Studies on the Chemical Synthesis of Potential Antimetabolites. 35.1 Synthesis of 2-Chloro-1-deazaadenosine and 2-Chloro-1-deazainosine as Candidate Inhibitors for S-Adenosylhomocysteinases and Methyltransferases

Abstract

The syntheses of 2-chloro-1-deazaadenosine (2) and 2-chloro-1-deazainosine (3) are described. Conversion of 7-ribosylated 6-chloro-1-deazapurine 3-oxide to the desired 2,6-disubstituted 9-ribosyl-1-deazapurines was effected by a series of reactions involving “deoxygenative chlorination” and transglycosylation in satisfactory yields.     

Photoreceptor optics of the honeybee and its eye colour mutants: the effect of screening pigments on the long-wave subsystem of colour vision

With the aim of clarifying the role of screening pigments in photoreceptor optics of the compound eye, a comparative study of the optical properties of the honeybee eye in the visible region of the spectrum was carried out using wild-type bees and eye colour mutantssnow, snow ^laranja, ivory^umberandchartreuse with total or partial blockage of the tryptophane-ommochrome pathway.

Plant response to variations in nitrogen availability in a desert shrubland community

Spatial variations in nitrogen availability were studied in a desert community codominated byLarrea tridentata (DC.) Cov. andProsopis glandulosa Torr. Measurements of natural ¹⁵N values in tissues suggested thatProsopis obtains approximately half of its nitrogen through direct symbiotic fixation. Soils were collected under 1)Prosopis shrubs, 2)Larrea shrubs 2 m fromProsopis (LP), and 3)Larrea 2 m from otherLarrea but> 5 m from the nearestProsopis (LL).Prosopis soils showed significantly higher rates of nitrogen mineralization than LL soils in both A and B horizons. Rates of mineralization in LP soils were significantly higher than rates in LL soils only in the B horizon and were not significantly different from rates inProsopis soils. Leaf nitrogen concentrations were significantly higher in LP shrubs (2.06%) than in LL shrubs (1.78%), although ¹⁵N values did not differ between the two shrub types. Nitrogen concentrations inPerezia nana Gray, a perennial herb, were greater in plants underProsopis shrubs (2.09%) than under LP shrubs (1.93%) or LL shrubs (1.67%). Despite apparent differences in nitrogen availability, biomass ofLarrea and density ofPerezia did not differ significantly among these sites.     

Changes in leaf water potential and CAM inSempervivum montanum andSedum album in response to water availability in the field

Summary The short term effects of irrigation on diurnal changes in leaf and titratable acidity were examined both inSempervivum montanum and inSedum album, a facultative CAM plant, in the Spanish Pyrenees. InSemperivivum, leaf responded rapidly to irrigation and, in both the control and irrigated plants, increased during the day and decreased during the night and early morning. By contrast, leaf inSedum responded more slowly to irrigation and showed a decrease during the day and an increase in the period between evening and early morning. Under the conditions of the short-term experiments, changes in acid metabolism were not observed in either species following irrigation. The results suggest that transpirational water loss together with redistribution of water within the plant are more important than the osmotic concentration of malic acid in determining leaf in both species and that daytime water loss is greater inSedum than inSempervivum.The effect of long-term water stress on leaf and acid levels was also assessed in both species over a 3-week period. Both leaf and acidification inSempervivum decreased over this time period but could, at least partially, be reversed by irrigation. InSedum, leaf also declined but a more gradual reduction in acidification occurred than inSempervivum. Irrigation inSedum at least partially reversed the decline in leaf but produced a complex pattern of acid metabolism. Nocturnal acidification in the irrigated plants was lower than in the non-irrigated control when preceded by a cool day but showed complete recovery following a hot day. It is suggested inSedum album that C₃ photosynthesis during the preceding light period, as determined by light intensity and leaf temperature, may be important in determining the extent of nocturnal acidification under field conditions.     

Stress response in two Antarctic teleosts (Notothenia coriiceps Richardson andChaenocephalus aceratus Lönnberg) following capture and surgery

Blood chemistry and haematological parameters have been determined in two Antarctic teleosts,Notothenia coriiceps Richardson andChaenocephalus aceratus Lönnberg, held at around 1°C.Notothenia coriiceps has a low haemoglobin content compared to tem-perate-zone species, whereasC. aceratus apparently lacks respiratory pigments. Blood samples were obtained by cardiac puncture following landing or using chronically implanted post-branchial arterial cannulae. Although both species showed a similar acidosis on capture (arterial pH as low as 7.5 versus the final recovery value of around 7.9),C. aceratus took 48 h to reestablish baseline values whileN. coriiceps recovered within 12 h, despite initially showing a greater degree of hypercapnic hypoxia. Surgery led to a more severe disturbance of acid-base regulation inN. coriiceps thanC. aceratus (arterial pH of 7.5 versus 7.8) but needed only half as long for recovery. A progressive decrease in arterial oxygen tension and increase in arterial carbon dioxide tension (both more pronounced inN. coriiceps) with level of acidosis was observed down to arteria pH 7.2 InC. aceratus this was accompanied by a rise in blood lactate (up to 10 mmol·1^-1 in some individuals), whileN. coriiceps showed only a modest and transient lactacidosis. Stress inN. coriiceps therefore induces primarily a respiratory, rather than a metabolic acidosis, whereas inC. aceratus both components are present. A differential response to stress is also indicated by an elevated, though low noradrenaline titre inN. coriiceps following surgery and capture, whileC. aceratus was little affected by surgery. However, both species show an unusually weak catecholamine response to induced stress.Abbreviations pH/T °C thermal sensitivity of pH - Ad adrenaline - bw body weight - C.CO₂ total carbon dioxide content - C.O₂ total oxygen content - ED ₅₀ Median effective dose - EDTA ethylenediaminetertra-acetic acid - Hb haemoglobin - Hct haematocrit - HPLC high-performance liquid chromatography - lac lactate - MCH mean corpuscular haemoglobin content - MCHC mean corpuscular haemoglobin concentration - MCV mean cell volume - MS222 tricaine methane sulphonate - NAd noradrenaline - P _aCO₂ arterial carbon dioxide tension - P _aO₂ arterial oxygen tension - pHa arterial blood pH - RBCC red blood cell count - SW sea water - T _a ambient air temperature - VO₂ oxygen consumption     

Nitrogen uptake in relation to excess supply and its effects on the lichens<Emphasis Type="Italic"> Evernia prunastri</Emphasis> (L.) Ach and<Emphasis Type="Italic"> Xanthoria parietina</Emphasis> (L.) Th. Fr.

The aim of this study was to compare the physiological responses to increased nitrogen (N) supply between the nitrophytic lichen Xanthoria parietina (L.) Th. Fr. and the acidophytic lichen Evernia prunastri (L.) Ach. The two lichens were exposed to a weekly dosage of 0.05, 0.1, 0.2, 0.6 or 2.4 g N m^–2 for 2 months, administered as NH₄NO₃ dissolved in artificial rainwater (1 l m^–2). After the treatments, in vivo chlorophyll a fluorescence was determined to assess vitality; concentrations of total N, ammonium, nitrate and dominant amino acids, including glutamate, glutamine and arginine, were quantified in order to follow changes in N status; and the polyols ribitol, arabitol and mannitol were quantified to follow changes in the lichens carbon (C) status. The uptake of N was quantified by labelling the fertiliser with ¹⁵N in the ammonium position; chlorophyll a was used as an indirect marker for algal activity, and ergosterol as an indirect marker of fungal activity. Nitrogen uptake was higher in E. prunastri than in X. parietina, although the latter species may have used the mannitol reserves to obtain C skeletons and energy for N assimilation. Chlorophyll a and ergosterol concentrations remained unaltered in X. parietina irrespective of N dosage while ergosterol decreased with increasing N uptake in E. prunastri. The latter species had accumulated a large pool of ammonium at the highest N dosage, whilst in X. parietina a significant nitrate pool was instead observed. Taken together, these short-term responses to high N supply observed in the two lichens, and the differences between them, can partly explain the higher tolerance of X. parietina towards increased atmospheric N levels.     

Effects of long scotophase and cold acclimation on heat production in two diurnal rodents

Summary Heat production of two diurnal rodents,Rhabdomys pumilio andLemniscomys griselda was measured in long scotophase-LS (8L: 16D; 25°C) acclimated and long scotophase and cold — LSAC (8L: 16D; 6°C) acclimated animals and compared to a control group (12L: 12D; 25°C).LS increased in both species. Further acclimation of LSAC increased inR. pumilio and decreased inL. griselda. LS increased body temperature (T _b) inL. griselda only. LS increased overall thermal conductance in both species. LSAC caused a further increase in this parameter inR. pumilio.A singificant (P<0.001) increase in the magnitude of maximal nonshivering thermogenesis (NST) was observed in both species due to LS acclimation. LSAC did not change this maximal NST but increased its obligatory part (minimal , P<0.05, inL. griselda, andP<0.001, inR. pumilio).The results of this study show that winter acclimatization of heat production mechanisms, in both species, may be due to extension of scotophase.Abbreviations LS long scotophase - LSAC long scotophase and cold - NA noradrenaline - NST nonshivering thermogenesis - RMR resting metabolic rate     

Mobile nucleolus organizing regions (NORs) inAllium (Liliaceae s. lat.)? — Inferences from the specifity of silver staining

NORs and interphase nucleoli have been silver stained inAllium cepa, A. fistulosum, reciprocal crosses between both species, and in different strains of top onions which originated from hybridization betweenA. cepa andA. fistulosum. The variability observed in size, number, and position of active NORs and correspondingly in number (and size) of interphase nucleoli is at least in part strain-specific. These data are taken to indicate that NORs inAllium behave like movable genetic elements.—With respect to the staining specifity of silver nitrate, it was found that AgNO₃ labels (1) nucleoli, (2) NORs (i.e., actively transcribed ribosomal genes) inside the achromatic secondary constrictions, and (3) sometimes (but less pronounced) centromeres; Giemsa banding labels heterochromatin surrounding the NOR but not the nucleolus organizing secondary constriction.     

Quantitative analysis of histochemical and immunohistochemical reactions in skeletal muscle fibres ofRana andXenopus

Summary Intensities of histochemical and immunohistochemical reactions in muscle fibres ofRana andXenopus have been estimated microphotometrically, and the data from serial sections statistically analysed. Quantitative validities of reactions and measurements have also been assessed against independent published evidence. It is concluded that NADH—tetrazolium reductase overestimates tonic-fibre aerobic capacities and the actomyosin ATPase reaction overestimates their contraction speeds. However, it appears that succinate dehydrogenase, despite being a near-equilibrium enzyme of particulate distribution, indicates the relative aerobic capacities of fibres with acceptable accuracy when lightly reacted. Capacities for aerobic and anaerobic metabolism are positively correlated over all types of fibre (r typically 0.6 for 200 fibres), perhaps as an adaptation to environmental hypoxia.Multivariate clusters (indicating fibre types) have been sought, using Ward's method with optimizing procedures (iterative relocation and multivariate-normal modelling). Cluster analysis confirms the subjective identifications of two slow/tonic types inXenopus (labelled T5 and S4) but of only one (T5) inRana. Division of the fast family twitch fibres into three types (F1–F3) in both genera, with metabolic capacity related inversely to apparent shortening velocity, is highly supportable by objective criteria. However, statistically significant subdivisions also present themselves.Rana F2 andXenopus F1 clusters can be bisected according to metabolic capacity; andXenopus F2 fibres fall into three subtypes reflecting different isomyosin contents. In the different types of twitch fibre, ratios of myofibrillar ATP consumption rate to aerobic capacity increase up to 30-fold with contraction speed, but anaerobic/aerobic ratios do so only 5-fold.     

Characterization of a novel cellulose synthesis inhibitor

The physiological effects of an experimental herbicide and cellulose synthesis inhibitor, N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine, called AE F150944, are described. In the aminotriazine molecular class, AE F150944 is structurally distinct from other known cellulose synthesis inhibitors. It specifically inhibits crystalline cellulose synthesis in plants without affecting other processes that were tested. The effects of AE F150944 on dicotyledonous plants were tested on cultured mesophyll cells of Zinnia elegans L. cv. Envy, which can be selectively induced to expand via primary wall synthesis or to differentiate into tracheary elements via secondary wall synthesis. The IC₅₀ values during primary and secondary wall synthesis in Z. elegans were 3.91×10^–8 M and 3.67×10^–9 M, respectively. The IC₅₀ in suspension cultures of the monocot Sorghum halapense (L.) Pers., which were dividing and synthesizing primary walls, was 1.67×10^–10 M. At maximally inhibitory concentrations, 18–33% residual crystalline cellulose synthesis activity remained, with the most residual activity observed during primary wall synthesis in Z. elegans. Addition to Z. elegans cells of two other cellulose synthesis inhibitors, 1 M 2,6-dichlorobenzonitrile and isoxaben, along with AE F150944 did not eliminate the residual cellulose synthesis, indicating little synergy between the three inhibitors. In differentiating tracheary elements, AE F150944 inhibited the deposition of detectable cellulose into patterned secondary wall thickenings, which was correlated with delocalization of lignin as described previously for 2, 6-dichlorobenzonitrile. Freeze-fracture electron microscopy showed that the plasma membrane below the patterned thickenings of AE F150944-treated tracheary elements was depleted of cellulose-synthase-containing rosettes, which appeared to be inserted intact into the plasma membrane followed by their rapid disaggregation. AE F150944 also inhibited cellulose-dependent growth in the rosette-containing alga, Spirogyra pratensis, but it did not inhibit cellulose synthesis in Acetobacter xylinum or Dictyostelium discoideum, both of which synthesize cellulose via linear terminal complexes. Therefore, AE F150944 may inhibit crystalline cellulose synthesis by destabilizing plasma membrane rosettes.Abbreviations AE F150944 N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine - CBI cellulose biosynthesis inhibiting - CGA CGA 325615, 1-cyclohexyl-5-(2,3,4,5,6-pentafluorophenoxy)-14,2,4,6-thiatriazin-3-amine - DCB 2,6-dichlorobenzonitrile - TE tracheary element     

Factors regulating copper uptake in a cyanobacterium

Copper uptake in the diazotrophic cyanobacteriumNostoc calcicola was found to be typically biphasic, comprising rapid binding of the cations to the cell wall (during the first 10 min) followed by the subsequent metabolism-dependent intracellular uptake for at least 1 h, with a curvilinear kinetics saturating at 40 µM (K_m 25.0 µM, V_max 3.0 nmol Cu mg^–1 protein min^–1). The cellular Cu uptake was light- and ATP-dependent, and the addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea or exogenous ATP proved that the energy to drive Cu transport was derived mainly through PS II reactions. The application of metabolic inhibitors and uncouplers like carbonylcyanidep-nitrofluoromethoxylphenyl hydrazone, N,N-dicyclohexycarbodiimide, azide, and p-chloromercuribenzoate revealed that -SH group(s), proton gradient across the cell membrane, and ATP hydrolysis were involved in the transmembrane movement of Cu inN. calcicola. While monothiol (2-mercaptoethanol) caused a twofold reduction in Cu uptake rate, dithiol (dithiothreitol) contributed towards a further drop in the cation uptake rate.     

Analyses of<Emphasis Type="Italic"> cis</Emphasis> -acting elements that affect the transposition of<Emphasis Type="Italic"> Mos1 mariner</Emphasis> transposons in vivo

The left (5) inverted terminal repeat (ITR) of the Mos1 mariner transposable element was altered by site-directed mutagenesis so that it exactly matched the nucleotide sequence of the right (3) ITR. The effects on the transposition frequency resulting from the use of two 3 ITRs, as well as those caused by the deletion of internal portions of the Mos1 element, were evaluated using plasmid-based transposition assays in Escherichia coli and Aedes aegypti. Donor constructs that utilized two 3 ITRs transposed with greater frequency in E. coli than did donor constructs with the wild-type ITR configuration. The lack of all but 10 bp of the internal sequence of Mos1 did not significantly affect the transposition frequency of a wild-type ITR donor. However, the lack of these internal sequences in a donor construct that utilized two 3 ITRs resulted in a further increase in transposition frequency. Conversely, the use of a donor construct with two 3 ITRs did not result in a significant increase in transposition in Ae. aegypti. Furthermore, deletion of a large portion of the internal Mos1 sequence resulted in the loss of transposition activity in the mosquito. The results of this study indicate the possible presence of a negative regulator of transposition located within the internal sequence, and suggest that the putative negative regulatory element may act to inhibit binding of the transposase to the left ITR. The results also indicate that host factors which are absent in E. coli, influence Mos1 transposition in Ae. aegypti.Communicated by G. P. Georgiev     

The effect of nitrate feeding levels on the pathway of nitrogen incorporation into photosynthesizing leaf metabolism ofDatura stramonium L

Datura stramonium leaves fed K¹⁵NO₃ at 25 g N ml^–1 and 200 g N ml^–1 for 7 and 17 min show that at both time courses the main route of newly reduced nitrogen is to glutamate at the low N-feeding level, and to glutamine at the high N-feeding level. At the high N-feeding level the amido-N of glutamine shows higher¹⁵N enrichment than the amino-N, whereas at the low N-feeding level the opposite is true. Feeding of glutamine (¹⁵N amido labelled) at the 25 and 200 g N ml^–1 level produces¹⁵N enrichment of leaf amino acids, the prime routing being to glutamate. This indicates the operation of glutamate synthase at both feeding levels.It is possible that inDatura stramonium leaves both the glutamate dehydrogenase and glutamine synthetase/GOGAT pathways are simultaneously operative, the former route being favoured at low N-feeding levels, the latter at high N-feeding levels.     

Efficiency of hydrogen photoproduction by photosystem I-enriched subchloroplast vesicles combined with Proteus mirabilis cells. Effects of some exogenous electron donors

High rates of hydrogen photoproduction are obtained when glutaraldehyde-fixed Photosystem I-enriched vesicles (Photosystem II-depleted) are added to hydrogenase-containing cells of Proteus mirabilis in the presence of the mediator methylviologen and a suitable electron donating system. This donor system includes ascorbate, dithioerythritol (DTE) and the mediator tetramethylphenylene-diamine (TMPD) and reduces the photosynthetic electron transfer chain at the level of plastocyanin. Both DTE and ascorbate are required for hydrogen photoproduction, DTE being the ultimate electron donor and ascorbate only having a catalytic function. Whereas the aerobic photoreduction of methylviologen is similar in the presence of DTE, ascorbate or both, under anaerobic conditions only combination of both compounds results in a high and stable amount of reduced methylviologen that can be utilized by the hydrogenase. It is concluded that oxidation reactions of reduced methylviologen, competing with the hydrogenase, rather than methylviologen photoreduction, limit hydrogen photoproduction in the presence of either DTE or ascorbate. These oxidation reactions are suggested to involve back reactions to the oxidized form(s) of ascorbate and DTE but backflow to the photosynthetic electron transfer chain (i.e. cyclic electron transfer) can not be excluded.Abbreviations Tes N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid - DTE 1,4-dithioerythritol - TMPD, N,N,N N-tetramethyl-p-phenylenediamine - DCMU 3-(3, 4-dichlorophenyl)-1, 1,-dimethylureum - EDAC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide - DNP-INT 2-iodo-6-isopropyl-3-methyl-2, 4, 4-trinitrodiphenyl ether - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone - PS photosystem - Chl chlorophyll     

Gramicidin toxicity in NG108-15 cells: protective effects of acetamidine and guanidine

Studies were conducted using a novel in vitro approach to investigate the efficacy of acetamidine hydrochloride (ACE) and guanidine hydrochloride (GUAN), previously shown to block gramicidin D (GRAM) channels in artificial membranes, in preventing the toxic effects of GRAM in NG108-15 (neuroblastoma×glioma hybrid) cells. Specifically, intracellular microelectrode techniques were employed to examine changes in membrane resting potential (V _m) and input resistance (R _in). At 1 mol/L, ACE significantly reduced loss of V _m induced by 1 or 10 g/ml GRAM, although higher concentrations of ACE did not afford enhanced antagonism. GUAN, in contrast, produced a concentration-dependent antagonism of GRAM-induced V _m and R _in loss, with high concentrations (10 or 100 mol/L) completely preventing diminutions in both V _m and R _in. In control cells superfused without GRAM, ACE produced a direct, concentration-dependent reduction in V _m and R _in, whereas GUAN hyperpolarized NG108-15 cells but did not alter R _in. These data represent the initial demonstration of the reversal of GRAM toxicity in an intact cell system.     

URF13, a ligand-gated,pore-forming receptor for T-toxin in the inner membrane ofcms-T mitochondria

URF13 is the product of a mitochondrial-encoded gene (T-urfl3) found only in maize plants containing the Texas male-sterile cytoplasm (cms-T), and it is thought to be responsible for both cytoplasmic male sterility and the susceptibility ofcms-T maize to the fungal pathogensBipolaris maydis race T andPhyllosticta maydis. Mitochondria isolated fromcms-T maize are uniquely sensitive to pathotoxins (T-toxin) produced by these fungi and to methomyl (a commercial insecticide). URF13 acts as a receptor that specifically binds T-toxin to produce hydrophilic pores in the inner mitochondrial membrane. When expressed inEscherichia coli cells, URF13 also forms hydrophilic pores in the plasma membrane if exposed to T-toxin or methomyl. Topological studies established that URF13 contains three membrane-spanning -helices, two of which are amphipathic and can contribute to pore formation. Chemical crosslinking of URF13 was used to demonstrate the existence of URF13 oligomers incms-T mitochondria andE. coli cells. The ability of the carboxylate-specific reagent,N,N-dicyclohexycarbodiimide, to cross-link URF13 was used in conjunction with site-directed mutagenesis to establish that the URF13 tetramer has a central core consisting of a four--helical bundle which undergoes a conformational change after interaction with T-toxin or methomyl. Overall, the experimental evidence indicates that URF13 functions as a ligand-gated, pore-forming T-toxin receptor incms-T mitochondria.     

Intracellular pH inSchizosaccharomyces pombe — Comparison withSaccharomyces cerevisiae

We examined cytoplasmic pH regulation inSchizosaccharomyces pombe andSaccharomyces cerevisiae using pH-sensitive fluorescent dyes. Of several different fluorescent compounds tested, carboxy-seminaphthorhodafluor-1 (C.SNARF-1) was the most effective. Leakage of C.SNARF-1 fromS. pombe was much slower than leakage fromC. cerevisiae. Using the pH-dependent fluorescence of C.SNARF-1 we showed that at an external pH of 7, mean resting internal pH was 7.0 forS. pombe and 6.6 forS. cerevisiae. We found that internal pH inS. pombe was maintained over a much narrower range in response to changes in external pH, especially at acidic pH. The addition of external glucose caused an intracellular alkalinization in both species, although the effect was much greater inS. cerevisiae than inS. pombe. The plasma membrane H⁺-ATPase inhibitor diethylstilbestrol reduced both the rate and extent of alkalinisation, with an IC₅₀ of approximately 35 M in both species. Amiloride also inhibited internal alkalinisation with IC₅₀'s of 745 M forS. cerevisiae and 490 M forS. pombe.Abbreviations C.SNARF-1 carboxy-seminaphthorhodafluor-1 (-AM-acetoxy-methylester) - DES diethylstilbestrol - IC₅₀ apparent inhibitory constant - BCECF 2,7-bis-(carboxyethyl)-5(6)-carboxyfluorescein (-AM--pentaacetoxymethyl ester) - FDA fluorescein diacetate     

Inactivation of Glucose-6-Phosphate Dehydrogenase in Solution by Low- and High-Frequency Ultrasound

We compared the kinetics of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) inactivation in 0.1 M phosphate buffer (pH 7.4) at 36–50° under conditions of exposure to low-frequency (LF, 27 kHz, 60 W/cm²) or high-frequency (HF, 880 kHz, 1.0 W/cm²) ultrasound (USD). The inactivation of G6PDH was characterized by effective first-order rate constants: k _in, total inactivation; k _in ^*, thermal inactivation; and k _in(usd), ultrasonic inactivation. Dilution of the enzyme solution from 20 to 3 nM was accompanied by a significant increase in the values of the three rate constants. The following inequality was valid in all cases: k _in > k _in ^*. The rate constants increased with temperature. The Arrhenius plots of the temperature dependences of k _in and k _in(usd) had an break point at 44°C. The activation energy ( _act) of the total inactivation of G6PDH was higher than _act for the process of ultrasonic inactivation of this enzyme. The two values were found to depend on USD frequency: _act was higher in the case of inactivation with low-frequency ultrasound (LF-USD) than high-frequency ultrasound (HF-USD). The rate of the ultrasonic inactivation of this enzyme substantially decreased in the presence of low concentrations of HO^. radical scavengers (dimethylformamide, ethanol, and mannitol). This fact supports the conclusion that free radicals are involved in the mechanism of G6PDH inactivation in solutions exposed to LF-USD and HF-USD. Ethanol was an effective protector of G6PDH inactivation in solutions exposed to USD.     

Inappropriate annotation of a key defence marker in<Emphasis Type="Italic"> Arabidopsis</Emphasis>: will the real<Emphasis Type="Italic"> PR-1</Emphasis> please stand up?

PR-1 has been extensively used as a marker for salicylic acid (SA)-mediated defence and systemic and local acquired resistance. The Arabidopsis Genome Project annotates At2g19990 as PR-1. This gene is also identified as PR-1 in two full genome Arabidopsis microarrays, and TAIR cites approximately 60 articles to describe its patterns of expression. However, most of these citations are incorrect; the probes used were not At2g19990, but a homologous gene At2g14610, which is annotated as PR-1-like. Because of the potential for confusion, we analyzed the expression of both genes in Arabidopsis thaliana (L.) Heynh. At2g14610 (PR-1-like) showed the archetypal patterns of SA-responsive expression: mRNA levels increased following SA-treatment, inoculation with an avirulent (but not a virulent) strain of Pseudomonas syringae, and in wild-type (but not NahG) Arabidopsis infected with cauliflower mosaic virus (CaMV). In cpr5 mutants it was expressed constitutively. In contrast, expression of At2g19990 (annotated as PR-1) was detectable in neither SA-treated Col-0 nor in cpr5. Infection by virulent and avirulent isolates of P. syringae up-regulated expression, but to a similar level, and infection by CaMV induced a modest increase in expression in both the wild type and NahG. At2g19990, although pathogen responsive, does not show the SA-dependent patterns of expression expected from a member of the PR-1 regulon, and its annotation as PR-1 is inappropriate. The annotations should identify At2g14610 as the authentic PR-1.     

Δ13C and tree-ring width reflect different drought responses in Quercus ilex and Pinus halepensis

Holm oak (Quercus ilex L.) and Aleppo pine (Pinus halepensis Mill) are representative of two different functional types of trees extensively found in the Mediterranean: evergreen sclerophyllous and drought-adapted conifers. The former is considered a partially drought-tolerant species, whereas the latter is a typically drought-avoiding, water-saving species. We postulated that contrasting strategies in response to water deficits in Q. ilex and P. halepensis would lead to a differential sensitivity to changes in water availability. To test this hypothesis, we compared the response of both species in growth rate (measured as radial increments) and intrinsic water use efficiency [WUE_i, as inferred from carbon isotope discrimination (¹³C) in wood samples] among sites from different provenance regions in NE Spain. We found significant differences in ¹³C and growth among provenance regions, partly explained by contrasting water availability. Wood ¹³C was positively related with precipitation and the ratio between precipitation and potential evapotranspiration (P / E). However, these relationships were stronger in P. halepensis (for P / E, r ²=0.67, P <0.001) than in Q. ilex (r ²=0.42, P <0.01). In addition, radial growth was positively related with precipitation and ¹³C in P. halepensis (r ²=0.32 and r ²=0.35, respectively, P <0.01), but not in Q. ilex. We concluded that P. halepensis was more sensitive than Q. ilex to water availability, showing faster increase in WUE_i in response to water stress. We also found that the effect of north/south aspect on ¹³C and growth was site-specific, and unrelated to climatic variables.Due to an error in the citation line, this revised PDF (published in December 2003) deviates from the printed version, and is the correct and authoritative version of the paper.