Bifemelane Hydrochloride Protects Against Cytotoxicity of Hydrogen Peroxide on Cultured Rat Neuroblastoma Cell Line

Free radicals are involved in neuronal damage. Bifemelane hydrochloride has been reported to protect neural tissues against ischemic damage and age-related neurodegeneration. We examined the protective effects of bifemelane HCl and the relation between its effectiveness and free radical formation in hydrogen peroxide (H₂O₂)-induced cytotoxicity using cultured rat neuroblastoma cell line (B50). Cytotoxicity was examined by using the lactate dehydrogenase (LDH) assay and cell viability by the WST-1 assay. H₂O₂ reduced the survival of B50 cells in a dose-dependent manner, and treatment of these cells with 75 M or 100 M H₂O₂ reduced their viability by 50% relative to the control group. B50 cells were treated with 5 or 10 M bifemelane for 2 days followed by treatment with 75 M or 100 M H₂O₂. H₂O₂ cytotoxicity was reduced by pretreatment with bifemelane. We also examined the effect of bifemelane on lipid peroxide formation in B50 cells using thiobarbituric acid reactive substances assay. Pretreatment of B50 cells with 10 M bifemelane for 2 days reduced lipid peroxide formation to approximately 54% of the control group. Our results suggest that bifemelane hydrochloride provides a protective effect against H₂O₂ cytotoxicity partly due to its anti-oxidative properties.     

Stimulation of β-carotene synthesis by hydrogen peroxide in Blakeslea trispora

-Carotene synthesis was increased from a negligible amount to 152 mg (g-dry cells)^–1 and H₂O₂ was accumulated up to 16.7 M during 2.5 day-culture of Blakeslea trispora. When cells were cultivated in 250 ml flasks containing various volumes (25–150 ml) of the medium, not only H₂O₂ accumulation but also -carotene synthesis increased as culture volume decreased. Addition of H₂O₂ (10 M) to the 1.5-day old cultures of B. trispora resulted in 46% higher -carotene synthesis than that without addition. All these results indicate that -carotene biosynthesis is stimulated by H₂O₂ in B. trispora.     

Respiration of the hydrogenosome-containing fungus Neocallimastix patriciarum

Mass spectrometric determinations of O₂ affinities by the rumen fungus Neocallimastix patriciarum indicated a stable respiration under liquid phase O₂ concentrations up to 10 M, the apparent K _m for O₂ under these conditions was 4.0 M. Exposure to O₂ concentrations in excess of 10 M resulted in rapid inactivation of the observed respiration. Calculated H₂ evolution rates for the organism are 8.1 nmol min^-1 per mg of protein. Exposure to liquid-phase O₂ concentrations in excess of 1.4 M caused 50% inhibition of H₂ production. That superoxide and peroxide are amongst the products of respiration was shown by the use of ESR spectroscopy with the spin trapping agent 5,5-dimethyl-l-pyrroline-N-oxide. An active superoxide dismutase was present, but catalase could not be detected.Abbreviations ESR electron spin resonance - DMPO 5,5-dimethyl-l-pyrroline-N-oxide - DETAPAC diethylene-triamine pentaacetic acid     

Proton translocation in corn coleoptiles: ATPase or redox chain?

The O₂ dependence of net H⁺ efflux of maize coleoptiles has been investigated. Below 100 M O₂, H⁺ efflux in young (1 cm long) coleoptiles is markedly decreased while old (7 cm long) coleoptiles show a decline only at 10 M O₂. Old coleoptiles show the same decrease in net H⁺ efflux as young ones if treated with fusicoccin. The ratio of alteration of CO₂ production to the change in net proton efflux is about 1:1 at 40–80 M O₂ but not at 10 M O₂. An influx can be observed at 10 M O₂ in young as well as in old coleoptiles if the H⁺ concentration is held at values below pH 6.5. Lower O₂ concentrations lead to an increase of net H⁺ efflux, which might be caused by leaching of organic acids resulting from anaerobic processes, but CO₂ production is not significantly changed at these values. It is proposed that more than one system is responsible for proton translocation across the plasmalemma. One of the systems has a high sensitivity to reduced O₂ concentration which is within the same range as the high K_m of the alternative path.Abbreviation FC fusicoccin     

A catalase microbiosensor for detecting hydrogen peroxide

A microbiosensor for hydrogen peroxide (H₂O₂) was constructed by immobilizing catalase in a polyacrylamide gel on the tip of a Clark-type oxygen microelectrode. The outer tip diameter was 15–40 m. The sensors had response times of 0.7–1.2 s, and could detect as little as 2–4 M H₂O₂. They could measure with a spatial resolution of about 100m and remained operational for up to three weeks.     

Effect of Inhibition of Cyclooxygenase on Pre- and Postjunctional Actions of Peroxides in the Iris-Ciliary Body

In the present study, we investigated the effect of inhibition of cyclooxygenase (COX) with flurbiprofen (FBF) on peroxide-induced enhancement of field-stimulated [³H]norepinephrine ([³H]NE) release from bovine isolated irides. Furthermore, the effect of FBF was examined on peroxide-induced attenuation of contractions evoked by carbachol on this tissue. Irides were prepared for studies of neurotransmitter release and for measurement of contractile tension in vitro. Pretreatment of tissues with FBF (10 M) caused significant (P < 0.001) rightward shifts of concentration-response curves to H₂O₂ and also decreased cumene hydroperoxide (cuOOH)-induced enhancement of evoked [³H]NE release. FBF (10 M) partially prevented the attenuation of carbachol-induced contractions induced by H₂O₂ (300 M) and cuOOH (300 M). We conclude that inhibition of the biosynthesis of prostanoids reduced both the prejunctional stimulatory effects of H₂O₂ and cuOOH on sympathetic neurotransmission and inhibitory effects of peroxides on carbachol-induced contractions the in the bovine isolated iris.     

Enhancement of somatic embryogenesis in orchardgrass leaf cultures by epinephrine

Epinephrine at 10–100 M stimulated somatic embryogenesis from orchardgrass (Dactylis glomerata L.) leaves cultured on SH medium with 30 M of indole-3-acetic acid (IAA). Ethylene emanation was increased at epinephrine concentrations greater than 10 M. Decarboxylation by the leaves of [1-¹⁴C]IAA included in the medium was decreased almost 3-fold by 10 M epinephrine. Epinephrine at 10 M enhanced the number of regenerated plants on SH medium with 30 M dicamba (SH-30). Ethylene emanation was increased by epinephrine concentrations of 500 M and greater included in SH-30 but somatic embryogenesis was decreased. Addition of 8 M CoCl₂, 6H₂O (an ethylene biosynthesis inhibitor) to medium with 500 M epinephrine decreased ethylene emanation to the control level but did not alleviate the decreased embryogenic response.     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Transient state characteristics of adaptation to changes in light conditions for the cyanobacterium Oscillatoria agardhii

Transitions in growth irradiance level from 92 to 7 Em^-2 s^-1 and vice versa caused changes in the pigment contents and photosynthesis of Oscillatoria agardhii. The changes in chlorophyll a and C-phycocyanin contents during the transition from high to low irradiance (HL) were reflected in photosynthetic parameters. In the LH transition light utilization efficiencies of the cells changed faster than pigment contents. This appeared to be related to the lowering of light utilization efficiencies of photosynthesis. As a possible explanation it was hypothesized that excess photosynthate production led to feed back inhibition of photosynthesis. Time-scales of changes in the maximal rate of O₂ evolution were discussed as changes in the number of reaction centers of photosystem II in relation to photosynthetic electron transport. Parameters that were subject to change during irradiance transitions obeyed first order kinetics, but hysteresis occurred when comparing HL with LH transients. Interpretation of first order kinetic analysis was discussed in terms of adaptive response vs changes in growth rate.Non-standard abbreviations Chla chlorophyll a - CPC C-phycocyanin - PS II photosystem II - PS I photosystem I - RC II reaction center of photosystem II - P photosynthetic O₂-evolution - I irradiance, Em^-2 s^-1 - light utilization efficiency of cells, mmol O₂·mg dry wt^-1·h^-1/Em^-2 s^-1 - light utilization efficiency of photosynthetic apparatus, mol O₂·mol Chla ^-1·h^-1/Em^-2 s^-1 - P_max maximal rate of O₂ evolution by cells, mol O₂·mg dry wt^-1·h^-1 - P_max maximal rate of O₂ evolution by photosynthetic apparatus, mol O₂·mol·Chla ^-1·h^-1 - LL low light, E m^-2 s^-1 - HL high light, E m^-2 s^-1 - LH low to high light transition - HL high to low light transition - k specific rate of adaptation, h^-1 - specific growth rate, h^-1 - Q pool size of cell constituent, mol·mg dry wt^-1 - q net synthesis rate of cell constituent, mol·mg dry wt^-1·h^-1     

Binding of [3H]muscimol to calf cerebrocortical synaptic membranes and the effects of sulphur-containing convulsant and non-convulsant compounds

Endogenous and xenobiotic sulphur-containing convulsant and non-convulsant compounds containing structural moieties of, or bearing a structural resemblance to, GABA and homocysteine were tested in binding studies for their potency in displacing the GABA-mimetic [³H]muscimol from specific, high-affinity sites (K _d=3.6 nM;B _max=3.94 pmol/mg protein) on freeze-thawed, Triton-treated calf-brain synaptic membranes. The xenobiotic convulsants, 4-mercaptobutyric acid (MBA), 3-mercaptopropionic acid (3-MPA) and 2-mercaptopropionic acid (2-MPA) were found to be two-site competitive inhibitors exhibiting apparent inhibition affinity constants (K _i ^app ) of 5000 M, 3750 M, and 4800 M, respectively; while homocysteic acid (K _i ^app =4800 M) was shown to be a one-site partial competitive inhibitor. Intermediary metabolites of methionine: S-adenosyl-l-homocysteine,l-cysteine, the convulsantl-homocysteine, and its non-convulsant disulphide oxidation product, homocystine, were found to be one-site partial competitive inhibitors exhibitingK _i ^app values of 5750 M, 8350 M, 5000 M, and 510 M, respectively. The endogenous anticonvulsant neuroeffector, taurine, and the tripeptide, reduced glutathione (GSH) were shown to be, respectively, one-site (K _i=20 M) and two-site (K _i ^app =4300 M) competitive inhibitors of [³H]muscimol binding. These findings are discussed with regard to a previously proposed mechanism for the convulsant action of homocysteine.     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.     

Regeneration from long-term cell suspension cultures of tepary bean (Phaseolus acutifolius)

Plant regeneration has been achieved from long-term cell suspension cultures established from leaf derived callus of tepary bean (Phaseolus acutifolius). The proportion of densely cytoplasmic cells in suspension culture increased when cultured in the L-6 medium with 54 M NAA and 2 M KN. Filtration of the cells at each of five consecutive subcultures resulted in the isolation of a plant regenerating cell line (TB 686), which is being maintained in L-6 medium with 4.5 M 2,4-D and 2.3 M zeatin. Differentiated green cell aggregates were obtained when cells from maintenance medium were transferred to the same medium with 10 M BA. Embryo-like structures developed from these aggregates on L-6 medium with 2.3 M zeatin, 0.69 M GA₃ and 1.5 M NAA. Plantlets regenerated from these structures when they were cultured on L-6 medium with 7.0 M NAA and 1.0 M KN. Plant regeneration from the cell line remained relatively constant for 270 days. Regenerated plants were grown to maturity in the greenhouse.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IPA Isopentenyladenine - KN Kinetin - NAA Naphthaleneacetic acid - AA Amino acid medium (Toriyama and Hinita, 1985) The research was sponsored by United States Agency for International Development, Washington D.C., Cooperative Agreement DAN-4137-A-00-4053-00     

Characterization of Mg2+-ATPase activity in isolated B16 murine melanoma melanosomes

In view of the accumulation of H₂O₂ in the myocardium due to ischemia-reperfusion and changes in -adrenoceptor mechanisms in the ischemic-reperfused heart, we investigated the effects of H₂O₂ on the -adrenoceptor, G-protein and adenylyl cyclase complex. Rat hearts were perfused with 1 mM H₂O₂ for 10 min before isolating membranes for measuring the biochemical activities. The stimulation of adenylyl cyclase by different concentrations of isoproterenol was depressed upon perfusing hearts with H₂O₂. Both the affinity and density of ₁-adrenoceptors as well as the density of the ₂-adrenoceptors were decreased whereas the affinity of ₂-adrenoceptors was increased by H₂O₂ perfusion. Competition curves did not reveal any effect of H₂O₂ on the proportion of coupled receptors in the high affinity state. The basal as well as forskolin-, NaF- and Gpp(NH)p-stimulated adenylyl cyclase activities were depressed by perfusing the heart with H₂O₂. Catalase alone or in combination with mannitol was able to significantly decrease the magnitude of alterations due to H₂O₂. The positive inotropic effect of 1 M isoproterenol was markedly attenuated upon perfusing hearts with 200-500 M H₂O₂ for 10 min. These results suggest that H₂O₂ may depress the ₁-adrenoceptor, G_s-proteins and catalytic subunit of the adenylyl cyclase enzyme and thus may play an important role in attenuating the -adrenoceptor linked signal transduction due to ischemia-reperfusion injury.     

Clozapine and Several Other Antipsychotic/Antidepressant Drugs Preferentially Block the Same ‘Core’ Fraction of GABAA Receptors

Clozapine and several other antipsychotic/antidepressant drugs that fully or partially block GABA_A receptors were tested at concentrations that reversed the inhibitory effect of 1 M GABA on ³⁵S-t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding to rat forebrain membranes only about 20–30%, here designated core fractions. Clozapine at 10 M reverses 1 M GABA 25 ± 4.0% (n = 23) (its core fraction). Fourty three compounds were tested alone, and pairwise together with 10 M Clozapine. The core fractions of some of the compounds yielded significant additive reversals together with 10 M Clozapine, while others did not. A group of 14 compounds of which 7 are clinically effective antipsychotic drugs, including Chlorprothixene, Clomacran, Clopipazan, Fluotracen, Sulforidazine, Thioproperazine, and cis-Thiothixene, were statistically non-additive with 10 M Clozapine, suggesting that all of these drugs selectively block the same core population of GABA_A receptors as Clozapine. These non-additivities also suggest that Clozapine at 10 M fully saturates a subset of GABA_A receptors blocked by 1 M GABA. Therefore, Clozapine probably blocks 2 or more types of GABA_A receptors, but only half of the receptors that are sensitive to 1 M GABA. A second group of 12 compounds of which 6 are clinically active antidepressant/antipsychotic drugs including Amoxapine, Clothiapine, Dibenzepine, Inkasan (Metralindole), Metiapine and Zimelidine were slightly, but significantly, additive with Clozapine suggesting that these compounds block most of Clozapine's core fraction, plus a small additional fraction. A third group consisted of ten compounds that yielded larger (R > 80) and statistically highly significant additivities with Clozapine. Complete additivity was obtained with Bathophenanthroline disulfonate, and Isocarboxazid, suggesting that they block GABA_A receptors other than those blocked by 10 M Clozapine. Seven classical GABA_A receptor blockers, also tested at concentrations yielding 21 to 33% reversal alone, were all significantly additive with 10 M Clozapine, but in no case was the additivity complete. The largest additivity was obtained with Pitrazepine (21%) and the smallest with Tubocurarine (9%). These results provide further support for the notion that selective blockade of the same subset of GABA_A receptors may contribute to the clinical antipsychotic/antidepressant effects of Clozapine. The B_opt values for Clozapine are 50 ± 1.7% and 26 ± 2.6% ( n = 3) in whole rat forebrain and cerebellum, respectively, confirming that clozapine-sensitive GABA_A receptors are unevenly distributed in the brain. The sedative and anxiolytic properties of Clozapine and other antipsychotic drugs may be due to selective blockade of GABergic disinhibition at certain interneurons.     

Modulation of specific [3H]phenytoin binding by benzodiazepines

We have shown that diazepam (ED₅₀ 2.4 M), flunitrazepam (ED₅₀ 10.2 M) and Ro5-4864 (ED₅₀ 5 M) are able to enhance both total and specific [³H]phenytoin binding. Picrotoxin (IC₅₀ 1.43 M) and chloride, either NaCl or KCl (IC₅₀ 42.4 M) inhibit both the increase in total and specific binding of [³H]phenytoin, Ro 15-1788 does not. The optimum time for this enhancement was 3–4 hours. While the ED₅₀'s for the benzodiazepines are high their order of potency suggests that an involvement of both the peripheral type benzodiazepine receptor and the GABA-chloride ionophore complex is likely. Clonazepam (IC₅₀ 23 M), oxazepam (IC₅₀ 12 M) chlordiazepoxide (IC₅₀ 35 M) and Ro8682-10, a convulsant benzodiazepine (IC₅₀ 16 M) all inhibit both total and specific [³H]phenytoin binding. These effects were not blocked by chloride ions, picrotoxin or Ro 15-1788, and reached equilibrium within 45 minutes. This order of potency also parallels that for the peripheral benzodiazepine receptor in rat brain. These data suggest the presence of a micromolar benzodiazepine receptor site which may play a role in the control of CNS excitability. Nitrazepam, medazepam, bromazepam and the tetralobenzodiazepines U38335, U42794, U43434, and U37834 had no effect on total or specific [³H]phenytoin binding nor on the actions of the other benzodiazepines described in concentrations up to 50 M.     

Antioxidant Properties of New Chalcogenides Against Lipid Peroxidation in Rat Brain

Ebselen (2-phenyl- 1,2-benzisoselenazole-3 (2H)-one) is a seleno-organic compound with antioxidant properties, and anti-inflammatory actions. Recently, ebselen improved the outcome of acute ischemic stroke in humans. In the present study, the potential antioxidant capacity of organochalcogenide compounds diphenyl diselenide (PhSe)₂, diphenyl ditelluride (PhTe)₂, diphenyl disulfide (PhS)₂, p-Cl-diphenyl diselenide (pCl-PhSe)₂, bis-[S-4-isopropyl 2-phenyl oxazoline] diselenide (AA-Se)₂, bis-[S-4-isopropyl 2-phenyl oxazoline] ditelluride (AA-Te)₂ and bis-[S-4-isopropyl 2-phenyl oxazoline] disulfide (AA-S)₂ was compared with that of ebselen (a classical antioxidant). Spontaneous and quinolinic acid (QA)- (2 mM) and sodium nitroprusside (SNP)- (5 M)-induced thiobarbituric reactive species (TBARS) production by rat brain homogenates was determined colorimetrically. TBARS formation was reduced by ebselen, (PhSe)₂, (PhTe)₂, (AA-Se)₂, (AA-S)₂ and (pCl- PhSe)₂ to basal rates. The concentrations of these compounds needed to inhibit TBARS formation by 50% (lC₅₀) are 1.71 M, 3.73 M, 1.63 M, 9.85 M, > 33.3 M, 23.2 M and 4.83 M, respectively for QA. For TBARS production induced by SNP the lC₅₀ was 2.02 M, 12.5 M, 2.80 M, > 33.3 M, 24.5 M and 7.55 M, respectively. The compounds (AA-Te)₂ and (PhS)₂ have no antioxidant activity and pro-oxidant activity, respectively. These results suggest that (AA-Se)₂ and (AA-S)₂ can be considered as potential pharmaceutical antioxidant agents.     

A lyophilized aqueous extract of<Emphasis Type="Italic"> Maytenus ilicifolia</Emphasis> leaves inhibits histamine-mediated acid secretion in isolated frog gastric mucosa

Lyophilized aqueous extract of Maytenus ilicifolia leaves (LAEMIL) is commonly used in Brazilian folk medicine in the treatment of dyspepsia as well as gastric ulcers. We have investigated the effect and the possible mechanism of action of the LAEMIL on acid secretion in isolated frog gastric mucosa incubated in an Ussing chamber. It was observed that LAEMIL (7–28 mg%) as well as cimetidine (125–4,000 M), a well-known histamine H₂ receptor antagonist, decreased basal acid secretion in a concentration-dependent manner. Similarly to cimetidine (190 M), LAEMIL (21 mg%) also inhibited gastric acid secretion induced by increasing concentrations of histamine (50–800 M). The EC₅₀ values for histamine alone and histamine in the presence of LAEMIL or cimetidine were 94.6 M (71.1–125.9 M), 244.9 M (209.4–286.4 M) and 142.2 M (23.6–855.0 M), respectively. LAEMIL, histamine and cimetidine were effective on acid secretion only when added to the serosal surface of the mucosa. Furthermore, simultaneous addition of LAEMIL and cimetidine at concentrations, per se, ineffective, caused a 16% reduction in the basal acid secretion [from 8.3±0.3 to 6.9±0.2 Eq g^–1 (15 min)^–1, n=4]. Although effects such as inhibition of histamine biosynthesis and/or histamine release can not be ruled out, our data suggest that LAEMIL, like cimetidine, reduces acid secretion in the isolated frog gastric mucosa by antagonising histamine H₂ receptors.Abbreviations LAEMIL Lyophilized aqueous extract of Maytenus ilicifolia leaves - Hist Histamine - Cim Cimetidine     

Calcium control of differentiation in Phytophthora palmivora

A method has been developed for the preparation of zoospores from Phytophthora palmivora which allows the ionic composition of the suspension medium to be closely controlled. Sub-micromolar concentrations of calcium ions have been shown to play a key role in maintaining the zoospore state and in the transition to the cyst stage. Restriction of free Ca²⁺ to between 0.2 and 1 M resulted in zoospores which could be maintained for several hours before they finally encysted and germinated. When exposed to citrus-pectin, or 3 mM SrCl₂, or to vigorous shaking, these zoospores underwent rapid synchronous encystment. At free Ca²⁺ concentrations below 0.1 M, zoospores lysed slowly. If exposed to inducers of encystment before lysis had occurred, the zoospores failed to respond to pectin or to vigorous shaking. However, they did differentiate in response to SrCl₂ addition. Provided the free Ca²⁺ was maintained between 0.02 and 0.2 M, zoospores survived gentle centrifugation, a procedure which previously had resulted in encystment.Abbreviations IM (ion-mix) release medium containing 100 M KCl, 10 M CaCl₂, and 10 M MgCl₂     

Interaction of Cytokinins and Abscisic Acid During Regulation of Stomatal Opening in Bean Leaves

Effects of benzyladenine (BA) and abscisic acid (ABA) applied separately or simultaneously on parameters of gas exchange of Phaseolus vulgaris L. leaves were studied. In the first two experimental sets) 100 M ABA and 10 M BA were applied to plants sufficiently supplied with water. Spraying of leaves with ABA decreased stomatal conductance (g _s) and in consequence transpiration rate (E) and net photosynthetic rate (P _N) already 1 h after application, but 24 h after application the effect almost disappeared. 10 M BA slightly decreased gas exchange parameters, but in simultaneous application with ABA reversed the effect of ABA. Immersion of roots into the same solutions markedly decreased gas exchange parameters and 24 h after ABA application the stomata were completely closed. The effect of ABA was ameliorated by simultaneous BA application, particularly after 1-h treatment. In the third experimental set, plants were pre-treated by immersing roots into water, 1 M BA, or 100 M ABA for 24 h and then the halves of split root system were dipped into different combinations of 1 M BA, 100 M ABA, and water. In plants pre-treated with ABA all gas exchange parameters were small and they did not differ in plants treated with H₂O+H₂O, H₂O+BA, or BA+BA. In plants pre-treated with BA or H₂O, markedly lower values of P _N were found when both halves of roots were immersed in ABA. Further, the effects of pre-treatment of plants with water, 1 M BA, 100 M ABA, or ABA+BA on the development of water stress induced by cessation of watering and on the recovery after rehydration were followed. ABA markedly decreased gas exchange parameters at the beginning of the experiment, but in its later phase the effect was compensated by delay in development of water stress. BA also delayed development of water stress and increased P _N in water-stressed leaves. BA reversed the effect of ABA at mild water stress. Positive effects of BA and ABA pre-treatments were observed also after rehydration.     

CR (VI) induces cell growth arrest through hydrogen peroxide‐mediated reactions

Cr (VI) compounds are widely used in industries and are recognized human carcinogens. The mechanism of carcinogenesis associated with these compounds is not well understood. The present study focused on Cr (VI)induced cell growth arrest in human lung epithelial A549 cells, using flow cytometric analysis of DNA content. Treatment of the cells with Cr (VI) at 1 M caused a growth arrest at G₂/M phase. An increase in Cr (VI) concentration enhanced the growth arrest. At a concentration of 25 M, Cr (VI)induced apoptosis became apparent. Superoxide dismutase (SOD) or sodium formate did not alter the Cr (VI)induced cell growth arrest. While catalase inhibited growth, indicating H₂O₂ is an important mediator in Cr (VI)induced G₂/M phase arrest. Electron spin resonance (ESR) spin trapping measurements showed that incubation of cells with Cr (VI) generated hydroxyl radical (OH). Catalase inhibited the OH radical generation, indicating that H₂O₂ was generated from cells stimulated by Cr (VI), and that H₂O₂ functioned as a precursor for OH radical generation. The formation of H₂O₂ from Cr (VI)stimulated cells was also measured by the change in fluorescence of scopoletin in the presence of horseradish peroxidase. The mechanism of reactive oxygen species generation involved the reduction of molecular oxygen as shown by oxygen consumption assay. These results support the following conclusions: (a) Reactive oxygen species are generated in Cr (VI)stimulated A549 cells through reduction of molecular oxygen, (b) Among the reactive oxygen species generated, H₂O₂ played a major role in causing G₂/M phase arrest in human lung epithelial cells.