highest efficiency of production of Hyg^r cell clusters was obtained when embryogenic cultures were inoculated with A. tumefaciens EHA101/pIG121Hm in the presence of 100 µM acetosyringone (AS) and 0.1% (v/v) of a surfactant (Tween20) followed by co-cultivation in the presence of 100 µM AS. Hyg^r embryogenic cultures developed into complete plants via somatic embryogenesis, and most of them were verified to be transgenic by GUS histochemical assay and polymerase chain reaction analysis. Southern blot analysis revealed the integration of one to five copies of the transgene into the genome of transgenic plants, but most of them had one or two copies.     

Primary production, light and vertical mixing in Potter Cove, a shallow bay in the maritime Antarctic

Phytoplankton photosynthesis was measured during spring-summer 1991-1992 in the inner and outer part of the shallow Potter Cove, King George Island. Strong winds characterise this area. Wind-induced turbulent mixing was quantified by means of the root-mean square expected vertical displacement depth of cells in the water column, Z_t. The light attenuation coefficient was used as a measure of the influence of the large amount of terrigenous particles usually present in the water column; 1% light penetration ranged between 30 and 9 m, and between 30 and 15 m for the inner and outer cove, respectively. Obvious differences between photosynthetic capacity [P*_max; averages 2.6 and 0.6 µg C (µg chlorophyll-a)^-1 h^-1] and photosynthetic efficiency {!*; 0.073 and 0.0018 µg C (µg chlorophyll-a)^-1 h^-1 [(µmol m^-2 s^-1)^-1]} values were obtained for both sites during low mixing conditions (Z_t from 10 to 20 m), while no differences were found for high mixing situations (Z_t>20 m). This suggests different photoacclimation of phytoplankton responses, induced by modifications of the light field, which in turn are controlled by physical forcing. Our results suggest that although in experimental work P*_max can be high, wind-induced mixing and low irradiance will prevent profuse phytoplankton development in the area.     

Transformation of elite white maize using the particle inflow gun and detailed analysis of a low-copy integration event

Elite white maize lines W506 and M37W were transformed with a selectable marker gene (bar) and a reporter gene (uidA) or the polygalacturonase-inhibiting protein (pgip) gene after bombardment of cultured immature zygotic embryos using the particle inflow gun. Successful transformation with this device did not require a narrow range of parameters, since transformants were obtained from a wide range of treatments, namely pre-culture of the embryos for 4-6 days, bombardment at helium pressures of 700-900 kPa, selection-free culture for 2-4 days after bombardment and selection on medium containing bialaphos at 0.5-2 mg l^-1. However, bombardments with helium pressures below 700 kPa yielded no transformants. The culture of immature zygotic embryos of selected elite white maize lines on medium containing 2 mg l^-1 2,4-dichlorophenoxyacetic acid and 20 mM L-proline proved to be most successful for the production of regenerable embryogenic calli and for the selection of putative transgenic calli on bialaphos-containing medium after transformation. Transgenic plants were obtained from four independent transformation events as confirmed by Southern blot analysis. Transmission of the bar and uidA genes to the T₄ progeny of one of these transformation events was demonstrated by Southern blot analysis and by transgene expression. In this event, the transgenes bar and uidA were inserted in tandem.     

Generation of Hydroxyl Radical in Isolated Pea Root Cell Wall, and the Role of Cell Wall-Bound Peroxidase, Mn-SOD and Phenolics in Their Production

The hydroxyl radical produced in the apoplast has been demonstratedto facilitate cell wall loosening during cell elongation. Cellwall-bound peroxidases (PODs) have been implicated in hydroxylradical formation. For this mechanism, the apoplast or cellwalls should contain the electron donors for (i) H₂O₂ formationfrom dioxygen; and (ii) the POD-catalyzed reduction of H₂O₂to the hydroxyl radical. The aim of the work was to identifythe electron donors in these reactions. In this report, hydroxylradical (·OH) generation in the cell wall isolated frompea roots was detected in the absence of any exogenous reductants,suggesting that the plant cell wall possesses the capacity togenerate ·OH in situ. Distinct POD and Mn-superoxidedismutase (Mn-SOD) isoforms different from other cellular isoformswere shown by native gel electropho-resis to be preferably boundto the cell walls. Electron paramagnetic resonance (EPR) spectroscopyof cell wall isolates containing the spin-trapping reagent,5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO),was used for detection of and differentiation between ·OHand the superoxide radical (O₂^–·). The data obtainedusing POD inhibitors confirmed that tightly bound cell wallPODs are involved in DEPMPO/OH adduct formation. A decreasein DEPMPO/OH adduct formation in the presence of H₂O₂ scavengersdemonstrated that this hydroxyl radical was derived from H₂O₂.During the generation of ·OH, the concentration of quinhydronestructures (as detected by EPR spectroscopy) increased, suggestingthat the H₂O₂ required for the formation of ·OH in isolatedcell walls is produced during the reduction of O₂ by hydroxycinnamicacids. Cell wall isolates in which the proteins have been denaturated(including the endogenous POD and SOD) did not produce ·OH.Addition of exogenous H₂O₂ again induced the production of ·OH,and these were shown to originate from the Fenton reaction withtightly bound metal ions. However, the appearance of the DEPMPO/OOHadduct could also be observed, due to the production of O₂^–·when endogenous SOD has been inactivated. Also, O₂^–·was converted to ·OH in an in vitro horseradish peroxidase(HRP)/H₂O₂ system to which exogenous SOD has been added. Takentogether with the discovery of the cell wall-bound Mn-SOD isoform,these results support the role of such a cell wall-bound SODin the formation of ·OH jointly with the cell wall-boundPOD. According to the above findings, it seems that the hydroxycinnamicacids from the cell wall, acting as reductants, contribute tothe formation of H₂O₂ in the presence of O₂ in an autocatalyticmanner, and that POD and Mn-SOD coupled together generate ·OHfrom such H₂O₂.     

Arguments against the significance of the Fenton reaction contributing to signal pathways under in vivo conditions

One of the common explanations for oxidative stress in the physiological milieu is based on the Fenton reaction, i.e. the assumption that radical chain reactions are initiated by metal-catalyzed electron transfer to hydrogen peroxide yielding hydroxyl radicals. On the other hand — especially in the context of so-called “iron switches” — it is postulated that cellular signaling pathways originate from the interaction of reduced iron with hydrogen peroxide.

Using fluorescence detection and EPR for identification of radical intermediates, we determined the rate of iron complexation by physiological buffer together with the reaction rate of concomitant hydroxylations of aromatic compounds under aerobic and anaerobic conditions. With the obtained overall reaction rate of 1,700 M^-1s^-1 for the buffer-dependent reactions and the known rates for Fenton reactions, we derive estimates for the relative reaction probabilities of both processes.

As a consequence we suggest that under in vivo conditions initiation of chain reactions by hydroxyl radicals generated by the Fenton reaction is of minor importance and hence metal-dependent oxidative stress must be rather independent of the so-called “peroxide tone”. Furthermore, it is proposed that — in the low (subtoxic) concentration range — hydroxylated compounds derived from reactions of “non-free” (crypto) OH radicals are better candidates for iron-dependent sensing of redox-states and for explaining the origin of cellular signals than the generation of “free” hydroxyl radicals.     

The role of hydroxyl radical as a messenger in Cr(VI)-induced p53 activation

The present study investigates whether reactive oxygen species (ROS)are involved in p53 activation, and if they are, which species isresponsible for the activation. Our hypothesis is that hydroxyl radical(·OH) functions as a messenger for the activation of this tumorsuppressor protein. Human lung epithelial cells (A549) were used totest this hypothesis. Cr(VI) was employed as the source of ROS due toits ability to generate a whole spectrum of ROS inside the cell. Cr(VI)is able to activate p53 by increasing the protein levels and enhancingboth the DNA binding activity and transactivation ability of theprotein. Increased cellular levels of superoxide radicals(O₂·), hydrogen peroxide(H₂O₂), and ·OH radicals were detected on theaddition of Cr(VI) to the cells. Superoxide dismutase, by enhancing theproduction of H₂O₂ from O₂·radicals, increased p53 activity. Catalase, anH₂O₂ scavenger, eliminated ·OH radicalgeneration and inhibited p53 activation. Sodium formate and aspirin,·OH radical scavengers, also suppressed p53 activation. Deferoxamine,a metal chelator, inhibited p53 activation by chelating Cr(V) to makeit incapable of generating radicals from H₂O₂.NADPH, which accelerated the one-electron reduction of Cr(VI) to Cr(V)and increased ·OH radical generation, dramatically enhanced p53activation. Thus ·OH radical generated from Cr(VI) reduction in A549cells is responsible for Cr(VI)-induced p53 activation.

     

Reductive addition of OH radicals to macrocyclic complexes

The reactions of OH radicals with complexes of the macrocycle Me₂-3,4,5-pyo-[14]trieneN₄ and Cu(II), Co(II), Co(III) have been investigated by pulse radiolysis. The addition of the OH radical to the macrocycle leads to metal-ligand radical intermediates which achieve stability by reduction of the metal center. The mechanism of such reactions is discussed in terms of the redox potentials of the complexes and known mechanisms of addition to organic aromatic compounds.     

Influence of climate-driven shifts in biomass allocation on water transport and storage in ponderosa pine

Conifers decrease the amount of biomass apportioned to leaves relative to sapwood in response to increasing atmospheric evaporative demand. We determined how these climate-driven shifts in allocation affect the aboveground water relations of ponderosa pine growing in contrasting arid (desert) and humid (montane) climates. To support higher transpiration rates, a low leaf:sapwood area ratio (A_L/A_S) in desert versus montane trees could increase leaf-specific hydraulic conductance (K_L). Alternatively, a high sapwood volume:leaf area ratio in the desert environment may increase the contribution of stored water to transpiration. Transpiration and hydraulic conductance were determined by measuring sap flow (J_S) and shoot water potential during the summer (June-July) and fall (August-September). The daily contribution of stored water to transpiration was determined using the lag between the beginning of transpiration from the crown at sunrise and J_S. In the summer, mean maximum J_S was 31.80LJ.74 and 24.34Dž.05 g m^-2 s^-1 for desert and montane trees (a 30.6% difference), respectively. In the fall, J_S was 25.33NJ.52 and 16.36dž.64 g m^-2 s^-1 in desert and montane trees (a 54.8% difference), respectively. J_S was significantly higher in desert relative to montane trees during summer and fall (P<0.05). Predawn and midday shoot water potential and sapwood relative water content did not differ between environments. Desert trees had a 129% higher K_L than montane trees in the summer (2.41᎒^-5 versus 1.05᎒^-5 kg m^-2 s^-1 MPa^-1, P<0.001) and a 162% higher K_L in the fall (1.97᎒^-5 versus 0.75᎒^-5 kg m^-2 s^-1 MPa^-1, P<0.001). Canopy conductance decreased with D in all trees at all measurement periods (P<0.05). Maximum g_C was 3.91 times higher in desert relative to montane trees averaged over the summer and fall. Water storage capacity accounted for 11 kg (11%) and 10.6 kg (17%) of daily transpiration in the summer and fall, respectively, and did not differ between desert and montane trees. By preventing xylem tensions from reaching levels that cause xylem cavitation, high K_L in desert ponderosa pine may facilitate its avoidance. Thus, the primary benefit of low leaf:sapwood allocation in progressively arid environments is to increase K_L and not to increase the contribution of stored water to transpiration.     

Generation of reactive oxygen species in the enzymatic reduction of PbCrO4 and related DNA damage

Free radical reactions are believed to play an important role in the mechanism of Cr(VI)-induced carcinogenesis. Most studies concerning the role of free radical reactions have been limited to soluble Cr(VI). Various studies have shown that solubility is an important factor contributing to the carcinogenic potential of Cr(VI) compounds. Here, we report that reduction of insoluble PbCrO4 by glutathione reductase in the presence of NADPH as a cofactor generated hydroxyl radicals (.OH) and caused DNA damage. The .OH radicals were detected by electron spin resonance (ESR) using 5,5-dimethyl-N-oxide as a spin trap. Addition of catalase, a specific H2O2 scavenger, inhibited the .OH radical generation, indicating the involvement of H2O2 in the mechanism of Cr(VI)-induced .OH generation. Catalase reduced .OH radicals measured by electron spin resonance and reduced DNA strand breaks, indicating .OH radicals are involved in the damage measured. The H2O2 formation was measured by change in fluorescence of scopoletin in the presence of horseradish peroxidase. Molecular oxygen was used in the system as measured by oxygen consumption assay. Chelation of PbCrO4 impaired the generation of .OH radical. The results obtained from this study show that reduction of insoluble PbCrO4 by glutathione reductase/NADPH generates .OH radicals. The mechanism of .OH generation involves reduction of molecular oxygen to H2O2, which generates .OH radicals through a Fenton-like reaction. The .OH radicals generated by PbCrO4 caused DNA strand breakage.