The influence of some inhibitors on the formation of caffeic acid in cultures ofPerilla cell suspensions

A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the 11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition of caffeic acid formation was caused by 10⁻⁴Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10⁻³M glyphosate concentration, while 10⁻³M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures.     

Polyamines in Helianthus annuus L. during Germination under Salt Stress

The level of the three main polyamines putrescine, spermidine, and spermine and the biosynthetic enzyme arginine decarboxylase (ADC) decreased in Helianthus annuus L. seedlings subjected to increasing (50, 100, and 150 mm) NaCl concentrations. The pattern of polyamines in control plants increased during the initial 72 h and then reached a plateau. The putrescine level showed an increase of 370% after 72 h of development. The lower salt treatment slightly diminished the overall polyamine content. The highest NaCl concentration (150 mm) induced a strong putrescine diminution (from 381 to 78.9 nmol g⁻¹ FW) at 72 h whereas a small decrease in ADC activity was detected. ODC was detected in neither control nor treated plantlets during the experimental period. The level of spermidine also decreased, but the magnitude of the decay was less pronounced than putrescine. The fact that ODC was not detected and ADC activity followed a pattern similar to that of putrescine led us to suppose that the variation in putrescine content could be attributed entirely to the decrease in ADC activity. α-Difluoromethylarginine and α-difluoromethylornithine (ADC and ODC inhibitor, respectively) did not inhibit but delayed the onset of germination of sunflower seeds, and α-difluoromethylornithine increased the content of spermidine and spermine. The present data suggest that polyamines could be involved in the germination process of H. annuus seeds and in response to salt stress. Received April 14, 1997; accepted July 10, 1997     

Influence of Polyamines on Growth of Hairy Root Cultures of Witloof Chicory (Cichorium intybus L. cv. Lucknow Local) and Formation of Coumarins

The effect of polyamines (putrescine, spermidine, and spermine) was examined for growth and production of two coumarins, esculetin and esculin, in the hairy roots of chicory (Cichorium intybus L. cv. Lucknow local). Of the polyamines administered, 1.5 mm putrescine alone resulted in a 2.3-fold higher increase in the growth of hairy roots as well as in the production of esculetin and esculin, which was 3.37 times more than that of the control on day 21. The endogenous level of conjugated putrescine was more than fivefold that of free putrescine levels in untreated samples. The production of esculetin and esculin in hairy root cultures strictly correlated with growth in all of the treatments. Putrescine at 1.5 mm resulted in a greater length of primary root (18.29 ± 1.37 cm) compared with the control (10.96 ± 0.82 cm) and more secondary and tertiary roots. This study also provides insight into the morphogenetic changes that occur in roots in response to the external supply of polyamines. Received July 20, 1998; accepted January 19, 1999     

L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K _mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na⁺ (l-methionine inhibition constant K_iA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (K_mB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na⁺ (K_iB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b ^0,+ and y⁺, respectively. Received: 14 August 1995/Revised: 2 April 1996     

Modification of l-phenylalanine ammonia-lyase in soybean cell suspension cultures by 2-aminooxyacetate and l-2-aminooxy-3-phenylpropionate

Suspension-cultured cells of soybean (Glycine max (L.) Merr. cv. Kanrich) produce large amounts of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), the first enzyme of phenylpropanoid metabolism, during growth. 2-Aminooxyacetic acid (AOA) and l-2-aminooxy-3-phenylpropionic acid (l-AOPP) inhibit the enzyme competitively in vitro and have been used for in vivo studies. The amount of extractable enzyme in the cells and their utilization of NO ₃ ^– and NH ₃ ⁺ are reduced upon the addition of AOA. When AOA was added at various times during growth, the appearance of additional enzyme activity was prevented but enzyme already formed was not inhibited. No evidence was obtained for the presence of an inhibitor in the extracts and AOA inhibition in vitro was readily reversible. It is conculded that AOA acts to inhibit the formation of PAL in suspension-cultured soy bean cells. In vitro inhibition of soybean PAL by l-AOPP could not be reversed; in contrast, the inhibition of maize (Zea mays L.) PAL was readily reversible. Added l-AOPP, which was rapidly taken up by the soybean cells, prevented the large increase in enzyme activity. Although PAL activity was blocked in the cultures, no appreciable increase in phenylalanine content could be detected in cell extracts. The response of soybean cell suspensions to l-AOPP addition thus differs from that of other tissues which in presence of l-AOPP show an increase in PAL activity and an accumulation of phenylalanine.Abbreviations AOA 2-aminooxyacetic acid - l-AOPP l-2-aminoxy-3-phenylpropionic acid - PAL l-phenylalanine ammonialyase (EC4.3.1.5)     

The Binding Specificity of Amino Acid Transport System y+L in Human Erythrocytes is Altered by Monovalent Cations

System y⁺L is a broad-scope amino acid transporter which binds and translocates cationic and neutral amino acids. Na⁺ replacement with K⁺ does not affect lysine transport, but markedly decreases the affinity of the transporter for l-leucine and l-glutamine. This observation suggests that the specificity of system y⁺L varies depending on the ionic composition of the medium. Here we have studied the interaction of the carrier with various amino acids in the presence of Na⁺, K⁺, Li⁺ and guanidinium ion. In agreement with the prediction, the specificity of system y⁺L was altered by the monovalent cations. In the presence of Na⁺, l-leucine was the neutral amino acid that interacted more powerfully. Elongation of the side chain (glycine - l-norleucine) strengthened binding. In contrast, bulkiness at the level of the β carbon was detrimental. In K⁺, the carrier behaved as a cationic amino acid specific carrier, interacting weakly with neutral amino acids. Li⁺ was found to potentiate neutral amino acid binding and in general the apparent affinities were higher than in Na⁺; elongation of the nonpolar side chain made a more important contribution to binding and the carrier was more tolerant towards β carbon substitution. Guanidinium stimulated the interaction of the carrier with neutral amino acids, but the effect was restricted to certain analogues (e.g., l-leucine, l-glutamine, l-methionine). Thus, in the presence of guanidinium, the carrier discriminates sharply among different neutral amino acids. The results suggest that the monovalent cations stabilize different carrier conformations. Received: 22 January 1996/Revised: 26 April 1996     

Role of polyamines in the cytokinin-dependent physiological processes II. Modulation of polyamine levels during cytokinin-stimulated expansion of cucumber cotyledons

The cucumber cotyledon expansion test was used as a model system to study a possible relationship between cytokinin and polyamines. When kinetin was applied to excised cotyledons incubated in the dark it caused a marked increase in the activity of arginine decarboxylase. As a result of ADC action, putrescine content also rose markedly, whereas the level of spermidine and spermine decreased. However, inhibition of putrescine biosynthesis with D-arginine did not affect cytokinin promotion growth. Applied alone, putrescine had no significant effect on growth. These results indicate that the large increase in putrescine content that derives from cytokinin treatment cotyledons is not essential for cytokinin-induced expansion of cotyledons. Addition of K⁺ and Ca²⁺ ions to the cotyledons incubated with cytokinin caused a marked reduction in the putrescine level and ADC activity. The higher level of putrescine (35 %) and spermine (62 %) bound to chromatin and the large increase (174 %) in spermidine content bound to ribosomes which derive from cytokinintreated cotyledons in relation to literature data can indicate that these polyamines may play an important role in gene expression during cytokinin-stimulated expansion of cucumber cotyledons. The inhibition of cytokinin effect, viz. enlargement of the cotyledons by inhibitors of spermidine biosynthesis, additionally suggessted a possible involvement of polyamines in cytokinin action.     

Rectification of the Olfactory Cyclic Nucleotide-Gated Channel by Intracellular Polyamines

Polyamine-induced inward rectification of cyclic nucleotide-gated channels was studied in inside-out patches from rat olfactory neurons. The polyamines, spermine, spermidine and putrescine, induced an `instantaneous' voltage-dependent inhibition with K <sub>d</sub> values at 0 mV of 39, 121 μm and 2.7 mm, respectively. Hill coefficients for inhibition were significantly < 1, suggesting an allosteric inhibitory mechanism. The Woodhull model for voltage-dependent block predicted that all 3 polyamines bound to a site 1/3 of the electrical distance through the membrane from the internal side. Instantaneous inhibition was relieved at positive potentials, implying significant polyamine permeation. Spermine also induced exponential current relaxations to a `steady-state' impermeant level. This inhibition was also mediated by a binding site 1/3 of the electrical distance through the pore, but with a K _d of 2.6 mm. Spermine inhibition was explained by postulating two spermine binding sites at a similar depth. Occupation of the first site occurs rapidly and with high affinity, but once a spermine molecule has bound, it inhibits spermine occupation of the second binding site via electrostatic repulsion. This repulsion is overcome at higher membrane potentials, but results in a lower apparent binding affinity for the second spermine molecule. The on-rate constant for the second spermine binding saturated at a low rate (∼200 sec⁻¹ at +120 mV), providing further evidence for an allosteric mechanism. Polyamine-induced inward rectification was significant at physiological concentrations. Received: 17 February 1999/Revised: 27 April 1999     

A Sequential Scenario for the Origin of Biological Chirality

A sequential model is proposed regarding the origin of biological chirality. Three major stages are presumed: a symmetry breaking (prebiotic chiral disruption in enantiomeric mixtures of monomers), a chiral amplification (prebiotic increase of the chiral character of the monomers affected first by the symmetry breaking), and a chiral expansion (proto biological increase of the chiral character and spread of the chirality to molecules which were less affected by prebiotic chiralizations). As a symmetry-breaking mechanism, the model proposed by Deutsch (1991) is used, which involves a dissymmetric exposure of amino acids (AA) to ultraviolet circularly polarized light (UV-CPL) on evaporative seashores. It is presumed that the chiral amplification, up to a protobiologic significance, was influenced by a periodic overlapping of two abiotic events, a synchronization between tidal-based hydrous–anhydrous cycles, and littoral asymmetric photolysis cycles. This long-term astronomic asymmetry acted around 3.8–4.2 billion years ago and was unique to the Earth in our solar system. It is also presumed that the abiotic symmetry breaking is heterogenous, that only a few l-AAs were used in the beginning, and that the chirality expanded later to all 20 AAs based on a coevolutionary strategy of the genetic code and on a physiological relationship between AAs. In this scenario the d-chirality of pentoses in polynucleotides was attributed to both d-pentose/l-AA relationships and to a structural evolution. Received: 10 May 1996 / Accepted: 13 August 1996     

Effect of Chlorsulfuron on Morphogenetic and Disordered Cell Division in Cultures of Passiflora edulis

We examined the effects of a sulfonylurea herbicide, chlorsulfuron, which is known as a potent inhibitor of plant cell division, on morphogenetic cell division and disorganized cell division using the culture system of multiple shoot primordia and callus of Passiflora edulis. The multiple shoot primordia tissue treated with chlorsulfuron failed to achieve shoot morphogenesis, and a large part of the tissue was necrotized during the posttreatment culture, even when it was washed and transferred to chlorsulfuron-free medium. The inhibition of Passiflora shoot morphogenesis by chlorsulfuron was not reversed by the simultaneous addition of branched amino acids, which are known to reverse the inhibitory effect of chlorsulfuron. In contrast, the same treatment of chlorsulfuron on the callus did not kill the cells, although the growth resumption was retarded by a prolonged lag period. The addition of branched amino acids enhanced the recovery growth of the chlorsulfuron-treated callus. These results suggest that the inhibition of disorganized cell division (callus growth) by chlorsulfuron is reversible, whereas morphogenetic cell division (shoot morphogenesis), which is under complex regulation, is inhibited irreversibly by chlorsulfuron. Qualitative differences between morphogenetic cell division and disordered simple proliferative cell division are discussed. Received November 17, 1997; accepted June 4, 1998     

Hormonal Control of Gene Expression During Reactivation of the Cell Cycle in Tobacco Mesophyll Protoplasts

The roles of auxin and cytokinin in cell cycle reactivation were studied during the first 48 h of culture of mesophyll protoplasts of Nicotiana tabacum. Using hormone delay and withdrawal studies we found that auxin was required by 0–4 h of culture, whereas cytokinin was not required until hour 10–12, which is 6–10 h before S phase. Cycloheximide blocks division, indicating that protein synthesis is required. In an effort to detect a molecular response to either hormone, we examined the expression of the cell cycle marker, cdc2. Cdc2 expression was detected by 12 h of culture, coincident with the timing of the cytokinin requirement and well before the entry into S. However, cdc2 was partially induced by either auxin or cytokinin alone, suggesting that cdc2 expression is not the primary target of either hormone. Our hormone delay experiments suggest that there are separate signal transduction pathways leading from auxin and from cytokinin to reactivation of the cell cycle and that these pathways converge before S. The underlying mechanisms for these distinct pathways remain to be elucidated. Received November 4, 1997; accepted October 7, 1998     

Differential Transmembrane Diffusion of Triiodothyronine and Thyroxine in Liposomes: Regulation by Lipid Composition

How thyroid hormones move across biological or model membranes is a subject of controversy. The passage of the 3,5,3′triiodo l-thyronine and 3,5,3′,5′ tetraiodo l-thyronine across model membranes was evaluated by the addition of the hormones to liposomes containing 2,4,6-trinitrobenzene sulfonic acid. Results indicate that hormones can react with an amino-reactive compound pre-encapsulated into phosphatidylcholine liposomes. The transversal motions of thyroid hormones were characterized by using physiological concentration levels of (¹²⁵I) 3,5,3′triiodo l-thyronine and (¹²⁵I) 3,5,3′,5′ tetraiodo l-thyronine. The hormone distribution between the two monolayers was time-dependent and kinetic data were fitted to a single exponential. Results obtained show that 3,5,3′ triiodo l-thyronine can permeate phospholipid membranes and the diffusion time increases in the gel and liquid-ordered phase. On the contrary, 3,5,3′, 5′ tetraiodo l-thyronine could not diffuse the liposomal membrane from dimyristoyl and dipalmitoyl phosphatidylcholine in gel phase and egg yolk phosphatidylcholine:cholesterol in the liquid-ordered phase. Our results in the liquid-ordered phase suggest that diffusion movement of thyroid hormones across cell membranes depends on the amount of cholesterol in the bilayer. Received: 1 June 1998/Revised: 14 October 1998     

System y+L-like Activities Account for High and Low Amino-Acid Transport Phenotypes in Chicken Erythrocytes

The functional properties of the transport of lysine across the chicken erythrocyte membrane were investigated. The animal population studied (male Leghorn chickens, 6–14 weeks old) was found to consist of two groups presenting either low (LT, 19 individuals) or high transport rates (HT, 20 individuals). The rates of influx in the two groups, measured at a concentration of l-lysine of 1 μm, differed by a factor of 34. The transport activities observed in LT and HT erythrocytes were compatible with the general features of system y⁺L, but showed some differences in specificity. The transporter in the LT group was found to bind l-lysine, l-leucine, l-methionine and l-glutamine with high affinity, in the presence of sodium, as described for system y⁺L in human erythrocytes. The activity present in HT erythrocytes exhibited a much lower affinity for l-leucine, but was able to interact strongly with l-glutamine and l-methionine. The specificity pattern of the HT transporter, has not been described in other cell types. In other respects, the properties of the two systems were similar. Sodium replacement with potassium, drastically reduced the affinity for l-leucine, without affecting lysine transport. Both transporters function as tightly coupled exchangers, are inactivated by p-chloromercuribenzene sulfonate and resistant to N-ethylmaleimide. These findings explain previous results obtained in selective breeding experiments of chicken with high and low amino-acid transport activity. Received: 12 February 2001/Revised: 11 June 2001     

Membrane Potassium Channels and Human Bladder Tumor Cells: II. Growth Properties

These experiments were done to determine the effect of glibenclamide and diazoxide on the growth of human bladder carcinoma (HTB-9) cells in vitro. Cell growth was assayed by cell counts, protein accumulation, and ³H-thymidine uptake. Glibenclamide added at 75 and 150 μm for 48 hr reduced cell proliferation. Dose-inhibition curves showed that glibenclamide added for 48 hr reduced cell growth at concentrations as low as 1 μm (IC₅₀= 73 μm) when growth was assayed in the absence of added serum. This μM-effect on cell growth was in agreement with the dose range in which glibenclamide decreased open probability of membrane K_ATP channels. Addition of glibenclamide for 48 hr also altered the distribution of cells within stages of the cell cycle as determined by flow cytometry using 10⁻⁵ m bromodeoxyuridine. Glibenclamide (100 μm) increased the percentage of cells in G₀/G₁ from 33.6% (vehicle control) to 38.3% (P < 0.05), and it reduced the percentage of cells in S phase from 38.3% to 30.6%. On the other hand, diazoxide, which opens membrane K_ATP channels in HTB-9 cells, stimulated growth measured by protein accumulation, but it did not increase the cell number. We conclude that the sulfonylurea receptor and the corresponding membrane K_ATP channel are involved in mechanisms controlling HTB-9 cell growth. However, K_ATP is not rate-limiting among the signaling mechanisms or molecular switches that regulate the cell cycle. Received: 12 June 1997/Revised: 21 October 1997     

Effect of polyamines and L-ornithine on the development of proembryogenic masses of Cryptomeria japonica

We examined the effects of polyamines, namely, putrescine, spermidine and spermine, and of amino acids, such as l-arginine and l-ornithine, as part of our efforts to identify factors that stimulate the development of proembryogenic masses (PEMs) of Cryptomeria japonica. We maintained two distinct types of PEM designated PEMs A, which consisted of normal embryogenic cells as single embryos with elongated suspensor cells, and PEMs B, which consisted of abnormal embryogenic cells with coalesced embryos on modified Campbell and Durzan medium (mCD) supplemented with individual polyamines at 0–100 μM or amino acids at 0–16.4 mM. All additives had a stimulatory/suppressive effect. Microscopy and image-processing techniques revealed that the regions of authentic embryos of PEMs that were treated with l-ornithine were remarkably enlarged and that the suspensor cells had elongated in the same direction. When all PEMs A were transferred to maturation medium (mCD that contained abscisic acid and maltose at various concentrations), only PEMs that had been treated with l-ornithine matured into somatic embryos and were able to germinate on hormone-free mCD. Our results indicate that l-ornithine is an important stimulator of the development of PEMs to the pre-filamentous stage in C. japonica.     

Putrescine Influences Growth and Production of Coumarins in Hairy Root Cultures of Witloof Chicory (Cichorium intybus L. cv. Lucknow Local)

The effect of putrescine (Put) on the growth and production of two coumarins, esculin and esculetin, in hairy roots of chicory (Cichorium intybus L. cv. Lucknow local) was examined. To study the role of Put on growth and production of coumarins, polyamine inhibitors, namely α-dl-difluromethylornithine and α-dl-difluromethylarginine were used at 1 mM concentration. Put treatment at 1.5 mM produced a 1.9-fold increase in the growth of hairy roots, as well as the production of esculin and esculetin. The treatments with polyamine (PA) inhibitors resulted in much lower growth and production of coumarins compared with both 1.5-mM Put treatment and the control. Both free and conjugated PAs were studied over the whole culture period, and conjugates of all three PAs, namely Put, spermidine, and spermine, were higher than free PAs throughout the culture period. The treatments with PA inhibitors showed lower levels of endogenous PAs compared with Put-treated samples. The treatment with 1.5 mM Put showed maximum accumulation of endogenous conjugated Put (2,098 ± 157 nmoles gm⁻¹ fresh weight). The production of esculin and esculetin was strictly correlated with growth in all treatments. Put at 1.5 mM resulted in greater length of primary root (18.3 ± 1.4 cm) as compared with the control (11 ± 0.9 cm) and larger numbers of secondary and tertiary roots. Received July 14, 1999; accepted October 5, 1999     

l-Arginine Effects on Na+ Transport in M-1 Mouse Cortical Collecting Duct Cells—A Cationic Amino Acid Absorbing Epithelium

The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using continuous short circuit current (I _SC ) measurements in HCO₃ ⁻/CO₂ buffered solution. Steady state I _SC averaged 73.8 ± 3.2 μA/cm² (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na⁺ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y⁺ and a basal amino acid transport system y⁺L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I _SC averaging 36.6 ± 5.4 μA/cm² (n= 19) and 32.0 ± 7.2 μA/cm² (n= 8), respectively. The response was preserved in the absence of bath Cl⁻ (n= 4), but was abolished either in the absence of apical Na⁺ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I _SC response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na⁺ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract passive cationic amino acid leak into the CCD in the presence of electrogenic Na⁺ transport, consistent with reports of stimulated expression of Na⁺ and cationic amino acid transporters by aldosterone. Received: 11 September 2000/Revised: 6 December 2000     

Effect of Gibberellin on Growth, Protein Secretion, and Starch Accumulation in Maize Endosperm Suspension Cells

The physiologic effect of gibberellins (GA) in seed development is poorly understood. We examined the effect of gibberellic acid (GA₃) on growth, protein secretion, and starch accumulation in cultured maize (Zea mays L.) endosperm suspension cells. GA₃ (5 and 30 μm) increased the fresh weight, dry weight, and protein content of the cultured cells, but the effect of GA₃ at 50 μm was not significantly different. However, the protein content in the culture medium was increased by these three concentrations of GA₃. The effect of GA₃ on the amount of cellular structural polysaccharides was not significant, but GA₃ had a dramatic effect on the starch content. At 5 μm, GA₃ caused an increase in the starch content, but at 50 μm the starch accumulation was reduced. Chlorocholine chloride (CCC), an inhibitor of GA biosynthesis, significantly increased the starch content and decreased the structural polysaccharide content of the cultured cells. The effects of CCC at 500 μm on the starch and polysaccharide content were partially reversed by 5 μm GA₃ applied exogenously. Based on these results we suggest that GA does not favor starch accumulation in the cell cultures and that the addition of lower concentrations of GA₃ in the medium may provide an improved balance among the endogenous GA in the cultured cells. Received October 31, 1995; accepted March 25, 1997     

Methylglyoxal Causes Swelling and Activation of a Volume-Sensitive Anion Conductance in Rat Pancreatic β-Cells

Membrane potential and whole-cell current were studied in rat pancreatic β-cells using the `perforated patch' technique and cell volume measured by a video-imaging method. Exposure of β-cells to the α-ketoaldehyde methylglyoxal (1 mm) resulted in depolarization and electrical activity. In cells voltage-clamped at −70 mV, this effect was accompanied by the development of inward current noise. In voltage-pulse experiments, methylglyoxal activated an outwardly rectifying conductance which was virtually identical to the volume-sensitive anion conductance previously described in these cells. Two inhibitors of this conductance, 4,4′-dithiocyanatostilbene-2,2′-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), also inhibited the depolarization and inward current evoked by methylglyoxal. Methylglyoxal increased β-cell volume to a relative value of 1.33 after 10 min with a gradual return towards basal levels following withdrawal of the α-ketoaldehyde. None of the effects of methylglyoxal was observed in response to t-butylglyoxal which, unlike methylglyoxal, is a poor substrate for the glyoxalase pathway. Methylglyoxal had no apparent effect on β-cell K⁺ channel activity. It is suggested that the metabolism of methylglyoxal to d-lactate causes β-cell swelling and activation of the volume-sensitive anion channel, leading to depolarization. These findings could be relevant to the stimulatory action of d-glucose, the metabolism of which generates significant quantities of l-lactate. Received: 15 May 1998/Revised: 25 September 1998     

Internal Zinc Accumulation Is Correlated with Increased Growth in Rice Suspension Culture

A high zinc concentration of 520 μm, approximately 100 times that used most often in standard plant tissue culture media, was found to be superior in liquid callus cultures of japonica rice, increasing growth to 146% compared with standard N6 medium. At the same time, the internal zinc concentration increased 40 times in fast growing cells; soluble protein doubled, and free amino acids decreased. Under zinc-free conditions the cultures slowed in growth, and several free amino acids such as aspartic acid, glutamic acid, asparagine, and glutamine accumulated. We suggest that zinc acts as a direct regulatory factor in inducing auxin activity, but not auxin levels, making high internal zinc accumulation mandatory if high auxin concentrations are required as in rice callus cultures. Received July 16, 1997; accepted September 22, 1997