Effect of phenylhydrazine on red blood cell metabolism

In addition to the well known effect of phenylhydrazine on red blood cells (methaemoglobin and Heinz body formation, autologous IgG binding, lipid peroxidation, etc.) an increased glucose utilization was observed. Measurement of 14CO2 formation from [1-14C]-glucose showed a maximum value at 2mM phenylhydrazine followed by a progressive inhibition on increasing the drug concentration to 16 mM. Concomitantly we found a reduction in the reduced glutathione concentration but not a corresponding increase in the level of oxidized glutathione. Phenylhydrazine also causes ATP depletion. The ATP is in part dephosphorylated to ADP and AMP and in part converted to inosine monophosphate and hypoxanthine. Measurement of the cell content of reduced and oxidized pyridine nucleotides was also performed and showed a progressive increase in the reduced forms of these coenzymes. Thus phenylhydrazine promotes cellular ATP depletion followed by adenine nucleotide catabolism that is not efficiently counteracted by an increase in glucose utilization. The relevance of these data to the mechanism of phenylhydrazine-induced anemia is discussed.     

Non-statistical isotope fractionation as a novel “retro-biosynthetic” approach to understanding alkaloid metabolic pathways

During the biosynthesis of natural products, isotope fractionation occurs due to the selectivity of enzymes for the heavier or lighter isotopomers. As only some of the positions in the molecule are implicated in a given reaction mechanism, position-specific fractionation occurs. Thus, the position-specific ¹³C/¹²C ratios in these compounds can be related to their known precursors and to the known isotope effects of enzymes involved in their biosynthesis, or similar reaction mechanisms. This can be accessed by isotope ratio monitoring NMR spectrometry. In this short review, how isotope fractionation occurs and when it is manifest is described. Then, the way that ¹³C NMR spectrometry has been applied to study certain aspects of the biosynthesis of the solanaceous alkaloids S-(−)-nicotine and tropine is outlined. Notably, it is shown how similar isotope fractionation is found in the steps of the pathway to the common intermediate, N-methyl-Δ¹-pyrrolinium, but that in the moieties derived from different origins no such similarity is found, the isotopic composition of these atoms reflecting their specific metabolic ancestry. In a second example, tramadol, it is shown how this technique can be used in retro-biosynthesis to give direction as to what precursors and pathway intermediates are probable. It is shown how the observed fractionation in the site-specific ¹³C/¹²C ratios can be effectively explained by known metabolism and the properties of enzymes proposed for the pathway. Furthermore, it can give indications of possible mechanisms of those enzymes that are as yet to be described for a number of key steps.     

Effects of ozone and elevated atmospheric carbon dioxide on carbohydrate metabolism of spruce needles. Catabolic and detoxification pathways

We have studied the effects of ozone, carbon dioxide and ozone combined with carbon dioxide fumigations on catabolic and detoxification pathways in spruce ( Picea abies [L.] Karst.) needles. The results obtained showed an increase in the activities of three enzymes involved in the detoxification pathway, superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (AscPOD, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) when trees were exposed to ozone and to ozone‐carbon dioxide treatments. In these two treatments, the fraction of SOD activity due to the chloroplastic isoform was increased (1.5‐fold). In the needles of trees exposed to ozone and to ozone‐carbon dioxide fumigation, an increase in the activities of glucose‐6‐phosphate dehydrogenase (G‐6‐PDH, EC 1.1.1.49) showed that the cell had the capacity to produce more NADPH necessary for the detoxification. Stimulation of other enzymes of catabolic pathways (fumarase [EC 4.2.1.2], phosphofructokinase [PFK, EC 2.7.1.1] and phosphoenolpyruvate carboxylase [PEPC, EC 4.1.1.31]), was also observed making it possible for the cell to provide the reducing power necessary for detoxification as well as energy and carbon skeletons involved in the repair processes.
When carbon dioxide alone was applied, no effects could be detected on these enzyme activities. However, when carbon dioxide was combined with ozone, the effect of ozone on trees was less than that induced by ozone alone, suggesting that elevated atmospheric carbon dioxide concentrations may to some extent protect plants from ozone injury.     

Characterization of Nicotine-Induced Desensitization of Evoked Dopamine Release from Rat Striatal Synaptosomes

Abstract: Nicotine has been shown to stimulate neurotransmitter release from brain tissue by acting on presynaptic receptors. In this study, the ability of nicotine pretreatment to produce functional desensitization was investigated in rat striatal synaptosomes in which the release of [³H]dopamine was measured with an in vitro superfusion system. Pretreatment of synaptosomes with low concentrations of l -nicotine resulted in a decrease in the ability of a subsequent nicotine challenge to evoke [³H]dopamine release. The IC₅₀ for nicotine-induced desensitization was found to be 12 n M with a maximum inhibition of >90% at 300 n M . Nicotine pretreatment did not affect the release evoked by amphetamine, veratridine, or 15 m M K⁺. The onset of nicotine-induced desensitization occurred with a t _1/2 of 43 s at 30 n M nicotine. The temperature dependence of onset yielded a Q10 of 1.2.Recovery from desensitization was slower ( t _1/2 = 4.33 min), and both the onset and recovery appeared to follow a single first-order process. Several intermittent schedules of nicotine treatment were found to be effective at inducing and maintaining desensitization. The results of this study show that nonstimulating concentrations of nicotine can produce a complete functional desensitization of subsequent nicotine-induced neurotransmitter release.     

Sulfhydryl Modification of Two Nicotinic Binding Sites in Mouse Brain

Two distinct binding sites with properties corresponding to those expected for nicotinic cholinergic receptors can be identified in brain by the specific binding of nicotine (or acetylcholine) and alpha-bungarotoxin. The effects of modification of these binding sites by treatment with the disulfide-reducing agent dithiothreitol were examined in tissue prepared from DBA mouse brains. Treatment with dithiothreitol reduced the binding measured with either ligand, and reoxidization of the disulfides fully restored binding. The effects of dithiothreitol treatment appeared to be due to a reduction in the maximal binding of nicotine and to a decrease in the binding affinity for alpha-bungarotoxin. Agonist affinity for the alpha-bungarotoxin binding site was reduced by treatment with low concentrations of dithiothreitol. The nicotine binding sites remaining after disulfide treatment displayed rates of ligand association and dissociation similar to those of unmodified tissue, but treatment of previously unmodified tissue with dithiothreitol accelerated the rate of nicotine dissociation. After reduction, both binding sites could be selectively alkylated with bromoacetylcholine. The results suggest that both putative nicotinic receptors in brain respond similarly to disulfide reduction and that their responses resemble those known for the nicotinic receptor of electric tissue.     

Arginine deiminase pathway provides ATP and boosts growth of the gas-fermenting acetogen Clostridium autoethanogenum

Acetogens are attractive organisms for the production of chemicals and fuels from inexpensive and non-food feedstocks such as syngas (CO, CO₂ and H₂). Expanding their product spectrum beyond native compounds is dictated by energetics, particularly ATP availability. Acetogens have evolved sophisticated strategies to conserve energy from reduction potential differences between major redox couples, however, this coupling is sensitive to small changes in thermodynamic equilibria. To accelerate the development of strains for energy-intensive products from gases, we used a genome-scale metabolic model (GEM) to explore alternative ATP-generating pathways in the gas-fermenting acetogen Clostridium autoethanogenum. Shadow price analysis revealed a preference of C. autoethanogenum for nine amino acids. This prediction was experimentally confirmed under heterotrophic conditions. Subsequent in silico simulations identified arginine (ARG) as a key enhancer for growth. Predictions were experimentally validated, and faster growth was measured in media containing ARG (t_D~4 h) compared to growth on yeast extract (t_D~9 h). The growth-boosting effect of ARG was confirmed during autotrophic growth. Metabolic modelling and experiments showed that acetate production is nearly abolished and fast growth is realised by a three-fold increase in ATP production through the arginine deiminase (ADI) pathway. The involvement of the ADI pathway was confirmed by metabolomics and RNA-sequencing which revealed a ~500-fold up-regulation of the ADI pathway with an unexpected down-regulation of the Wood-Ljungdahl pathway. The data presented here offer a potential route for supplying cells with ATP, while demonstrating the usefulness of metabolic modelling for the discovery of native pathways for stimulating growth or enhancing energy availability.     

Managing anabolic steroids in pre-hibernating Arctic ground squirrels: obtaining their benefits and avoiding their costs

Androgens have benefits, such as promoting muscle growth, but also significant costs, including suppression of immune function. In many species, these trade-offs in androgen action are reflected in regulated androgen production, which is typically highest only in reproductive males. However, all non-reproductive Arctic ground squirrels, irrespective of age and sex, have high levels of androgens prior to hibernating at sub-zero temperatures. Androgens appear to be required to make muscle in summer, which, together with lipid, is then catabolized during overwinter. By contrast, most hibernating mammals catabolize only lipid. We tested the hypothesis that androgen action is selectively enhanced in Arctic ground squirrel muscle because of an upregulation of androgen receptors (ARs). Using Western blot analysis, we found that Arctic ground squirrels have AR in skeletal muscle more than four times that of Columbian ground squirrels, a related southern species that overwinters at approximately 0°C and has low pre-hibernation androgen levels. By contrast, AR in lymph nodes was equivalent in both species. Brain AR was also modestly but significantly increased in Arctic ground squirrel relative to Columbian ground squirrel. These results are consistent with the hypothesis that tissue-specific AR regulation prior to hibernation provides a mechanism whereby Arctic ground squirrels obtain the life-history benefits and mitigate the costs associated with high androgen production.     

Development of fluorescence imaging probes for nicotinic acetylcholine α4β21 receptors

Nicotinic acetylcholine α4β2¹ receptors (nAChRs) are implicated in various neurodegenerative diseases and smoking addiction. Imaging of brain high-affinity α4β2¹ nAChRs at the cellular and subcellular levels would greatly enhance our understanding of their functional role. Since better resolution could be achieved with fluorescent probes, using our previously developed positron emission tomography (PET) imaging agent [¹⁸F]nifrolidine, we report here design, synthesis and evaluation of two fluorescent probes, nifrodansyl and nifrofam for imaging α4β2¹ nAChRs. The nifrodansyl and nifrofam exhibited nanomolar affinities for the α4β2¹ nAChRs in [³H]cytisine-radiolabeled rat brain slices. Nifrofam labeling was observed in α4β2¹ nAChR-expressing HEK cells and was upregulated by nicotine exposure. Nifrofam co-labeled cell-surface α4β2¹ nAChRs, labeled with antibodies specific for a β2 subunit extracellular epitope indicating that nifrofam labels α4β2¹ nAChR high-affinity binding sites. Mouse brain slices exhibited discrete binding of nifrofam in the auditory cortex showing promise for examining cellular distribution of α4β2¹ nAChRs in brain regions.     

Novel liquid chromatographic–tandem mass spectrometric methods using silica columns and aqueous–organic mobile phases for quantitative analysis of polar ionic analytes in biological fluids

Use of silica stationary phase and aqueous–organic mobile phases could significantly enhance LC–MS–MS method sensitivity. The LC conditions were compatible with MS detection. Analytes with basic functional groups were eluted with acidic mobile phases and detected by MS in the positive ion mode. Analytes with acid functional groups were eluted with mobile phases at neutral pH and detected by MS in the negative ion mode. Analytes poorly retained on reversed-phase columns showed good retention on silica columns. Compared with reversed-phase LC–MS–MS, 5–8-fold sensitivity increases were observed for basic polar ionic compounds when using silica columns and aqueous–organic mobile phase. Up to a 20-fold sensitivity increase was observed for acidic polar ionic compounds. Silica columns and aqueous–organic mobile phases were used for assaying nicotine, cotinine, and albuterol in biological fluids.     

Patterns of induced and constitutive monoterpene production in conifer needles in relation to insect herbivory

Studies were conducted to determine whether herbivore-induced synthesis of monoterpenes occurs in the needles of ponderosa pine (Pinus ponderosa Lawson), lodgepole pine (P. contorta Douglas var. latifolia Engelmann), white fir (Abies concolor Lindl. and Gordon) and Engelmann spruce [Picea engelmanii (Parry) Engelm.]. In the needles of all species except Engelmann spruce, simulated herbivory significantly induced the activity of monoterpene cyclases 4–8 days after wounding. In ponderosa pine, real herbivory by last-instar tiger moth larvae (Halisdota ingens Hy. Edwards: Lepidoptera) induced a significantly larger response (4.5-fold increase in monoterpene cyclase activity) than did simulated herbivory (2.5-fold increase). To our knowledge, this is the first report of herbivore-induced increases in monoterpene synthesis in needle tissue. Despite this increase in monoterpene synthesis, we observed no significant increase in total monoterpene pool size in wounded needles compared to controls. Large increases in the rate of monoterpene volatilization were observed in response to wounding. We conclude that the volatile losses caused by tissue damage compensate for herbivore-induced monoterpene synthesis, resulting in no change in pool size. Tiger moth larvae consume ponderosa pine needles in a pattern that begins at the tip and proceeds downward to midway along the needle, at which point they move to an undamaged needle. Constitutive monoterpene concentrations and monoterpene cyclase activities were highest in the lower half of ponderosa pine needles. The monoterpene profile also differed between the upper and lower needle halves, the lower half possessing an additional one to four monoterpene forms. We propose that the increasing gradient in monoterpene concentrations and number of monoterpenes along the needle from tip to base deters feeding beyond the midway point and provides time for the induction of increased cyclase activity and production of new monoterpenes. The induction of new monoterpene synthesis may have a role in replacing monoterpenes lost through damage-induced volatilization and preventing extreme compromise of the constitutive defense system. Received: 4 June 1997 / Accepted: 2 December 1997