Benzoylphenyl ureas inhibit chitin synthesis without interfering with amino sugar uptake in imaginal wing discs of Plodia interpunctella (Hübner)

We tested the hypothesis that the inhibition of chitin synthesis by benzoylphenyl ureas could be explained by their effect on the uptake of GlcNAc into chitin. Our test system consisted of organ cultures of wing imaginal discs from Plodia interpunctella. These wing discs synthesize chitin in response to 20-hydroxyecdysone or RH 5849, a non-steroidal ecdysteroid mimic. Two benzoylphenyl ureas, diflubenzuron and teflubenzuron, inhibited ecdysteroid-dependent chitin synthesis in the wing discs. However, although chitin synthesis was inhibited, there was no corresponding diminution of amino sugar uptake by the imaginal discs. In another experiment 20-hydroxyecdysone stimulated uptake of two sugars, 2-deoxy-D-glucose and 3-O-methyl-D-glucose, which are not synthesized into chitin. Transport of these non-metabolized sugars was unaffected by the inhibitors. In an additional test of the effects on precursor transport, the action of ecdysone (alpha-ecdysone) was examined. Ecdysone stimulated amino sugar uptake, but not chitin synthesis. Neither diflubenzuron nor teflubenzuron inhibited ecdysone-dependent precursor transport. Finally, we examined ecdysteroid-induced uptake of amino sugars by an imaginal disc derived cell line IAL-PID2. In this case, also, GlcNAc transport was not inhibited significantly by either diflubenzuron or teflubenzuron. From these observations we conclude that inhibition of uptake of amino sugars does not account for the ability of teflubenzuron and diflubenzuron to inhibit chitin synthesis in P. interpunctella wing discs.     

Specificity of the Glucose Transport System in Leishmania tropica Promastigotes*

SYNOPSIS. The glucose transport system in Leishmania tropica promastigotes was characterized by the use of labeled 2-deoxy-D-glucose (2-DOG), a nonmetabolizable glucose analog. The uptake system has a Q₁₀ of 2 and a heat of activation of 10.2 kcal/mole. The glucose transport system is subject to competitive inhibition by 2-DOG, glucosamine, N-acetyl glucosamine, mannose, galactose, and fructose which suggests that substitutions in the hexose chain at carbons 2 and 4 do not affect carrier specificity. In contrast, changes at carbon 1 (α-methyl-D-glucoside, 1,5-anhydroglucitol) and carbon 3 (3–0-methyl glucose) lead to loss of carrier affinity since these sugars do not compete for the glucose carrier. Sugars that compete with the glucose carrier have one common feature—they all exist in the pyranose form in solution. The carrier for D-glucose does not interact with L-glucose or any of the pentose sugars tested. Uptake of 2-DOG is inhibited by glycerol. This inhibition, however, is noncompetitive; it is evident, therefore, that glucose and glycerol do not compete for the same carrier. Glycerol does not repress the glucose carrier since cells grown in presence of glycerol transport the sugar normally.     

Sugar Transport and Metal Binding in Yeast

The uptake of sugars by yeast can be separated into two classes. The first involves the uptake of sorbose or galactose by starved cells, and the uptake of glucose by iodoacetate-poisoned cells. These uptakes do not involve any changes in Ni⁺⁺- or Co⁺⁺-binding by the cell surface, are not inhibited by Ni⁺⁺, are inhibited by UO₂ ⁺⁺ in relatively high concentrations, are characterized by high Michaelis constants and low maximal rates and by a final equilibrium distribution of the sugars. The second involves the uptake of glucose in unpoisoned cells and galactose in induced cells. These uptakes are characterized by a reduction of Ni⁺⁺- and Co⁺⁺-binding, by a partial inhibition by Ni⁺⁺, by an inhibition with UO₂ ⁺⁺ in relatively low concentrations, and by a low Km and a high Vm. In the case of galactose in induced cells, previous studies demonstrate that the sugar is accumulated against a concentration gradient. It is suggested that the first class of uptakes involves a "facilitated diffusion" via a relatively non-specific carrier system, but the second represents an "uphill" transport involving the highly specific carriers, and phosphoryl groups (cation-binding sites) of the outer surface of the cell membrane.     

Glucose uptake by Cellulomonas fimi

Glucose uptake by whole-cell suspension of the facultative anaerobe Cellulomonas fimi, which was two-fold higher under aerobic conditions than under N₂ or H₂, was inhibited by inhibitors of electron transport and ATP synthesis and, particularly, by proton and metal ion ionophores. A variety of sugars, including 2-deoxyglucose, did not inhibit glucose uptake but cellobiose was a non-competitive inhibitor. Cells grown on cellobiose medium transported glucose at one half the rate of glucose-grown cells. Cellulomonas fimi has a highly specific active system for glucose transport.     

Expression of GFAT1 and OGT in podocytes: Transport of glucosamine and the implications for glucose uptake into these cells

Glutamine:fructose‐6‐phosphate amidotransferase (GFAT) and N‐acetylglucosaminyltransferase (OGT) participate in glucosamine (GlcN) production and its utilization in O‐glycosylation, one of key post‐translational modifications of nuclear and cytoplasmic proteins. For this purpose, cells require a high rate of intracellular production of GlcN and/or significant GlcN delivery. We studied the expression of GFAT1 and OGT and measured uptake of glucose and GlcN in cultured rat podocytes, the main cellular component of glomerular filtration barrier. RT‐PCR revealed the presence of both GFAT1 and OGT mRNA. Immunofluorescence of GFAT1 has shown staining signal diffused within the cytoplasm of the cell body and processes. However, OGT was distinctly visible around the nucleus and, in diffuse form, within the cytoplasm of cell bodies and processes. Glucose was transported (1.3 ± 0.2 nmol/min/mg protein) mainly by facilitative transporter systems whilst GlcN uptake (1.1 ± 0.2 nmol/min/mg protein) in a significant part, involved a sodium‐dependent transporter. There was interplay between glucose and GlcN uptake. In the presence of GlcN (50 µM), the rate of glucose uptake decreased by about 50%. The rate of GlcN uptake decreased by 28% in the presence of 5.6 mM glucose. Our results suggest that cultured podocytes possess limited ability to synthesize GlcN internally and therefore may need to receive GlcN from the extracellular environment. J. Cell. Physiol. 225: 577–584, 2010. © 2010 Wiley‐Liss, Inc.     

Regulation of pentosan biosynthesis in barley aleurone tissue by gibberellic acid

Summary Treatment of isolated barley aleurone layers with gibberellic acid (GA₃) resulted in a progressive inhibition of cell-wall synthesis after a 4-h lag period. The incorporation of both [¹⁴C]arabinose and [¹⁴C]glucose into the cell wall was inhibited by GA₃, but analysis of the labelled sugars in the polymerized product showed that the process most affected by the hormone treatment was pentosan biosynthesis. Labelling kinetics and pulse-chase analysis indicated that the pentosans were synthesized in the cytoplasm and subsequently transferred to the cell wall; GA₃ did not significantly affect the latter step. The GA₃-inhibited labelling of the cell-wall pentosans cannot be explained on the basis of an effect on uptake of radioactive cell-wall precursor, expansion of the free pentose pool, or degradation of newly-formed pentosan. GA₃ inhibited the activity of a membrane-bound arabinosyl transferase present in the aleurone layers. This inhibition may explain the inhibition of cell-wall pentosan synthesis by GA₃.Abbreviations GA gibberellin - GA₃ gibberellic acid     

Influence of 2-deoxy-D-glucose upon growth and invertase activity of carrot (Daucus carota L.) cell suspension cultures

Carrot (Daucus carota L.) cell suspension cultures grew well when provided with glucose, fructose, sucrose or raffinose. Galactose and melibiose supported less growth unless supplemented with glucose or fructose. In combination with ten different sugar mixtures, 2-deoxy-D-glucose (dGlc) inhibited culture growth. Inhibitory effects of dGlc were more marked with fructose, melibiose, raffinose or mixtures of these sugars in the culture medium. The presence of glucose or galactose reduced the inhibitory effects of dGlc on culture growth. Experiments with radioactive labelled sugars demonstrated that dGLc uptake was greater in the presence of fructose than glucose, and that growth inhibition of dGlc coincided with its uptake. Reduced protein content was also associated with the inhibitory effects of dGlc. Cultured cells contained lower levels of invertase (EC 3.2.1.26) activity during the active phase of culture growth (up to 25 days after subculture) than when growth had peaked and subsequently declined. Acid and alkaline invertase activities were not greatly reduced by exogenous hexoses. Invertase activity was greatest during periods of low protein content in all cultures and was inhibited by dGlc during the latter phases of the culture period. Free intracellular sugars throughout the culture period consisted mainly of glucose and fructose.     

Fatty Acid Toxicity and Methyl Ketone Production in Aspergillus niger

Vegetative hyphae of Aspergillus niger rapidly converted caproic acid into 2-pentanone. More caproic acid was required for maximal ketone production at alkaline as compared to acidic pH values. Further increases in caproate concentrations at each pH value tested (4.5, 5.5, 6.5, 7.5, and 8.5) resulted in inhibition of ketone production and O₂ uptake. At alkaline pH values (8.5 and 7.5), oxygen uptake above the endogenous level and the production of 2-pentanone were parallel. This relationship did not hold at acidic pH values. At these pH values, ketone production continued (pH 6.5) or attained a maximum (pH 5.5 and 4.5) at caproate concentrations at which oxygen uptake was inhibited below endogenous levels. These data indicate that endogenous oxygen uptake was not inhibited by caproate at alkaline pH values at concentrations which did inhibit caproate oxidation and 2-pentanone production. Conversely, at acidic pH values, endogenous oxygen uptake was vigorously inhibited by caproate at concentrations at which exogenous fatty acid oxidation and 2-pentanone production were less affected. Simon-Beevers plots of these data showed that the undissociated acid was the permeant form of caproic acid. The fatty anion appeared to be the active or inhibitory form of caproate within the cell. Vegetative hyphae of A. niger were poorly buffered. Once the hyphae were washed and resuspended in phosphate buffer, they were well buffered towards inhibitory concentrations of caproic acid. These findings suggest that the primary mechanism(s) by which caproate inhibits oxygen uptake and ketone formation does not involve a change in the intracellular pH.     

Turgor regulation of sucrose transport in sugar beet taproot tissue

Sink tissues that store osmotically active compounds must osmoregulate to prevent excessively high turgor. The ability to regulate turgor may be related to membrane transport of solutes and thus sink strength. To study this possibility, the kinetics of sugar uptake were determined in sugar beet (Beta vulgaris L.) taproot tissue discs over a range of cell turgors. Sucrose uptake followed biphasic kinetics with a high affinity saturating component below 20 millimolar and a low affinity linear component at higher concentrations. Glucose uptake exhibited only simple saturation type kinetics. The high affinity saturating component of sucrose and glucose uptake was inhibited by increasing cell turgor (decreasing external mannitol concentrations). The inhibition was evident as a decrease in V_max but no effect on K_m. Sucrose uptake by tissue equilibrated in dilute buffer exhibited no saturating component. Ethylene glycol, a permeant osmoticum, had no effect on uptake kinetics, suggesting that the effect was due to changes in cell turgor and not due to decreased water potential per se. p-(Chloromercuri)benzene sulfonic acid (PCMBS) inhibited sucrose uptake at low but not high cell turgor. High cell turgor caused the tissue to become generally leaky to potassium, sucrose, amino acids, and reducing sugars. PCMBS had no effect on sucrose leakage, an indication that the turgor-induced leakage of sucrose was not via back flow through the carrier. The ability of the tissue to acidify the external media was turgor dependent with an optimum at 300 kilopascals. Acidification was sharply reduced at cell turgors above or below the optimum. The results suggest that the secondary transport of sucrose is reduced at high turgor as a result of inhibition of the plasma membrane ATPase. This inhibition of ATPase activity would explain the reduced V_max and leakiness to low molecular weight solutes. Cell turgor is an important regulator of sucrose uptake in this tissue and thus may be an important determinant of sink strength in tissues that store sucrose.     

Sugar utilization by yeast during fermentation

Summary When glucose and fructose are fermented separately, the uptake profiles indicate that both sugars are utilized at similar rates. However, when fermentations are conducted in media containing an equal concentration of glucose and fructose, glucose is utilized at approximately twice the rate of fructose. The preferential uptake of glucose also occurred when sucrose, which was first rapidly hydrolyzed into glucose and fructose by the action of the enzyme invertase, was employed as a substrate. Similar results were observed in the fermentation of brewer's wort and wort containing 30% sucrose and 30% glucose as adjuncts. In addition, the high levels of glucose in the wort exerted severe catabolite repression on maltose utilization in theSaccharmyces uvarum (carlsbergensis) brewing strain. Kinetic analysis of glucose and fructose uptake inSaccharomyces cerevisiae revealed aK _m of 1.6 mM for glucose and 20 mM for fructose. Thus, the yeast strain has a higher affinity for glucose than fructose. Growth on glucose or fructose had no repressible effect on the uptake of either sugar. In addition, glucose inhibited fructose uptake by 60% and likewise fructose inhibited, glucose uptake by 40%. These results indicate that glucose and fructose share the same membrane transport components.     

Optimization of glucose feeding approaches for enhanced glucosamine and N-acetylglucosamine production by an engineered Escherichia coli

In this work, a recombinant Escherichia coli was constructed by overexpressing glucosamine (GlcN) synthase and GlcN-6-P N-acetyltransferase for highly efficient production of GlcN and N-acetylglucosamine (GlcNAc). For further enhancement of GlcN and GlcNAc production, the effects of different glucose feeding strategies including constant-rate feeding, interval feeding, and exponential feeding on GlcN and GlcNAc production were investigated. The results indicated that exponential feeding resulted in relatively high cell growth rate and low acetate formation rate, while constant feeding contributed to the highest specific GlcN and GlcNAc production rate. Based on this, a multistage glucose supply approach was proposed to enhance GlcN and GlcNAc production. In the first stage (0–2 h), batch culture with initial glucose concentration of 27 g/l was conducted, whereas the second culture stage (2–10 h) was performed with exponential feeding at μ _set = 0.20 h⁻¹, followed by feeding concentrated glucose (300 g/l) at constant rate of 32 ml/h in the third stage (10–16 h). With this time-variant glucose feeding strategy, the total GlcN and GlcNAc yield reached 69.66 g/l, which was enhanced by 1.59-fold in comparison with that of batch culture with the same total glucose concentration. The time-dependent glucose feeding approach developed here may be useful for production of other fine chemicals by recombinant E. coli.     

Glucose uptake by the cellulolytic ruminal anaerobe Bacteroides succinogenes.

Glucose uptake by Bacteroides succinogenes S85 was measured under conditions that maintained anaerobiosis and osmotic stability. Uptake was inhibited by compounds which interfere with electron transport systems, maintenance of proton or metal ion gradients, or ATP synthesis. The most potent inhibitors were proton and metal ionophores. Oxygen strongly inhibited glucose uptake. Na+ and Li+, but not K+, stimulated glucose uptake. A variety of sugars, including alpha-methylglucoside, did not inhibit glucose uptake. Only cellobiose and 2-deoxy-D-glucose were inhibitory, but neither behaved as a competitive inhibitor. Metabolism of both sugars appeared to be responsible for the inhibition. Cells grown in cellobiose medium transported glucose at one-half the rate of glucose-grown cells. Spheroplasts transported glucose as well as whole cells, indicating glucose uptake is not dependent on a periplasmic glucose-binding protein. Differences in glucose uptake patterns were detected in cells harvested during the transition from the lag to the log phase of growth compared with cells obtained during the log phase. These differences were not due to different mechanisms for glucose uptake in the cell types. Based on the results of this study, B. succinogenes contains a highly specific, active transport system for glucose. Evidence of a phosphoenolpyruvate-glucose phosphotransferase system was not found.     

Mechanism of sugar retention by roots of intact maize and field bean plants

The re-uptake of sugars driven by the proton gradient was studied in sugar net-release and net-uptake experiments using roots of intact maize (Zea mays cv. Blizzard) and field bean (Vicia faba L. cv. Alfred) plants. The net release of sugars into the root medium (0.1 mM CaSO₄) was stimulated by: the protonophore CCCP (10 M); the sulfhydryl reagent NEM (300 M); the specific inhibitor of plasmalemma ATPase vanadate (0.5 mM); and the inhibitor of the glucose carrier phlorizin (2 mM). Net uptake of glucose, fructose and arabinose from 10 M external concentrations was also inhibited by these substances. Surprisingly fusicoccin, a stimulator of net proton release did not effect net sugar uptake. Medium pH values only influenced sugar net uptake if the pH was above 7. It is concluded that a degradation of the proton gradient across the plasmalemma stimulates net sugar release because of disturbed re-uptake of sugars (in particular glucose) via a proton/sugar cotransport system. Thus, the retention of sugars by root cells not only depends on the plasmalemma permeability but also on the electro-chemical proton gradient. If an electro-chemical proton gradient is established by plasmalemma ATPase activity the re-uptake of sugars by proton/sugar cotransport minimizes the release of sugars into the rhizosphere.     

Studies on marine occurring yeasts: Respiration,fermentation and salt tolerance

Summary The effect of various NaCl concentrations on respiration and fermentation rates in cells with or without added glucose as exogenous substrate as well as on respiratory quotients was determined for Debaryomyces hansenii, Saccharomyces cerevisiae, Cryptococcus albidus, and Candida zeylanoides, all yeasts isolated from marine environment. A given strain had about the same respiratory and fermentatory intensity at 0% and 4% NaCl (w/v). A further increase considerably reduced the oxygen uptake or CO₂-evolution. D. hansenii was the most NaCl tolerant yeast tested, giving about 10% activity still at a concentration of 24% NaCl, whether the activities of whole cells or cell homogenates were determined. For S. cerevisiae or Cr. albidus the respiratory activity was reduced to about the same degree at 16% NaCl for whole cells, at 12% NaCl for homogenates of Cr. albidus. A somewhat higher NaCl concentration was evidently tolerated for respiration and fermentation than for growth, very obvious in the case of C. zeylanoides.The minimum values for water activity (a _w) permitting 10% respiration activity were higher when produced by electrolytes (NaCl, KCl, or Na₂SO₄), lower when due to sugars (metabolizable glucose or non-metabolizable lactose) and lowest when due to glycerol. The a _w per se was evidently not solely decisive for the limitation of respiration activity.Attempts were made to assess an effect of high NaCl concentrations on the glucose uptake.The potassium content was higher in cells of the highly halotolerant D. hansenii than in those of the other yeasts and decreased with the increase in external, consequently in internal, Na⁺ concentration. The decrease in K⁺ content can presumably only proceed to a certain extent, below which the ability for growth and respiration was lost.     

Factors affecting glucose uptake by the ruminal bacterium Bacteroides ruminicola

Summary Glucose uptake by whole cells of Bacteroides ruminicola B₁4 is constitutive. Potassium concentrations between 10 and 150 mm stimulated uptake over fourfold, while sodium had little effect on uptake. The involvement of potassium in glucose uptake by B. ruminicola was supported by strong inhibition of uptake by the ionophores valinomycin, lasalocid, and monensin. The electron transport inhibitor antimycin A had little effect on uptake, but menadione and acriflavine inhibited uptake by 30 and 48%, respectively. Potent inhibitors of uptake included oxygen, p-chloromercuribenzoate, HgCl₂, and o-phenanthroline. Sodium arsenate decreased uptake by 40%, suggesting that a high-energy phosphate compound and possibly a binding protein may be involved in glucose uptake. The protonophores carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol inhibited glucose uptake by 37 and 22%, respectively. Little change in uptake activity was observed at extracellular pH values between 4.0 and 8.0. Excess (10 mm) cellobiose, maltose, and sucrose inhibited glucose uptake less than 15%. High levels (0.15% w/v) of p-coumaric acid and vanillin decreased uptake by 32 and 37%, respectively, while 0.15% ferulic acid decreased uptake by 15%.     

The Glucose Transport System in Leishmania tropica Promastigotes*

Transport of glucose by Leishmania tropica promastigotes was measured by the uptake of the nonutilizable glucose analog, 2-deoxy-D-glucose (2-DOG), using the rapid filtration method. Both D-glucose and 2-DOG show identical rates of initial uptake. Intracellular 2-DOG readily exchanges with extracellular D-glucose and 2-DOG uptake is competitively inhibited by D-glucose. These observations suggest that both sugars are taken up by the same system. Neither the glucose analog α-methyl-D-glucoside (α-MG) nor 3-0-methyl glucose (3-0-MG) is taken up to any appreciable extent. Transport of 2-DOG shows saturation kinetics with a V_max of 3.2 nmoles/mg cells/min and a K_m of 0.16 mM. There is thus a stereospecific, carrier-mediated transport system for glucose uptake in L. tropica. About 2/3 of the intracellular pool following transport consists of 2-deoxy-D-glucose phosphate (2-DOG-P) and the remainder is free, unaltered 2-DOG.     

Glucosamine regulates hepatic lipid accumulation by sensing glucose levels or feeding states of normal and excess

Dose-dependent lipid accumulation was induced by glucose in HepG2 cells. GlcN also exerted a promotory effect on lipid accumulation in HepG2 cells under normal glucose conditions (NG, 5 mM) and liver of normal fed zebrafish larvae. High glucose (HG, 25 mM)-induced lipid accumulation was suppressed by l-glutamine-d-fructose 6-phosphate amidotransferase inhibitors. ER stress inhibitors did not suppress HG or GlcN-mediated lipid accumulation. HG and GlcN stimulated protein expression, DNA binding and O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP). Furthermore, both HG and GlcN increased nuclear sterol regulatory element-binding protein-1 (SREBP-1) levels in HepG2 cells. In contrast to its stimulatory effect under NG, GlcN suppressed lipid accumulation in HepG2 cells under HG conditions. Similarly, GlcN suppressed lipid accumulation in livers of overfed zebrafish. In addition, GlcN activity on DNA binding and O-GlcNAcylation of ChREBP was stimulatory under NG and inhibitory under HG conditions. Moreover, GlcN enhanced ChREBP, SREBP-1c, ACC, FAS, L-PK and SCD-1 mRNA expression under NG but inhibited HG-induced upregulation in HepG2 cells. The O-GlcNAc transferase inhibitor, alloxan, reduced lipid accumulation by HG or GlcN while the O-GlcNAcase inhibitor, PUGNAc, enhanced lipid accumulation in HepG2 cells and liver of zebrafish larvae. GlcN-induced lipid accumulation was inhibited by the AMPK activator, AICAR. Phosphorylation of AMPK (p-AMPK) was suppressed by GlcN under NG while increased by GlcN under HG. PUGNAc downregulated p-AMPK while alloxan restored GlcN- or HG-induced p-AMPK inhibition. Our results collectively suggest that GlcN regulates lipogenesis by sensing the glucose or energy states of normal and excess fuel through AMPK modulation.     

THE GLUCOSE TRANSPORT SYSTEM OF AMPHORA COFFEAEFORMIS (BACILLARIOPHYCEAE)1

Amphora coffeaeformis (Ag.) Kütz. var. perpusilla (Grun.) Cleve took up glucose by an inducible transport system. The system was induced by d -fructose, d -mannose, as well as glucose. Some d -pentoses also induced a glucose uptake system but it may not be the same one as that induced by hexose. d -fructose, d -mannose and 2-deoxy-d -glucose inhibited 2 mM glucose uptake at equimolar concentration, but d -pentoses did not. The uptake system decayed in ca. 5 h in the absence of glucose. The half-saturation constant for uptake, K₈ was ca. 0.1 mM glucose with a maximum uptake rate, V_max= 0.4 nmol/10⁶ cells-min^?1.     

Sucrose uptake in isolated phloem of celery is a single saturable transport system

The uptake of different sugars was studied in segments of isolated phloem from petioles of celery (Apium graveolens L.) in order to determine the kinetics and specificity of phloem loading in this highly uniform conductive tissue. The uptake kinetics of sucrose and the sugar alcohol, mannitol, which are both phloem-translocated, indicated presence of a single saturable system, while uptake of non-phloem sugars (glucose and 3-O-methylglucose) exhibited biphasic kinetics with lower uptake rates than those for sucrose and mannitol. The presence of unlabeled mannitol, 3-O-methylglucose and maltose in the incubation solution did not cause inhibition of labeled-sucrose uptake, indicating high carrier specificity and lack of sucrose hydrolysis in vivo. The pH optimum for sucrose uptake was 5–6. Furthermore, a rapid and transient alkalinization of the external media by sucrose indicated a sugar/H⁺-cotransport mechanism. Dual-labeling experiments showed that sucrose influx continued at a constant rate (V _max=15 mol·h^-1·(g FW)^-1), whereas sucrose efflux was low and insensitive to external concentration. Therefore, the saturable uptake kinetics for sucrose did not appear to be the result of an equilibrium between rates of sucrose influx and efflux.Abbreviations 3-OMG 3-O-methylglucose - PCMBS p-chloromercuribenzene sulfonate - SE-CC sieve element-companion cell - VB vascular bundle     

Leucine Uptake and Incorporation by Convolvulus Tissue Culture Cells and Protoplasts under Severe Osmotic Stress

The uptake and incorporation of radioactive leucine by Convolvulus arvensis L. suspension culture cells were studied under various osmotic conditions to provide information about the effects of osmotic stress at the cellular level and about the suitability of various osmotica for stabilizing protoplasts. When manitol, sorbitol, sucrose, or a mixture of CaCl₂ and KCl was added to the cells at a concentration normally used to stabilize protoplasts, the uptake of leucine was inhibited by 50 to 60% and incorporation by 37% with no major differences detected among these osmotica. NaNO₃ of a similar osmotic strength exerted considerably more inhibition, an inhibition that was reversed by as little as 10 mM simultaneous CaCl₂. None of the osmotica altered leucine or protein leakage from the tissue. In general, external solute concentrations below 0.36 osmolal slightly enhanced uptake and incorporation. At successively higher concentrations, uptake and incorporation decreased in a linear fashion, with no apparent discontinuity in the rate of decrease as the cells plasmolyzed. Cycloheximide inhibited both the uptake and the incorporation of leucine in all osmotic situations tested, exerting a much stronger inhibition upon the uptake by control tissue than upon that by cells in osmotica. Different cellulase enzyme preparations varied considerably in their effects on subsequent leucine uptake and incorporation.