Using of metal-affinity chromatography for identification of alkylated rat globin

Rat globin peptides alkylated by sulfur mustard on amino-acid residues C-126, C-93 and E-27 with MH+ 1444.62 Da, 1561.66 Da, 1676.78 Da, respectively, were concentrated using metal-affinity chromatography on Cu2+. The peptides were received by trypic digestion after in vitro incubation of rat globin with 60 microM HD. Aklylated peptide with MH+ 1444.62 Da is the most sensitive biomarker, which can be concentrated from globin trypic digest, incubated with 3 microM sulfur mustard.     

The use of human epidermal keratinocytes in culture as a model for studying the biochemical mechanisms of sulfur mustard toxicity

Human epidermal keratinocytes in culture were studied to evaluate their usefulness in demonstrating toxic events following exposure to sulfur mustard. Exposure of keratinocytes to sulfur mustard over a concentration range of 1–1000 μM HD, reduced NAD+ levels from 96% to 32% of control levels. When keratinocytes were exposed to a concentration of 300 μM HD, NAD+ levels began to fall at 1 hour and reached a plateau of 47% of control levels at 4 hours. Niacinamide, an inhibitor of the enzyme poly(ADP-ribose) polymerase, partially protected mustard-exposed cells against NAD+ depletion. It also protected cellular viability as assessed by vital staining 24 hours after exposure. This protection was not seen in long-term (72 hr) cultures. These studies suggest that human epidermal keratinocytes in culture can serve as a usefulin vitro model for research into the biochemical mechanisms of sulfur mustard-induced cutaneous injury.     

Evidence for Involvement of a Zymogen Granule Na+/H+ Exchanger in Enzyme Secretion from Rat Pancreatic Acinar Cells

We have characterized a Na⁺/H⁺ exchanger in the membrane of isolated zymogen granules (ZG) from rat exocrine pancreas and investigated its role in secretagogue-induced enzyme secretion. ZG Na⁺/H⁺ exchanger activity was estimated by measuring Na⁺ or Li⁺ influx and consequent osmotic swelling and lysis of ZG incubated in Na- or Li-acetate. Alternatively, intragranule pH was investigated by measuring absorbance changes in ZG which had been preloaded with the weak base acridine orange. Na⁺- or Li⁺-dependent ZG lysis was enhanced by increasing inward to outward directed H⁺ gradients. Na⁺-dependent ZG lysis was not prevented by an inside-positive K⁺ diffusion potential generated by valinomycin which argues against parallel operation of separate electrogenic Na⁺ and H⁺ permeabilities and for coupled Na⁺/H⁺ exchange through an electroneutral carrier. Na⁺- and Li⁺-dependent ZG lysis was inhibited by EIPA (EC₅₀∼25 μm) and benzamil (EC₅₀∼100 μm), but only weakly by amiloride. Similarly, absorbance changes due to release of acridine orange from acidic granules into the medium were obtained with Na⁺ and Li⁺ salts only, and were inhibited by EIPA, suggesting the presence of a Na⁺/H⁺ exchanger in the membrane. Na⁺ dependent lysis of ZG was inhibited by 0.5 mm MgATP and MgATP-γ-S by about 60% and 35%, respectively. Inhibition by MgATP was prevented by incubation of ZG with alkaline phosphatase (100 U/ml), or by the calmodulin antagonists calmidazolium (0.75 μm), trifluoperazine (100 μm) and W-7 (500 μm), suggesting that the ZG Na⁺/H⁺ exchanger is regulated by a ZG membrane-bound calmodulin-dependent protein kinase. Na⁺ dependence of secretagogue (CCK-OP)-stimulated amylase secretion was investigated in digitonin permeabilized rat pancreatic acini and was higher in acini incubated in Na⁺ containing buffer (30 mm NaCl/105 mm KCl buffer; 6.4 ± 0.4% of total amylase above basal) compared to buffer without Na⁺ (0 mm NaCl/135 mm KCl buffer; 4.7 ± 0.4% of total amylase above basal, P < 0.03). EIPA (50 μm) reduced CCK-OP-induced amylase secretion in Na⁺ containing buffer from 7.5 ± 0.6% to 4.1 ± 0.8% (P < 0.02). In the absence of Na⁺ in the buffer, CCK-OP-stimulated amylase release was not inhibited by 50 μm EIPA. The data suggest that an amiloride insensitive, EIPA inhibitable Na⁺/H⁺ exchanger is present in ZG membranes, which is stimulated by calmodulin antagonists and could be involved in secretagogue-induced enzyme secretion from rat pancreatic acini. Received: 7 December 1995/Revised: 2 April 1996     

Optimization of culture conditions for determining hepatobiliary disposition of taurocholate in sandwich-cultured rat hepatocytes

Summary This study was undertaken to examine the influence of time and volume of collagen overlay, type of media, and media additives on taurocholate (TC) accumulation and biliary excretion in hepatocytes cultured in a collagen-sandwich configuration. Hepatocytes were isolated from male Wistar rats by in situ perfusion with collagenase, seeded onto collagencoated 60-mm dishes, overlaid with gelled collagen, and cultured for 4 d. Experiments to examine the influence of time and volume of collagen overlay were conducted in Dulbecco's modified Eagle's medium (DMEM)+1.0μM dexamethasone (DEX)+5% fetal bovine serum (FBS). Hepatocytes were overlaid at 0 h with 0.1 or 0.2 ml collagen, or at 24 h with 0.1 or 0.2 ml collagen. The influence of media type and additives was examined in hepatocytes overlaid at 0 h with 0.2 ml collagen and incubated in DMEM+0.1μM DEX, DMEM+0.1μM DEX+5% FBS, Williams' medium E+0.1μM DEX+1% ITS^Θ+, DMEM +1.0μM DEX, DMEM+1.0 μM DEX+5% FBS, or modified Chee's medium (MCM)+0.1 μM DEX+1% ITS^Г+. [³H] TC accumulation by hepatocytes in Hank's balanced salt solution (HBSS) and Ca²⁺-free HBSS was measured, and the biliary-excretion index (BEI: percentage of accumulated TC localized in the canalicular compartment) was calculated. Light microscopy and carboxydichlorofluorescein fluorescence were employed to examine the cellular and canalicular morphologies. The volume of collagen used for both the substratum and the overlay did not affect TC accumulation or biliary excretion. The BEI tended to be higher in cells overlaid at 24 h (BEI=0.649 [0.1 ml collagen]; BEI=0.659 [0.2 ml collagen]) compared with those overlaid at 0 h after seeding (BEI=0.538 [0.1 ml collagen]; BEI=0.517 [0.2 ml collagen]), although the differences were not statistically significant. Hepatocytes cultured in MCM produced consistently the lowest BEI of TC (BEI=0.396). Differing DEX concentration (0.1 μM versus 1.0 μM) with or without 5% FBS did not appear to have a significant effect on the BEI of TC.     

Glutathione reductase fromSaccharomyces cerevisiae undergoes redox interconversionin situ andin vivo

Redox interconversion of glutathione reductase was studiedin situ withS. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 μM NADPH or 100 μM NADH caused 50% inactivation in normal cells in 30 min, 0.75 μM NADPH or 50 μM NADH promoted a similar effect in starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP⁺ concentrations. The inactivation of yeast glutathione reductase has also been studiedin vivo. The activity decreased to 75% after 2 hours of growth with glucono-δ-lactone as carbon source, while NADPH rose to 144% and NADP⁺ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 μM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD⁺ and NADPH/NADP⁺ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-δ-lactone is oxidized promotesin vivo inactivation of glutathione reductase.     

Purification and characterization of a calcium-dependent protein kinase from beetroot plasma membranes

Several calcium-dependent protein kinases (CDPKs) are located in plant plasma membranes where they phosphorylate enzymes and transporters, like the H⁺-ATPase and water channels, thereby regulating their activities. In order to determine which kinases phosphorylate the H⁺-ATPase, a calcium-dependent kinase was purified from beetroot (Beta vulgaris L.) plasma membranes by anion-exchange chromatography, centrifugation in glycerol gradients and hydrophobic interaction chromatography. The kinetic parameters of this kinase were determined (V _max: 3.5 μmol mg⁻¹ min⁻¹, K _m for ATP: 67 μM, K _m for syntide 2: 15 μM). The kinase showed an optimum pH of 6.8 and a marked dependence on low-micromolar Ca²⁺ concentrations (K _d : 0.77 μM). During the purification procedure, a 63-kDa protein with an isoelectric point of 4.7 was enriched. However, this protein was shown not to be a kinase by mass spectrometry. Kinase activity gels showed that a 50-kDa protein could be responsible for most of the activity in purified kinase preparations. This protein was confirmed to be a CDPK by mass spectrometry, possibly the red beet ortholog of rice CDPK2 and Arabidopsis thaliana CPK9, both found associated with membranes. This kinase was able to phosphorylate purified H⁺-ATPase in a Ca²⁺-dependent manner.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

<Emphasis Type="Italic">In vitro</Emphasis> selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (<Emphasis Type="Italic">Dendrocalamus strictus</Emphasis> Nees)

Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l⁻¹ (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l⁻¹ (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na⁺, sugar, free amino acids, and proline but a slight decline was recorded in K⁺ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l⁻¹ (9.0 μM) 2,4-D+0.5 mg l⁻¹ (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l⁻¹ (0.1 μM) α-naphthaleneacetic acid +0.1 mg l⁻¹ (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.     

Response of the plant root to aluminum stress: Analysis of the inhibition of the root elongation and changes in membrane function

Pea root elongation was strongly inhibited in the presence of a low concentration of Al (5 μM). In Al-treated root, the epidermis was markedly injured and characterized by an irregular layer of cells of the root surface. Approximately 30% of total absorbed Al accumulated in the root tip and Al therein was found to cause the inhibition of whole root elongation. Increasing concentrations of Ca²⁺ effectively ameliorated the inhibition of root elongation by Al and 1 mM of CaCl₂ completely repressed the inhibition of root elongation by 50 μM Al. The ameliorating effect of Ca²⁺ was due to the reduction of Al uptake. H⁺-ATPase and H⁺-PPase activity as well as ATP and PPidependent H⁺ transport activity of vacuolar membrane vesicles prepared from barley roots increased to a similar extent by the treatment with 50 μM AlCl₃. The rate of increase of the amount of H⁺-ATPase and H⁺-PPase was proportional to that of protein content measured by immunoblot analysis with antibodies against the catalytic subunit of the vacuolar H⁺-ATPase and H⁺-PPase of mung bean. The increase of both activities was discussed in relation to the physiological tolerance mechanism of barley root against Al stress.     

Energization of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> membrane vesicles increases catalytic activity of succinate: Menaquinone oxidoreductase

In this work, high ΔμH⁺-dependent succinate oxidase activity has been demonstrated for the first time with membrane vesicles isolated from Bacillus subtilis. The maximal specific rate of succinate oxidation by coupled inside-out membrane vesicles isolated from a B. subtilis strain overproducing succinate:menaquinone oxidoreductase approaches the specific rate observed with the intact cells. Deenergization of the membrane vesicles with ionophores or alamethicin brings about an almost complete inhibition of succinate oxidation. An apparent K _m for succinate during the energy-dependent succinate oxidase activity of the vesicles (2.2 mM) is higher by an order of magnitude than the K _m value measured for the energy-independent reduction of 2,6-dichlorophenol indophenol. The data reveal critical importance of ΔμH⁺ for maintaining active electron transfer by succinate:menaquinone oxidoreductase. The role of ΔμH⁺ might consist in providing energy for thermodynamically unfavorable menaquinone reduction by succinate by virtue of transmembrane electron transport within the enzyme down the electric field; alternatively, ΔμH⁺ could play a regulatory role by maintaining the electroneutrally operating enzyme in a catalytically active conformation.     

Dose-related influence of sodium selenite on apoptosis in human thyroid follicles in vitro induced by iodine, EGF, TGF-β, and H2O2

Apoptosis of thyroid follicular cells is induced by high doses of iodide, epidermal growth factor (EGF), transforming growth factor-β (TGF-β), as well as H₂O₂ and might be attenuated by antioxidants. Therefore, we examined the apoptotic index induced by these substances in selenium-treated vs untreated human thyroid follicular cells. Reconstituted human thyroid follicles were incubated with sodium selenite (10 or 100 nM) for 72 h; controls received none. The follicles were then distributed to 24-well plates and incubated with potassium iodide (5, 10, or 20 nM), EGF (5 ng/mL), TGF-β (5 ng/mL), or H₂O₂ (100 μM). Apoptosis was determined by a mitochondrial potential assay and the number of apoptotic cells counted by two independent, experienced technicians and the glutathione peroxidase (GPx) activity was determined. A significant increase of apoptic cells was obtained in control thyroid follicles treated with iodine (5, 10, or 20 μM), thyroid-stimulating hormone (TSH) 1, or 10 mU/mL in combination with 5 and 10 μM iodine, EGF (5 ng/mL) and TGF-β (5 ng/mL), or H₂O₂ (100 μM) (p<0.001). In contrast, in thyroid follicles preincubated with 10 or 100 nM sodium selenite, the apoptototic index was identical to the basal rate. In H₂O₂-treated follicles, the apoptotic index was still significantly elevated but 50% lower compared to control cells. The GPx activity increased from 1.4±0.2 to 2.25±0.4 mU/μg DNA with 10 nM selenite and 2.6+0.4 mU/μg DNA with 100 nM selenite. Sodium selenite might increase the antioxidative potential in human thyroid follicles in vitro and therefore diminish the apoptosis induced by TGF-β, EGF, iodide, and even H₂O₂     

Electrogenic H<Superscript>+</Superscript> Transport and pH Gradients Generated by a V-H<Superscript>+</Superscript>-ATPase in the Isolated Perfused Larval <Emphasis Type="Italic">Drosophila</Emphasis> Midgut

A method for microperfusion of isolated segments of the midgut epithelium of Drosophila larvae has been developed to characterize cellular transport pathways and membrane transporters. Stereological ultrastructural morphometry shows that this epithelium has unusually long tight junctions, with little or no lateral intercellular volume normally found in most epithelia. Amplification of the apical and basal aspects of the cells, by ≈ 17-fold and ≈ 7-fold, respectively, predicts an almost exclusively transcellular transport system for solutes. This correlates with the high lumen-negative transepithelial potential (V_t) of 38 to 45 mV and high resistance (R_t) of 800 to 1400 Ω • cm² measured by terminated cable analysis, in contrast to other microperfused epithelia like the renal proximal tubule. Several blockers (amiloride 10⁻⁴ M, ouabain 10⁻⁴ M, bumetanide 10⁻⁴ M), K⁺-free solutions, or organic solutes such as D-glucose 10 mM or DL-alanine 0.5 mM failed to affect V_t or R_t. Bafilomycin-A₁ (3 to 5 μM) decreased V_t by ≈ 40% and short-circuit current (I_sc) by ≈ 50%, and decreased intracellular pH when applied from the basal side only, consistent with an inhibition of an electrogenic V-H⁺-ATPase located in the basal membrane. Gradients of H⁺ were detected by pH microelectrodes close to the basal aspect of the cells or within the basal extracellular labyrinth. The apical membrane is more conductive than the basal membrane, facilitating secretion of base (presumably HCO₃⁻), driven by the basal V-H⁺-ATPase.     

Biochemical manipulation of intracellular glutathione levels influences cytotoxicity to isolated human lymphocytes by sulfur mustard

Glutathione (GSH) is the major nonprotein thiol that can protect cells from damage due to electrophilic alkylating agents by forming conjugates with the agent. Sulfur mustard (HD) is an electrophilic alkylating agent that has potent mutagenic, carcinogenic, cytotoxic, and vesicant properties. Compounds that elevate or reduce intracellular levels of GSH may produce changes in cytotoxicity induced by sulfur mustard. Pretreatment of human peripheral blood lymphocytes (PBL) for 72 hr with 1 mM buthionine sulfoximine (BSO), which reduces intracellular GSH content to approximately 26% of control, appears to sensitize these in vitro cells to the cytotoxic effects of 10 M HD but not to higher HD concentrations. Pretreatment of PBL for 48 hr with 10 mM N-acetyl cysteine (NAC), which elevates intracellular glutathione levels to 122% of control, appears to partially protect these in vitro cells from the cytotoxic effects of 10 M HD but not to higher HD concentrations. Augmentation of intracellular levels of glutathione may provide partial protection against cytotoxicity of sulfur mustard.Abbreviations BSO L-buthionine (S,R)-sulfoximine - GSH glutathione - HD sulfur mustard - NAC N-acetyl-L-cysteine - PBL peripheral blood lymphocytes The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.     

Clonal propagation of kurrat (Allium ampeloprasum var. kurrat)

Summary Two protocols for clonal propagation of kurrat (Allium ampeloprasum var.kurrat) using explants from the basal plates of mature plants are described. In direct formation, explants were cultured in Murashige and Skoog (MS) medium and supplemented with either benzyladenine at 0.0 or 4.4 μM, or supplemented with 7.0 μM benzyladenine and 0.1 μM naphthaleneacetic acid. Shoots appeared after 4 wk of culture. In the two-step procedure, explants were cultured first on MS medium supplemented with 1.4 μM 2,4-dichlorophenoxyacetic acid and 1.4 μM kinetin, and incubated in the dark for 4 wk. They were then transferred to MS medium supplemented with 4.4 μM benzyladenine for shoot formation. All shoots were rooted on MS medium containing 5 g·liter⁻¹ activated charcoal. Normal viable plants were successfully established in soil.     

Regulation of the Pleuronectes americanus Na+/H+ exchanger by osmotic shrinkage, β-adrenergic stimuli, and inhibition of Ser/Thr protein phosphatases

The ubiquitous Na⁺/H⁺ exchanger NHE1 is regulated by protein phosphorylation events, but the mechanisms involved are incompletely understood. We recently cloned NHE1 from the red blood cells of the winter flounder, Pleuronectes americanus (paNHE1), and demonstrated its activation by osmotic cell shrinkage, β-adrenergic stimuli, and the Ser/Thr protein phosphatase PP1 and PP2A inhibitor calyculin A (CLA) (Pedersen et al. [2003] Am. J. Physiol. 284, C1561–C1576). Here, we investigate the mechanisms involved in paNHE1 activation by these stimuli. Osmotic shrinkage and CLA were only partially additive in their effects on paNHE1 activity, and CLA-mediated paNHE1 activation was inhibited by osmotic cell swelling. Activation by the β-adrenergic agonist isoproterenol (IP) was fully additive to activation by osmotic shrinkage or CLA. IP-mediated, but neither shrinkage-nor CLA-mediated paNHE1 activation were associated with an increase in cellular cyclic adenosine monophosphate (cAMP) level. IP-mediated activation was partially blocked by the protein kinase A (PKA) inhibitor H89 (10μM), wherease shrinkage- and CLA-mediated activation were unaffected. All three stimuli activated paNHE1 in a manner unaffected by inhibitors of protein kinase C (calphostin C, 5 μM) and protein kinase G (KT5823, 10 μM) as well as of myosin light chain kinase (ML-7, 10 μM). IP-mediated, but not shrinkage-mediated, paNHE1 activation was associated with an increase in serine phosphorylation of the paNHE1 protein. It is suggested that paNHE1 activation by osmotic shrinkage and by PP1/PP2A inhibition involves partially convergent signaling pathways, whereas activation of paNHE1 by β-adrenergic stimuli is mediated by a separate pathway.     

Biophysical and Pharmacological Characteristics of Native Two-Pore Domain TASK Channels in Rat Adrenal Glomerulosa Cells

Multiple genes of the TASK subfamily of two-pore domain K⁺ channels are reported to be expressed in rat glomerulosa cells. To determine which TASK isoforms contribute to native leak channels controlling resting membrane potential, patch-clamp studies were performed to identify biophysical and pharmacological characteristics of macroscopic and unitary K⁺ currents diagnostic of recombinant TASK channel isoforms. Results indicate K⁺ conductance (gK⁺) is mediated almost exclusively by a weakly voltage-dependent (leak) K⁺ channel closely resembling TASK-3. Leak channels exhibited a unitary conductance approximating that expected for TASK-3 under the recording conditions employed, brief mean open times and a voltage-dependent open probability. Extracellular H⁺ induced voltage-independent inhibition of gK⁺, exhibiting an IC₅₀ of 56 nM (pH 7.25) and a Hill coefficient of 0.75. Protons inhibited leak channel open probability (P_o) by promoting a long-lived closed state (τ > 500 ms). Extracellular Zn²⁺ mimicked the effects of H⁺; inhibition of gK⁺ exhibited an IC₅₀ of 41 μM with a Hill coefficient of 1.26, inhibiting channel gating by promoting a long-lived closed state. Ruthenium red (5 μM) inhibited gK⁺ by 75.6% at 0 mV. Extracellular Mg²⁺ induced voltage-dependent block of gK⁺, inhibiting unitary current amplitude without affecting mean open time. Bupivacaine induced voltage-dependent block of gK⁺, exhibiting IC₅₀ values of 116 μM at −100 mV and 28 μM at 40 mV with Hill coefficients of 1 at both potentials. Halothane induced a voltage-independent stimulation of gK⁺ primarily by decreasing the leak channel closed-state dwell time.     

Somatic embryogenesis and plant regeneration in Medicago arborea L.

Summary An efficient plant regeneration system employing cotyledons, hypocotyls, petioles and leaves as explants and characterized by continuous and prolific production of somatic embryos, has been developed with Medicago arborea ssp. arborea. The optimal somatic embryogenic response was obtained using a two-step protocol, where explants were incubated under a 16 h photoperiod for 2 mo. on Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 9 μM) and kinetin (9 μM), and followed by transfer to kinetin-free MS medium with 2,4-D (2.25 μM). Removal of the cytokinin and a reduction in the concentration of auxin (2.25 μM) in the second step of culture were critical for enhanced production of somatic embryos. The best explants proved to be cotyledons and petioles (i.e. a mean of 18.0±0.70 somatic embryos at 3 mo. for petiole culture). Somatic embryos were converted into normal plantlets (8.0±0.89%) when cultured on basal MS medium with 5 μM indolebutyric acid. No somatic embryos were obtained when thidiazuron was used in the culture media. Using petioles as explants and N⁶-benzyladenine (BA), embryogenesis was induced in the second step of culture when BA was removed from the medium and the concentration of 2,4-D was decreased to 2.25 μM.     

Allelochemical effects on net nitrate uptake and plasma membrane H+-ATPase activity in maize seedlings

Seven-day-old maize seedlings grown in a nitrogen-free hydroponic culture were exposed for 48 h to 0, 100 and 300 μM trans-cinnamic, p-coumaric, ferulic, caffeic acids, umbelliferone and 200 μM KNO₃. Net nitrate uptake was affected by trans-cinnamic, ferulic and p-coumaric acids in a concentration-dependent manner, and trans-cinnamic acid appeared to be the strongest inhibitor. Conversely, at low concentrations, caffeic acid stimulated net nitrate uptake while umbelliferone did not influence it. After 24 h of treatment, plasma membrane H⁺-ATPase activity significantly decreased in a concentration-dependent manner in response to trans-cinnamic, ferulic and p-coumaric acids, while umbelliferone and caffeic acid had no effect on H⁺-ATPase activity.     

K+/H+ antiporter in alkaliphilic Bacillus sp. no. 66 (JCM 9763)

A K⁺/H⁺ antiport system was detected for the first time in right-side-out membrane vesicles prepared from alkaliphilic Bacillus sp. no. 66 (JCM 9763). An outwardly directed K⁺ gradient (intravesicular K⁺ concentration, K_in, 100 mM; extravesicular K⁺ concentration, K_out, 0.25 mM) stimulated uphill H⁺ influx into right-side-out vesicles and created the inside-acidic pH gradient (ΔpH). This H⁺ influx was pH-dependent and increased as the pH increased from 6.8 to 8.4. Addition of 100 μM quinine inhibited the H⁺ influx by 75%. This exchange process was electroneutral, and the H⁺ influx was not stimulated by the imposition of the membrane potential (interior negative). Addition of K⁺ at the point of maximum ΔpH caused a rapid K⁺-dependent H⁺ eflux consistent with the inward exchange of external K⁺ for internal H⁺ by a K⁺/H⁺ antiporter. Rb⁺ and Cs⁺ could replace K⁺ but Na⁺ and Li⁺ could not. The H⁺ efflux rate was a hyperbolic function of K⁺ and increased with increasing extravesicular pH (pH_out) from 7.5 to 8.5. These findings were consistent with the presence of K⁺/H⁺ antiport activity in these membrane vesicles. Received: March 20, 1997 / Accepted: May 22, 1997     

Citrate Uptake into Tonoplast Vesicles from Acid Lime (Citrus aurantifolia) Juice Cells

Citrate transport into the vacuoles of acid lime juice cells was investigated using isolated tonoplast vesicles. ATP stimulated citrate uptake in the presence or in the absence of a ΔμH⁺. Energization of the vesicles only by an artificial K⁺ gradient (establishing an inside-positive Δψ) also resulted in citrate uptake as was the case of a ΔpH dominated ΔμH⁺. Addition of inhibitors to endomembrane ATPases showed no direct correlation between the inhibition to the tonoplast bound H⁺/ATPase and citrate uptake. The data indicated that, although some citrate uptake can be accounted for by Δψ and by a direct primary active transport mechanism involving ATP, under in vivo conditions of vacuolar pH of 2.0, citrate uptake is driven by ΔpH. Received: 27 April 1998/Revised: 8 September 1998     

Tissue culture and plant regeneration of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. Ex Steud

Summary As a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H. B. K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram (4-amino-3, 5,6-trichloropicolinic acid), N⁶-benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations containing 1 mg l⁻¹ (4.52 μM) 2,4-D plus 0.5 mg l⁻¹ (2.22 μM) BA. and 2 mg l⁻¹ (8.88 μM) BA plus 1 mg l⁻¹ (4.14 μM) Picloram with or without 40 mg l⁻¹ (296.08 μM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient treatments for induction of embryogenic callus contained 2 mg l⁻¹ (9.05 μM) 2,4-D combined with 0.25 (1.11 μM) or 0.50 mg l⁻¹ (2.22 μM) BA, or 1 mg l⁻¹ (4.52 μM) 2,4-D with 0.50 mg l⁻¹ (2.22 μM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus 1 mg l⁻¹ (1.44 μM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg l⁻¹ (9.05 μM) 2,4-D to 62.2 for induction medium containing 2 mg l⁻¹ (8,28 μM) Picloram, 1 mg l⁻¹ (4.44 μM) BA and 40 mg l⁻¹ (296.08 μM) adenine. Regnerated plants grown in soil under greenhouse conditions reached maturity and produced seeds.