Effect Of Aminooxyacetic Acid Research Articles

Protective Effects of Aminooxyacetic Acid on Colitis Induced in Mice with Dextran Sulfate Sodium.

As a known inhibitor of pyridoxal phosphate-dependent transaminase glutamic-oxaloacetic transaminase 1 (GOT1), aminooxyacetic acid (AOAA) has been pointed out to have potential pharmacological effects in antiepileptic, anticonvulsant, antibacterial, cancer cell proliferation inhibition, and acute myocardial infarction (MI) relief. However, its role in inflammatory bowel disease (IBD) has not been reported. Through the in vivo experiment of dextran sulfate sodium- (DSS-) induced colitis in mice, it was found that AOAA significantly attenuated the symptoms, signs, and pathological changes of colitis. In addition, AOAA treatment prevented gut barrier damages by enhancing the expression of zona occludens- (ZO-) 1, occludin, claudin-1, and E-cadherin and recovering the upregulation of the most abundant intermediate filament protein (vimentin). Moreover, the release of interleukin- (IL-) 1β, IL-6, and tumour necrosis factor- (TNF-) α was suppressed, yet the level of IL-10 was upregulated by AOAA treatment compared to the model group. Furthermore, it was shown that AOAA administration boosted M2-like phenotype and effectively reduced M1 macrophage phenotype in the lamina propria of mouse colonic epithelium. Similarly, the effect of AOAA was verified in vitro. AOAA effectively inhibited the classically activated M1 macrophage phenotype and proinflammatory cytokine (IL-1β, TNF-α, and IL-6) expression induced by lipopolysaccharide (LPS) and promoted M2-like phenotype. Collectively, this study reveals for the first time that short-term treatment of AOAA can significantly alleviate DSS-induced acute colitis by regulating intestinal barrier function and macrophage polarization, which provides a theoretical basis for the potential use of AOAA in the treatment of IBD.

H2S induces NO in the regulation of AsA-GSH cycle in wheat seedlings by water stress.

In current study, we investigated the relationship between hydrogen sulfide (H2S) and nitric oxide (NO) in the regulation of ascorbate-glutathione (AsA-GSH) cycle in wheat seedlings by water stress. Findings showed that water stress significantly stimulated the production of H2S and NO, the transcript levels and activities of enzymes in AsA-GSH cycle, as well as malondialdehyde (MDA) production and electrolyte leakage, but significantly decreased AsA/DHA and GSH/GSSG. Meanwhile, water stress significantly decreased plant height and dry biomass. Except MDA and electrolyte leakage, above changes induced by water stress were reversed by H2S synthesis inhibitor aminooxyacetic acid (AOA) and NO synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME). However, AOA and L-NAME significantly enhanced MDA production and electrolyte leakage, which further decreased plant height and dry biomass of wheat seedlings under water stress. Application of exogenous H2S donor sodium hydrosulfide (NaHS) to AOA-treated plants and application of exogenous NO donor sodium nitroprusside (SNP) to L-NAME-treated plants reversed above effects of AOA and L-NAME, respectively. Application of L-NAME plus water stress significantly decreased NO production induced by water stress. However, application of L-NAME plus water stress had no obvious influence on H2S production induced by water stress, while application of AOA plus water stress significantly reduced the production of H2S and NO induced by water stress. Current findings suggested that H2S acted upstream of NO in the regulation of AsA-GSH cycle in wheat seedlings by water stress.

Aminooxyacetic acid (AOAA) sensitizes colon cancer cells to oxaliplatin via exaggerating apoptosis induced by ROS.

Background: As the third confirmed gaseous transmitter, the role of hydrogen sulfide (H2S) in the pathogenesis of multiple types of cancer has been attracting increasing attention. Increased expression of cystathionine β-synthase (CBS) and H2S in colon cancer tissue samples has been validated and tumor-derived H2S, mainly produced by CBS, stimulates bioenergetics, cell proliferation, and angiogenesis in colon cancer. Recently, the therapeutic manipulation of H2S has been proposed as a promising anticancer approach. However, the effect of aminooxyacetic acid (AOAA), which has been widely used as an inhibitor of CBS dependent synthesis of H2S, on the chemotherapeutic effect of oxaliplatin (OXA) and the underlying mechanisms remain to be illustrated.Methods: We examined the expression of CBS in human colorectal cancer specimens and matched normal mucosa by immunohistochemistry. The effect of AOAA on the sensitivity of colon cancer cells to OXA and the level of apoptosis induced by caspase cascade was investigated in both HCT116 and HT29 cell lines utilizing CCK-8 assays, flow cytometry analysis and western blot analysis. The endogenous levels of reactive oxygen species (ROS) were detected fluorescently by DCF-DA, and glutathione (GSH) levels were measured by a Total GSH Detection Kit. Tumor bearing xenograft mouse models and in vivo imaging systems were further used to investigate the effect of AOAA in vivo and immunohistochemistry (IHC) and TUNEL analysis were performed.Results: In the current study, we confirmed CBS, the main target of AOAA, is overexpressed in human colorectal cancer by immunohistochemistry. The inhibitory effect of AOAA on the synthesis of H2S was validated utilizing fluorescent probe and specific electrode. AOAA significantly reduced the IC50 values of OXA in both colon cancer cell lines. Co-incubation with AOAA elicited increased apoptosis induced by OXA, featured by increased activation of caspase cascade. Besides, AOAA further increased the levels of ROS induced by OXA and attenuated the synthesis of glutathione (GSH), which is a vital antioxidant. Besides, the results of in vivo imaging and following IHC and TUNEL analysis were in accordance with cellular experiments, indicating that AOAA sensitizes colon cancer cells to OXA via exaggerating intrinsic apoptosis.Conclusion: The results suggested that CBS is overexpressed in colorectal cancer tissues and AOAA sensitizes colon cancer cells to OXA via exaggerating apoptosis both in vitro and in vivo. Decreasing the endogenous level of GSH and consequently impaired detoxification of ROS might be one of the mechanisms underlying the effect of AOAA.

Abstract 3824: Increased production of endogenous hydrogen sulfide (H2S) induced resistance to 5-FU and Oxaliplatin in colon cancer cells

Abstract The combination of 5-FU and Oxaliplatin has been widely adopted as one of the first line chemotherapeutic regimens for colon cancer patients. However, the development of chemoresistance can lead to early recurrence and metastasis to distant organs, thus remaining a leading cause of death from colon cancer. In the present study, the increased production of hydrogen sulfide (H2S) in colon cancer tissues was validated and the effect of inhibition of production of endogenous H2S on the responses of colon cancer cells to 5-FU and Oxaliplatin was further analyzed. The results suggested that inhibition of the enzymatic activity of cystathionine-β-synthase (CBS), a major producer of H2S in colon cancer cells, significantly decreased the IC50s of both 5-FU and Oxaliplatin. Inhibition of CBS leads to decreased PI3K-Akt signaling and consequently decreased expression of thymidylate synthetase (TYMS), which is the target of 5-FU. Inhibition of CBS utilizing aminooxyacetic acid (AOAA) leads to increased production of reactive oxygen species (ROS) and exaggerated mitochondrial damages in colon cancer cells. Analysis performed on CompuSyn confirmed the synergetic effect of AOAA with both 5-FU and Oxaliplatin. In vivo imaging of tumorigenesis in nude mice further confirmed the effect of inhibition of CBS on the therapeutic effect of both 5-FU and Oxaliplatin. Together, these results suggested that inhibition of production of endogenous H2S might be a promising target for chemoresistance in colon cancer. Citation Format: Shanwen Chen, Shuai Zuo. Increased production of endogenous hydrogen sulfide (H2S) induced resistance to 5-FU and Oxaliplatin in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3824.

Effect of aminooxyacetic acid (AOA) on ACS and ACO genes expression and the vase life of alstroemeria cut flower

Aging in the limbs is divided into two categories: ethylene-sensitive limbs, and ethylene-insensitive limbs. Extracellular synthesis of ethylene in the first group is carried out through a specific biochemical pathway that results from flower development and pollination. The present study aims to explore the effects of aminooxyacetic acid (AOA) on the expression of ethylene biosynthesis pathway genes (1-aminocyclopropane-1- carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO)) and the vase life of the Alstroemeria cut flowers ‘Mayfair’ and ‘Frosty’ varieties. Also, the effect of AOA on at the rates of 0, 50, 100 and 150 mg/l was studied on the morphological and physiological traits. The comparison of the vase life of Mayfair and Frosty showed that Frosty had a longer vase life than Mayfair. Frosty exhibited lower levels of cell degradation, higher relative water content, more chlorophyll content, more total soluble solids, and higher peroxidase level than Mayfair. Higher AOA levels were significantly associated with higher activity of catalase and peroxidase, so that the 150 mg/l AOA had the greatest effect on the activity of these two enzymes. Also, the increase in AOArate to 150 mg/l resulted in the retention of leaf chlorophyll and the reduction of leaf chlorosis percentage in both studied varieties.The results indicated that the highest ACO and ACS gene transcripts were related to the 7th flower opening stage. The use of AOA was effective in reducing the gene expression in Frosty. Also, the application of 150 mg/l AOA had a significant reducing effect on the expression of the ACS gene in Frosty.

The roles of H2S and H2O2 in regulating AsA-GSH cycle in the leaves of wheat seedlings under drought stress.

This paper investigated the roles of hydrogen sulfide (H2S) and hydrogen peroxide (H2O2) and the possible relationship between them in regulating the AsA-GSH cycle in wheat leaves under drought stress (DS). Results showed that DS markedly increased the production of H2S and H2O2, the transcript levels and activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR); malondialdehyde (MDA) content; and electrolyte leakage (EL). Meanwhile, DS markedly reduced plant height and biomass. Above increases induced by drought stress except MDA content and EL were all suppressed by pretreatments with H2S synthesis inhibitor aminooxyaceticacid (AOA) and H2O2 synthesis inhibitor diphenylene iodonium (DPI). Besides, pretreatments with AOA and DPI further significantly increased MDA content and EL and significantly reduced plant height and biomass under DS. DPI reduced the production of H2O2 and H2S induced by DS. AOA also reduced the production of H2S and H2O2 induced by DS. Pretreatments with NaHS + AOA and H2O2 + DPI reversed above effects of AOA and DPI. Our results suggested that H2S and H2O2 all participated in the up-regulation of AsA-GSH cycle in wheat leaves by DS and possibly affected each other.

Hydrogen Sulfide is Involved in the Regulation of Ascorbate-glutathione Cycle by Exogenous ABA in Wheat Seedling Leaves under Osmotic Stress

This study investigated the role of hydrogen sulfide (H2S) in the regulation of ascorbateglutathione (AsA-GSH) cycle by exogenous ABA in wheat leaves under osmotic stress. The results showed that osmotic stress significantly increased the activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR), the ratio of reduced ascorbate to oxidized ascorbate (AsA/DHA) and reduced glutathione to oxidized glutathione (GSH/GSSG), the malondialdehyde content and electrolyte leakage, and the H2S content, compared to control. Exogenous ABA significantly increased above indicators under osmotic stress, compared to osmotic stress alone. Above activity increases except MDHAR activity were suppressed by application of H2S scavenger hypotaurine (HT) and synthesis inhibitor aminooxyacetic acid (AOA). Meanwhile, exogenous ABA significantly decreased malondialdehyde content and electrolyte leakage induced by osmotic stress. Application of HT and AOA reversed above effects of application of exogenous ABA. Application of NaHS can reversed above effects of HT and AOA. Our results suggested that H2S induced by exogenous ABA is a signal that leads to the up-regulation of AsA-GSH cycle.

L-cysteine/d,L-homocysteine-regulated ileum motility via system L and B°,+ transporter: Modification by inhibitors of hydrogen sulfide synthesis and dietary treatments

Previous studies including ours demonstrated that l-cysteine treatments decreased motility in gastrointestinal tissues including the ileum via hydrogen sulfide (H2S), which is formed from sulfur-containing amino acids such as l-cysteine and l-homocysteine. However, the amino acid transport systems involved in l-cysteine/l-homocysteine-induced responses have not yet been elucidated in detail; therefore, we investigated these systems pharmacologically by measuring electrical stimulation (ES)-induced contractions with amino acids in mouse ileum preparations. The treatments with l-cysteine and d,l-homocysteine inhibited ES-induced contractions in ileum preparations from fasted mice, and these responses were decreased by the treatment with 2-aminobicyclo[2.2.1]heptane-2-carboxylate (BCH), an inhibitor of systems L and B°,+. The results obtained using ileum preparations and a model cell line (PC12 cells) with various amino acids and BCH showed that not only l-cysteine, but also aminooxyacetic acid and d,l-propargylglycine, which act as H2S synthesis inhibitors, appeared to be taken up by these preparations/cells in L and B°,+ system-dependent manners. The l-cysteine and d,l-homocysteine responses were delayed and abolished, respectively, in ileum preparations from fed mice. Our results suggested that the regulation of ileum motility by l-cysteine and d,l-homocysteine was dependent on BCH-sensitive systems, and varied depending on feeding in mice. Therefore, the effects of aminooxyacetic acid and d,l-propargylglycine on transport systems need to be considered in pharmacological analyses.

Effects of Amino Oxyacetic Acid (AOA) and Sucrose on the Longevity of Bougainvillea Flower Bract

Effects of Amino Oxyacetic Acid (AOA) and Sucrose on the Longevity of Bougainvillea Flower Bract

Stress and salicylic acid induce the expression of PnFT2 in the regulation of the stress-induced flowering of Pharbitis nil

Poor nutrition and low temperature stress treatments induced flowering in the Japanese morning glory Pharbitis nil (synonym Ipomoea nil) cv. Violet. The expression of PnFT2, one of two homologs of the floral pathway integrator gene FLOWERING LOCUS T (FT), was induced by stress, whereas the expression of both PnFT1 and PnFT2 was induced by a short-day treatment. There was no positive correlation between the flowering response and the homolog expression of another floral pathway integrator gene SUPPRESSOR OF OVEREXPRESSION OF CO1 and genes upstream of PnFT, such as CONSTANS. In another cultivar, Tendan, flowering and PnFT2 expression were not induced by poor nutrition stress. Aminooxyacetic acid (AOA), a phenylalanine ammonia-lyase inhibitor, inhibited the flowering and PnFT2 expression induced by poor nutrition stress in Violet. Salicylic acid (SA) eliminated the inhibitory effects of AOA. SA enhanced PnFT2 expression under the poor nutrition stress but not under non-stress conditions. These results suggest that SA induces PnFT2 expression, which in turn induces flowering; SA on its own, however, may not be sufficient for induction.

Increasing plant longevity and associated metabolic events in potted carnation (Dianthus caryophyllus L. Clove Pink)

The effects of aminooxyacetic acid, benzyladenine, and 1-methylcyclopropene treatments on the post-production flower quality of potted carnation plants (Dianthus caryophyllus L. Clove Pink) were investigated considering ethylene production and antioxidant metabolism. Maximum plant longevity (17 days) was obtained using 70 ppb of 1-methylcyclopropene. As compared to control plants, ethylene production was significantly decreased by aminooxyacetic acid at concentrations over 100 mg L-1, benzyladenine at 20 or 30 mg L-1, and 1-methylcyclopropene at 70 and 140 ppb. A significant increase in 1-aminocyclopropane-1-carboxylic-acid concentration was observed in 1-methylcyclopropene treated plants compared with the control ones. On the other hand, decline in 1-aminocyclopropane-1-carboxylic-acid concentration was observed after using 100 or 150 mg L-1 of aminooxyacetic acid. Use of 1-methylcyclopropene (70 or 140 ppb), aminooxyacetic acid (100 or 150 mg L-1), and benzyladenine (20 or 30 mg L-1) significantly decreased H2O2 concentration and superoxide radical when compared with the untreated control. Significant increases in activities of superoxide dismutase, catalase, and peroxidase were noticed when plants were treated with 70 ppb 1-methylcyclopropene. In conclusion, aminooxyacetic acid, benzyladenine (at high concentrations), and 1-methylcyclopropene treatments can be suitable candidates for extending plant longevity, maintaining the visual quality, and reducing the loss of flower anthocyanin.

Involvement of ethylene in growth induction of stationary tobacco pith tissue in vitro

The effects of ethylene on growth initiation of tobacco pith tissue in vitro were investigated. Pith explants were incubated on a double inorganic modified White’s media containing 0.2 mg l−1 kinetin with and without indole-3-acetic acid (IAA) and the ethylene synthesis inhibitor aminooxyacetic acid (AOA). The burst of wound ethylene had no effect on growth initiation, was not affected by the AOA, and decreased to its minimum level during the initial 24 h in culture. Tissue growth was initiated after 72 h and continued on IAA-containing media only. A marked increase in ethylene evolution occurred only in tissues subjected to an IAA-containing medium prior to growth initiation. AOA inhibited this ethylene synthesis and the following growth of the tissues. The initial water uptake by the pith explants occurring even in the absence of IAA was also inhibited by AOA. The metabolic indicators for growth initiation such as enhanced respiration, increased activity of nitrate reductase, and initiation of cathodic isoperoxidases were all inhibited by AOA. It was concluded that the primer function of IAA in growth initiation is via inducing the biosynthesis of a marked ethylene signal, which in the absence of which active growth will not occur. The inhibiting effect of AOA is continuous and a transfer of the pith explants to fresh IAA-containing media did not result in a new ethylene burst nor tissue growth induction. The morphological changes in the tissues and cells during the initial stages of their development on the different media are demonstrated.

Effects of Aminooxyacetic Acid and Sugar on the Longevity of Pollinated Dendrobium Pompadour

Experiments were carried out to investigate the effects of different types and concentration of sugars and Aminooxyacetic acid (AOA) and a combination of both sugar and AOA in extending the vase lives of pollinated Dendrobium Pompadour flowers. Post pollination symptoms such as weight loss, water uptake, petal thickness and discoloration were monitored. The results obtained showed that the most effective treatments in extending the vase life of pollinated Dendrobium Pompadour flowers and delaying the post pollination symptoms were 0.05 mM AOA and the treatment which combined 0.05 mM AOA with 4 glucose. Both treatments showed a longer vase life than that of the control. Furthermore positive effects were also observed in a delay of weight loss, improved water uptake, change in thickness and colour. A combination of 0.05 mM AOA and 2 sucrose extended the vase life of flowers up to 5 days. Inclusion of 4 glucose and 2 sucrose alone only prolonged the vase life of flowers two days more compared to that of the control. Holding solutions containing sugars alone maintained pH values of 5-7 while solutions containing AOA and a combination of AOA and sugars showed more acidic values of pH of 3-5 throughout. AOA and a combination of AOA and glucose can therefore extend the vase life of pollinated Dendrobium Pompadour flowers and also delay the post pollination syndrome. © 2008 Asian Network for Scientific Information.

L-Methionine Toxicity in Freshly Isolated Mouse Hepatocytes Is Gender-Dependent and Mediated in Part by Transamination

L-methionine (Met) has been implicated in parenteral nutrition-associated cholestasis in infants and, at high levels, it causes liver toxicity by mechanisms that are not clear. In this study, Met toxicity was characterized in freshly isolated male and female mouse hepatocytes incubated with 5 to 30 mM Met for 0 to 5 h. In male hepatocytes, 20 mM Met was cytotoxic at 4 h as indicated by trypan blue exclusion and lactate dehydrogenase leakage assays. Cytotoxicity was preceded by reduced glutathione (GSH) depletion at 3 h without glutathione disulfide formation. Exposure to 30 mM Met resulted in increased cytotoxicity and GSH depletion. It is interesting to note that female hepatocytes were resistant to Met-induced cytotoxicity at these concentrations and showed increased cellular GSH levels compared with hepatocytes exposed to medium alone. The effects of amino-oxyacetic acid (AOAA), an inhibitor of Met transamination, and 3-deazaadenosine (3-DA), an inhibitor of the Met transmethylation pathway enzyme S-adenosylhomocysteine hydrolase, on Met toxicity in male hepatocytes were then examined. Addition of 0.2 mM AOAA partially blocked Met-induced GSH depletion and cytotoxicity, whereas 0.1 mM 3-DA potentiated Met-induced toxicity. Exposure of male hepatocytes to 0.3 mM 3-methylthiopropionic acid (3-MTP), a known Met transamination metabolite, resulted in cytotoxicity and cellular GSH depletion similar to that observed with 30 mM Met, whereas incubations with D-methionine resulted in no toxicity. Female hepatocytes were less sensitive to 3-MTP toxicity than males, which may partially explain their resistance to Met toxicity. Taken together, these results suggest that Met transamination and not transmethylation plays a major role in Met toxicity in male mouse hepatocytes.

Effect of aminooxyacetic acid and sugars on the vase life of Dendrobium flowers

We investigated the effect of sugars and aminooxyacetic acid (AOA) on Dendrobium ‘Jew Yuay Tew’ inflorescences bearing open flowers and flower buds. Glucose or sucrose in the vase water had no effect on vase life. AOA alone also did not improve vase life, but AOA together with sugars considerably increased it. The treatment suppressed bud drop, promoted bud opening, and delayed the time to senescence of open flowers. Its effect was counteracted by raising solution pH (which was 3.2) to 4.2 or higher. Bacterial counts in the vase solution were low at pH 3.2, but increased when the pH was raised. The pH of 0.5 mM AOA alone was initially about 3.0, but started to increase by day 8 of vase life and had reached pH 5 by day 12. When 4% glucose was added to this solution, the pH remained around 3 until day 20 of vase life. The combination of 0.5 mM AOA and glucose thus allows continuous uptake of water and dissolved sugars by the flower. It is concluded that the vase life of Dendrobium ‘Jew Yuay Tew’ flowers is limited by adverse water relations and can be improved by sugars if water uptake is maintained. # 2003 Elsevier Science B.V. All rights reserved.

The metabolic pathway of salicylic acid rather than of chlorogenic acid is involved in the stress-induced flowering of Pharbitis nil

We examined the involvement of chlorogenic acid (CGA) and salicylic acid (SA) in the stress-induced flowering of Pharbitis nil (synonym Ipomoea nil). The incorporation efficiency of exogenously applied CGA and the deactivation rate of incorporated CGA were determined in cotyledons by high-performance liquid chromatography. The assay plants could not incorporate a sufficient amount of CGA via roots. The perfusion technique by which the assay solution was forced into the plant from the cut end of the hypocotyl improved the efficiency of CGA incorporation. However, no flower-inducing activity was detected, indicating that CGA was not involved in flowering. It was concluded that the close correlation between CGA content and flowering response is merely coincidence or a parallelism. Flowering under long-day conditions induced by low-temperature stress was completely inhibited by aminooxyacetic acid (AOA), an inhibitor of phenylalanine ammonialyase. The flower-inhibiting effect of AOA was nullified by co-applied t-cinnamic acid and by benzoic acid. This indicates that the metabolic pathway from t-cinnamic acid to SA via benzoic acid is involved in the stress-induced flowering. The results indicate that the metabolic pathway of SA is involved in the stress-induced flowering of P. nil not the metabolic pathway of CGA.

Concentration- and time-dependent effect of aminooxyacetic acid on cortical epileptogenicity.

In the present electrophysiological study the effect of aminooxyacetic acid (AOAA) on the cortical epileptogenicity, and on the basic electro-cortical activity was investigated in anesthetized rats. AOAA did not induce spontaneous epileptiform discharges but modified the somato-sensory evoked responses and the cortical epileptogenicity (induced by 4-aminopyridine) in the same manner depending on its concentration. AOAA at low concentrations increased the amplitude of evoked responses and the ipsilateral manifestation of epileptiform activity, however, at high concentrations significantly suppressed both the evoked responses and the induction and expression of seizures discharges. The anticonvulsive effect of AOAA was time-dependent (reached its maximum after 2h AOAA pre-treatment) and reversible. AOAA at low concentrations probably increases the efficacy of the NMDA excitatory system and decreases GABA-synthesis, resulting neuronal hyperexcitation. However, AOAA at high concentrations can lead to an effective cortical inhibition through intra- and extracellular accumulation of GABA. The gradual GABA accumulation - up to a certain level - at the synapses could also explain the time-dependency of the anticonvulsive effect of AOAA.

Involvement of ethylene in the maturation of black spruce embryogenic cell lines with different maturation capacities.

To examine the possible relationship between ethylene and the capacity of embryogenic cell lines to produce mature somatic embryos of black spruce (Picea mariana (Mill.) B.S.P.), two embryogenic cell lines which exhibit different maturation capacities were used to analyse ethylene biosynthesis and that of its immediate precursor, 1-aminocyclopropane-1- carboxylic acid (ACC). Several compounds known to alter ethylene metabolism were also evaluated for their effect on the number of mature somatic embryos produced. The results showed that in the high capacity cell line, ethylene production and endogenous ACC pools were less than in the low capacity cell line. It was also demonstrated that limiting ethylene biosynthesis by adding inhibitors of ethylene biosynthesis or its physiological action to the maturation medium promoted somatic embryo production for the low capacity cell line. Conversely, lowering ethylene biosynthesis reduced the number of somatic embryos in the high capacity cell line. These results were further substantiated by the finding that the effects of amino-oxyacetic acid (AOA), an inhibitor of ethylene biosynthesis, were partially reversed by adding ethylene to both embryogenic cell lines. It is concluded that ethylene is implicated in somatic embryogenesis of black spruce and that the low capacity cell line had excess, i.e. supraoptimal, ethylene production, whereas the high capacity cell line had nearly optimal ethylene production. The relationship between ethylene and other phytohormones, and the possible effects of the interaction between ethylene and polyamines on the maturation of the somatic embryos are discussed.

Malate-Aspartate Shuttle, Cytoplasmic NADH Redox Potential, and Energetics in Vascular Smooth Muscle

The effect of inhibition of the malate-aspartate shuttle on the cytoplasmic NADH/NAD ratio and NADH redox state and its corresponding effects on mitochondrial energetics in vascular smooth muscle were examined. Incubation of porcine carotid arteries with 0.4 mmol amino-oxyacetic acid an inhibitor of glutamate-oxaloacetate transaminase and, hence the malate-aspartate shuttle, inhibited O2consumption by 21%, decreased the content of phosphocreatine and inhibited activity of the tricarboxylic acid cycle. The rate of glycolysis and lactate production was increased but glucose oxidation was inhibited. These effects of amino-oxyacetic acid were accompanied by evidence of inhibition of the malate-aspartate shuttle and elevation in the cytoplasmic redox potential and NADH/NAD ratio as indicated by elevation of the concentration ratios of the lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate metabolite redox couples. Addition of the fatty acid octanoate normalized the adverse energetic effects of malate-aspartate shuttle inhibition. It is concluded that the malate-aspartate shuttle is a primary mode of clearance of NADH reducing equivalents from the cytoplasm in vascular smooth muscle. Glucose oxidation and lactate production are influenced by the activity of the shuttle. The results support the hypothesis that an increased cytoplasmic NADH redox potential impairs mitochondrial energy metabolism.

Auxin-Induced Epinasty of Tobacco Leaf Tissues (A Nonethylene-Mediated Response).

Interveinal strips (10 x 1.5 mm) excised from growing tobacco (Nicotiana tabacum L. cv Xanthi) leaves curled >300[deg] when incubated for 20 h in 5 to 500 [mu]M [alpha]-naphthalene acetic acid or 50 to 500 [mu]M indole-3-acetic acid. Epinasty was not induced without auxin or by the auxin analog [beta]-naphthalene acetic acid, and less substantial epinasty was induced in midrib and vein segments. Auxin treatment increased the length of both surfaces of strips. Curvature resulted from greater growth on the adaxial side. Epinastic sensitivity of strips to auxin appeared first in the distal third of young leaves (blade 4.5-6.0 cm). In older leaves (8-10 and 12-14 cm), the interveinal tissues throughout were sensitive, whereas in leaves 16- to 18-cm long, sensitivity was reduced in the distal two-thirds. Amino-oxyacetic acid (AOA), an ethylene biosynthesis inhibitor, partially inhibited epinasty at 100 [mu]M. However, a poor correlation between inhibition of ethylene biosynthesis by AOA and its inhibition of curvature and the inability of ethylene to produce epinasty or to reverse the effects of AOA suggests that auxin-induced epinasty is not caused by auxin-induced ethylene production.