Enzyme-catalysed synthesis of galactosylated 1D- and 1L-chiro-inositol, 1D-pinitol, myo-inositol and selected derivatives using the beta-galactosidase from the thermophile Thermoanaerobacter sp. strain TP6-B1

The products from the enzymatic beta-D-galactopyranosylation of 1D-chiro-inositol, 1D-pinitol, 1D-3-O-allyl-4-O-methyl-chiro-inositol, 1D-3,4-di-O-methyl-chiro-inositol, 1L-chiro-inositol and myo-inositol in combined yields ranging from 46% to 64% have been obtained using the beta-galactosidase isolated from an anaerobic extreme thermophile, Thermoanaerobacter sp. strain TP6-B1 and p-nitrophenyl beta-D-galactopyranoside as the donor. Analysis of the products from these reactions reveals information about the acceptor preferences of the enzyme.     

Relationships of plasma C-peptide and gender to the urinary excretion of inositols in older people.

PURPOSE: The urinary excretions of myo-inositol and D-chiro-inositol are elevated in diabetes, and have been suggested as possible markers or effectors of insulin action. The aim of the present study was to measure the urinary excretion of these compounds, and to assess possible relationships with the metabolic control of glucose, in older, non-diabetic men and women. SUBJECTS: 32 older (age range 54-71 yrs), moderately overweight (body mass index 29.1 +/- 0.4 kg/m2, mean +/- SEM), non-diabetic men (n = 17) and women (n = 15). METHODS: 75 g oral glucose tolerance testing was done the day after all subjects had consumed nutrient-defined menus for five days. Plasma samples were analyzed for the concentrations of glucose, insulin, and C-peptide, and the 180-minute area under the curve (AUC) for each of these compounds was calculated. Samples from 24-hour urine collections were analyzed for the concentrations of myo-inositol, D-chiro-inositol, L-chiro-inositol, and pinitol. RESULTS: The fasting glucose, insulin, and C-peptide, and the AUC for glucose and insulin, were not different between men and women. C-peptide AUC was greater in the men versus the women (p < 0.001). The median urinary excretions (micromol/g creatinine) of myo-inositol (p < 0.001), D-chiro-inositol (p < 0.001), L-chiro-inositol (p < 0.05), and pinitol (p < 0.001) were higher, and the myo-inositol:D-chiro-inositol ratio was lower (p < 0.001), in the men versus women. For all subjects combined, C-peptide AUC was positively correlated with the urinary excretion of each of the measured inositols, as well as the myo-inositol:D-chiro-inositol ratio. The correlations between C-peptide AUC and these inositols were strongly influenced by the co-linear relationship between C-peptide AUC and gender. CONCLUSIONS: Collectively, these data show that older, moderately overweight, non-diabetic men and women with gender-related differences in glucose-stimulated C-peptide AUC, an indirect indicator of insulin secretion, also display differences in the urinary excretion of myo-inositol, D-chiro-inositol, L-chiro-inositol, and pinitol. The gender-related difference in the myo-inositol:D-chiro-inositol ratio suggests that, while the urinary excretion of all of the inositols measured were higher in the men than the women, the difference was more pronounced for D-chiro-inositol.     

Purification and characterization of stachyose synthase from lentil (Lens culinaris) seeds: galactopinitol and stachyose synthesis.

Stachyose synthase (STS) (EC 2.4.1.67) was purified 313-fold from mature seeds of lentil. The final preparation had a specific activity of 9.09 nkat stachyose formed per milligram of protein. The enzyme was a monomeric protein with a molecular mass of 88.6 kDa (SDS-PAGE) and an isoelectric point of 4.8 (chromatofocusing). Western analysis revealed cross-reactivity of polyclonal antibodies raised against STS from adzuki bean with the lentil enzyme. The purified enzyme catalyzed a range of different galactosyl transfer reactions. In addition to the genuine STS reaction (raffinose + galactinol --> stachyose + myo-inositol), the enzyme catalyzed the reversible galactosyl transfer from galactinol to d-pinitol (1d-3-O-methyl-chiro-inositol), yielding galactopinitol A (O-alpha-d-galactopyranosyl-(1 --> 2)-4-O-methyl-d-chiro-inositol) and myo-inositol. Galactopinitol A could be further galactosylated by STS to give ciceritol (O-alpha-d-galactopyranosyl-(1 --> 6)-O-alpha-d-galactopyranosyl-(1 --> 2)-4-O-methyl-d-chiro-inositol). Enzymatic synthesis of galactopinitol A and ciceritol is a new observation. However, STS was not only able to utilize galactopinitol A as galactosyl acceptor, but also as galactosyl donor to form stachyose from raffinose. The role of STS in the metabolism of galactosyl cyclitols and oligosaccharides in plant seeds is discussed.     

The role of the phosphatidylinositol cycle in mitosis in sea urchin zygotes. Lithium inhibition is overcome by myo-inositol but not by other cyclitols or sugars

We have investigated the role of the phosphatidylinositol (PI) cycle in cellular events between fertilization and first cleavage in zygotes of the sea urchin Lytechinus pictus. The effects of lithium were studied: The lithium-induced changes due to effects on the PI cycle were reversed by myo-inositol, the next step in the cycle after the lithium block, but were not reversed by scyllo-inositol or other cyclitols or sugars. In this way we implicated the PI cycle in the formation of streak birefringence, in nuclear membrane breakdown, in onset of anaphase, and in cytokinesis. With respect to karyokinesis, mitotic apparatus (MA) structure often was altered when the PI cycle was blocked, and anaphase was blocked when the PI cycle was blocked. For all stages, the effects of 400 mM lithium were overcome by 10-100 microM myo-inositol. Excess myo-inositol potentiated the effect of lithium on MA structure (and on cytokinesis), suggesting that there is a negative feedback loop in the control of the PI cycle.     

Gas chromatographic analysis of in situ cyclitol utilization by Klebsielleae growing in redwood extracts.

Gas chromatographic analysis was employed to demonstrate in situ cyclitol utilization in aqueous extracts of redwood by isolates of Klebsiella, Enterobacter, and several other genera of gram-negative bacteria. In aqueous redwood extracts, all but one of the Klebsiella and Enterobacter isolates tested reached densities exceeding 5.0 x 10(6) cells/ml within 4 days, and all utilized pinitol and sequoyitol. Other enteric bacteria did not utilize cyclitols in this extract. A defined minimal medium, containing the carbohydrates and cyclitols (including myo-inositol) in redwood, was used to determine which carbon sources are preferentially utilized by Klebsielleae and other bacteria. It was found that D-glucose and L-arabinose were consumed by Klebsiella before the three cyclitols were utilized. Pinitol utilization proceeded in more slowly than that of sequoyitol and myo-inositol. Cyclitol utilization in the defined medium was also observed for Yersinia, Erwinia, and Salmonella. Escherichia coli isolates did not utilize cyclitol compounds. The ability to use cyclitols as a sole source of carbon can explain the high cell densities of Klebsielleae in redwood water reservoirs and in redwood lumber.     

Gas chromatographic analysis of in situ cyclitol utilization by Klebsielleae growing in redwood extracts

Gas chromatographic analysis was employed to demonstrate in situ cyclitol utilization in aqueous extracts of redwood by isolates of Klebsiella, Enterobacter, and several other genera of gram-negative bacteria. In aqueous redwood extracts, all but one of the Klebsiella and Enterobacter isolates tested reached densities exceeding 5.0 x 10(6) cells/ml within 4 days, and all utilized pinitol and sequoyitol. Other enteric bacteria did not utilize cyclitols in this extract. A defined minimal medium, containing the carbohydrates and cyclitols (including myo-inositol) in redwood, was used to determine which carbon sources are preferentially utilized by Klebsielleae and other bacteria. It was found that D-glucose and L-arabinose were consumed by Klebsiella before the three cyclitols were utilized. Pinitol utilization proceeded in more slowly than that of sequoyitol and myo-inositol. Cyclitol utilization in the defined medium was also observed for Yersinia, Erwinia, and Salmonella. Escherichia coli isolates did not utilize cyclitol compounds. The ability to use cyclitols as a sole source of carbon can explain the high cell densities of Klebsielleae in redwood water reservoirs and in redwood lumber.     

The Compatibility of d-Pinitol and 1d-1-O-Methyl-Muco-Inositol with Malate Dehydrogenase Activity

Pinitol (1d -3-O-methyl-chiro-inositol) and 1d -1-O-methyl-muco-inositol, two cyclitols wide-spread in the plant kingdom, were isolated from plant sources in order to test their compatibility with malate dehydrogenase activity. Both compounds had no inhibitory effect on malate dehydrogenase from Rhizophora mangle in a range of 100 to 1000 mol . m^?3. Their influence on malate dehydrogenase activity from different plant sources (Rh. mangle L., Mesembryanthemum crystallinum L., Cicer arietinum L. and Spinacia oleracea L.) was also small and similar to that observed for a number of well established compatible solutes (e.g. proline, glycine betaine). A possible role of cyclitols as cryoprotectants or radical scavengers is discussed.     

Fluorinated cyclitols as useful biological probes of phosphatidylinositol metabolism

A number of deoxyfluoro cyclitols have been synthesized and evaluated as probes of the phosphatidylinositol pathway (PtdIns pathway), most notably 5-deoxy-5-fluoro-myo-inositol, which is incorporated into the pathway at about 25% the level of myo-inositol itself. Unfortunately, none of the cyclitols have proved effective in limiting cell proliferation, as the cells are able to 'synthesize around' the fraudulent cyclitols using natural myo-inositol as substrate. Inhibitors for 3-phosphatidylinositol kinase, which has importance in a number of pathological conditions, including cancer, have been intensively investigated. 3-Deoxy-3-fluoro-myo-inositol is incorporated into the PtdIns pathway; however, only related phosphatidyl derivatives, for example, a methyl ether derivative of the 3-deoxy inositol, showed significant antiproliferative activity. Synthesis of the deoxyfluoro analogues most often has been accomplished by DAST fluoro-de-hydroxylation of the appropriate cyclitol, generally leading to products of inversion.     

New syntheses of 1D- and 1L-1,2-anhydro-myo-inositol and assessment of their glycosidase inhibitory activities

The 1D and 1L enantiomers of 1,2-anhydro-myo-inositol (conduritol B epoxide) were synthesised from 1D-pinitol and 1L-quebrachitol, respectively, and their activities were compared in selected glycosidase inhibition assays. The 1D enantiomer was found to be the active isomer, functioning as an irreversible inhibitor of sweet almond beta-D-glucosidase. Neither isomer was active against the alpha-D-glucosidase from Bacillus stearothermophilus or the beta-D-galactosidase from Aspergillus oryzae.     

Characterization of the myo-inositol transport system in Trypanosoma cruzi.

myo-inositol is a growth factor for mammalian cells as well as for the pathogenic protozoa Trypanosoma cruzi. Most of the cell surface molecules in this organism rely on myo-inositol as the biosynthetic precursor for phosphoinositides and glycosylated phosphatidylinositols. The aim of this work was to investigate the process of myo-inositol translocation across the parasite cell membrane. myo-Inositol uptake was concentration-dependent in the concentration range 0.1-10 microM with maximal transport obtained at 8 microM. Using sodium-free buffers, where Na+ was replaced by choline or K+, myo-inositol uptake was inhibited by 50%. Furosemide, an inhibitor of the ouabain-insensitive Na+-ATPase, inhibited the Na+-dependent and Na+-independent myo-inositol uptake by 68 and 33%, respectively. In contrast, ouabain, an (Na++/K+) ATPase inhibitor, did not affect transport. Part of the myo-inositol uptake is mediated by active transport as it was inhibited when energy metabolism inhibitors such as carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (34%), 2,4-dinitrophenol (50%), KCN (71%) and NaN3 (69%) were added to the medium, or the temperature of the medium was lowered to 4 degrees C. The addition of glucose (5-50 mM) or mannose (10 mM) did not change the myo-inositol uptake, whereas the addition of 10 mM nonlabeled myo-inositol totally inhibited this transport, indicating that the transporter is specific for myo-inositol. Phloretin (0.3 mM) and phoridzin (5 mM), but not cytochalasin B, were efficient inhibitors of myo-inositol uptake. A portion of the accumulated myo-inositol is converted to inositol phosphates and phosphoinositides. These data show that myo-inositol transport in T. cruzi epimastigotes is mediated by at least two specific transporters - one Na+-dependent and the other Na+-independent.     

myo-Inositol Transport in Mouse Astroglia-Rich Primary Cultures

Uptake of radiolabeled myo-inositol was studied in astroglia-rich primary cultures derived from neonatal mouse brains. The uptake was saturable in the presence of Na+ with a Km of 25 microM and a Vmax of 60 pmol.min-1.(mg protein)-1, suggesting a high-affinity transport system for myo-inositol in astroglial cells. In addition, a Na(+)-independent, nonsaturable component was found. Carrier-mediated uptake was not inhibited by cytochalasin B (50 microM), but was reduced by depolarizing concentrations of K+ and, to different extents, in the presence of phloretin, ouabain, or amiloride (1 mM each). scyllo-Inositol, glucose, and galactose also reduced myo-inositol uptake; inhibition by the two hexoses was not reversed in the presence of 0.4 mM sorbinil. On the other hand, uptake of 2-deoxyglucose was not inhibited by high concentrations of myo-inositol. Preincubation of the cells with glucose-free or inositol-free medium stimulated uptake of myo-inositol and preincubation with 25 mM glucose in the presence of 0.4 mM sorbinil had no effect on the rate of uptake. The results suggest that myo-inositol is taken up into the astroglial cells by a transport mechanism that is distinct from that of glucose and probably is an active one. Sorbitol pathway activity does not interfere with myo-inositol uptake.     

Temperature dependence and mechanism of local anesthetic effects on mitochondrial adenosinetriphosphatase

Chloroform-released ATPase prepared from beef heart mitochondria is inhibited by tetracaine and dibucaine over the entire temperature range in which the enzyme is active. The temperature of maximal activity is at 60 degrees C in the absence of anesthetic and is shifted upward by 2-3 degrees C by the addition of 0.3 mM dibucaine. Local anesthetics protect ATPase from irreversible cold inactivation. The kinetics of this protective effect are analyzed by a thermodynamic model in which the associated/dissociated subunit equilibrium is shifted toward the associated state by the preferential binding of anesthetic to the associated state. The accessibility of buried sulhydryl groups to reaction with 5,5'-dithiobis(2-nitrobenzoic acid) is increased by local anesthetics; this is interpreted to mean that the anesthetics increase the conformational flexibility of the protein. It is proposed that the hydrophobic moieties of local anesthetics and related compounds bind to numerous hydrophobic sites or crevices on ATPase; this binding induces a perturbation of the protein conformation, which in turn causes a decrease of enzyme activity. This model is sufficiently general to encompass the diversity of molecules which have similar anesthetic-like effects, and since it relates to common fundamental features of protein structure, it may also be the mechanism of the nonspecific effects of these molecules on other proteins.     

Seasonal Distribution of Carbohydrates in Nodules and Stem Exudate from Field-grown Soya Bean Plants

The concentration of carbohydrates in tap root nodules fromfield-grown soya bean [Glycine max (L.) Merr.] plants was verysimilar to the concentration of compounds previously reportedin greenhouse-grown nodules during vegetative growth of seedlings.The concentration of D-pinitol, sucrose and starch in nodulesdeclined during rapid fruit growth, but the concentration ofother compounds did not decline. The availability of carbohydratein nodules during fruit growth did not seem likely to be thecause of the decline in nitrogen-fixing activity of noduleswhich has been reported by others. All compounds except glucoseand , -trehalose declined to concentrations near zero duringa 10-day period of nodule decay. However, the decline in carbohydratedid not appear to cause nodule senescence because it did notprecede the period of decay and because decayed nodules containedsubstantial quantities of glucose and , -trehalose. Seasonalmean concentrations (72 samples from 24 dates) of compounds,in mg carbohydrate per g f. wt of nodule, were: sucrose, 2.84;D-pinitol, 1.14; D-chiro-inositol, 1.27; glucose, 1.40; , -trehalose,1.34; myo-inositol, 0.65; maltose, 0.31; and fructose, 0.21. Quantities of sugars and cyclitols in stem exudate collectedin the field on 13 dates were small (< 10 percent) relativeto the quantity of nitrogenous compounds transported from rootsto shoots. The seasonal pattern of pinitol transport in thexylem was very similar to the seasonal pinitol concentrationin nodules. A large increase in sugar concentration in stemexudate subsequent to 80 days after planting supports the viewthat lack of carbohydrate was not a cause of nodule senescence. Glycine max (L.) Merr, soya bean, cyclitols, , -trehalose, starch, D-pinitol, carbohydrates, root nodules, senescence     

D-Chiro-Inositol – Its Functional Role in Insulin Action and its Deficit in Insulin Resistance

In this review we discuss the biological significance of D-chiro-inositol, originally discovered as a component of a putative mediator of intracellular insulin action, where as a putative mediator, it accelerates the dephosphorylation of glycogen synthase and pyruvate dehydrogenase, rate limiting enzymes of non-oxidative and oxidative glucose disposal.Early studies demonstrated a linear relationship between its decreased urinary excretion and the degree of insulin resistance present. When tissue contents, including muscle, of type 2 diabetic subjects were assayed, they demonstrated a more general body deficiency. Administration of D-chiro-inositol to diabetic rats, Rhesus monkeys and now to humans accelerated glucose disposal and sensitized insulin action.A defect in vivo in the epimerization of myoinositol to chiro-inositol in insulin sensitive tissues of the GK type 2 diabetic rat has been elucidated. Thus, administered D-chiro-inositol may act to bypass a defective normal epimerization of myo-inositol to D-chiro-inositol associated with insulin resistance and act to at least partially restore insulin sensitivity and glucose disposal.     

Differential behavior of the two free sulfhydryl groups of human plasma fibronectin: effects of environmental factors.

We report here a novel approach to label specifically one of the two cryptic, free sulfhydryl groups per subunit of human plasma fibronectin with either an 15N,2H-maleimide spin label or a coumarinylphenyl maleimide fluorescent label. This permits the use of electron spin resonance (ESR) or fluorescence techniques to study molecular dynamics of fibronectin with the label attached to a single site per chain on the protein molecule. The method is based on our observation that upon adsorption of fibronectin to a gelatin-coated surface, the SH1 site, located between the DNA-binding and the cell-binding domains, is partially exposed, while the SH2 site, located within the carboxyl-terminal fibrin-binding domain, remains buried and unreactive. The procedures for the preparation of the selectively labeled fibronectins are described in detail. The physicochemical properties of these single-site labeled fibronectins, particularly as affected by high salt, heparin, surface binding, and temperature, were characterized by ESR spin-label and steady-state fluorescence techniques. The steady-state fluorescence measurement indicates that both local environments of SH1 and SH2 sites are relatively hydrophobic, and that the SH2 site is more hydrophobic than the SH1 site. The ESR results show that heparin or high salt induces an increase in the domainal flexibility in both SH1 and SH2 regions, perhaps through the disruption of domain-domain interactions in the fibronectin molecule, and that the former is more effective than the latter in producing such an effect. The observed heparin effect is reversible by addition of calcium ions in the SH2 regions but not in the SH1 regions. In addition, at temperatures above 44 degrees C, both type III homologous regions containing the free sulfhydryl groups are shown to undergo denaturation and aggregation processes. The data presented here suggest that the newly developed method for differential labeling of the free sulfhydryl groups in fibronectin should be useful for mapping the spatial arrangement of structural domains in the protein molecule using spin-label-spin-probe and fluorescence energy transfer techniques.     

Carbohydrates from Detarium microcarpum bark extract

The bark extract of the medicinal plant Detarium microcarpum was analysed for its carbohydrate content by GLC-CIMS. Preparative HPLC of the benzoylated carbohydrate fraction led to the isolation of L-quino-1,5-lactone, D-(-)-bornesitol, D-pinitol, myo-inositol, sucrose, D-glucose, and D-fructose benzoates, which were characterised by NMR spectroscopy experiments.     

Modeling the structure of the respiratory syncytial virus small hydrophobic protein by silent-mutation analysis of global searching molecular dynamics

Human respiratory syncytial virus (RSV) encodes a small hydrophobic (SH) protein, whose function in the life cycle of the virus is unknown. Recent channel activity measurements of the protein suggest that like other viroporins, SH may assemble into a homo-oligomeric ion channel. To further our understanding of this potentially important protein, a new strategy was implemented in order to model the transmembrane oligomeric bundle of the protein. Global searching molecular dynamic simulations of SH proteins from eight different viral strains, each at different oligomeric states, as well as different lengths of the putative transmembrane domain, were undertaken. Taken together, a total of 45 different global molecular dynamic simulations pointed to a single pentameric structure for the protein that was found in all of the different variants. The model of the structure obtained is a channel-like homopentamer whose minimal transmembrane pore diameter is 1.46 A.     

Temperature dependence of benzyl alcohol- and 8-anilinonaphthalene-1-sulfonate-induced aggregation of recombinant human interleukin-1 receptor antagonist

The critical role played by temperature in ligand-induced protein aggregation was investigated. Recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and the ligands benzyl alcohol and 8-anilinonaphthalene-1-sulfonate (ANS) were used. We investigated aggregation kinetics and the conformation and cysteine reactivity of rhIL-1ra in buffer alone or in the presence of 0.9% (w/v) benzyl alcohol or 4.2 or 21 mM ANS at 25 and 37 degrees C. In buffer, protein aggregation was not detected at 25 degrees C but occurred at 37 degrees C. At 25 degrees C, neither benzyl alcohol nor 4.2 mM ANS enhanced aggregation. However, at 37 degrees C, both compounds greatly accelerated protein aggregation. With 21 mM ANS, rhIL-1ra aggregation was accelerated at both temperatures, but the effect was more pronounced at 37 degrees C than at 25 degrees C. Increasing the temperature from 25 to 37 degrees C caused a minor perturbation in the tertiary structure of rhIL-1ra in buffer but no detectable alteration in secondary structure. Benzyl alcohol enhanced the tertiary structural perturbation at 37 degrees C, but the secondary structure was not affected by the ligand. The reactivity of buried free cysteines of rhIL-1ra was enhanced by benzyl alcohol at 37 degrees C but not at 25 degrees C, consistent with the structural results. Isothermal titration calorimetry documented that the interaction of benzyl alcohol with rhIL-1ra was hydrophobic and that the degree of hydrophobic interactions increased with temperature. At 25 degrees C, the interaction of ANS with rhIL-1ra was electrostatic, but at 37 degrees C, both electrostatic and hydrophobic interactions were important. Taken together, our results support the conclusion that benzyl alcohol and ANS interact hydrophobically with partially unfolded aggregation-prone protein molecules, resulting in temperature-dependent increases in their levels and acceleration of protein aggregation.     

Reducing sugars can induce the oxidative inactivation of rhodanese.

The enzyme rhodanese (thiosulfate sulfurtransferase, EC 2.8.1.1) is inactivated on incubation with reducing sugars such as glucose, mannose, or fructose, but is stable with non-reducing sugars or related polyhydroxy compounds. The enzyme is inactivated with (ES) or without (E) the transferable sulfur atom, although E is considerably more sensitive, and inactivation is accentuated by cyanide. Inactivation of E is accompanied by increased proteolytic susceptibility, a decreased sulfhydryl titer, a red-shift and quenching of the protein fluorescence, and the appearance of hydrophobic surfaces. Superoxide dismutase and/or catalase protect rhodanese. Inactive enzyme can be partially reactivated during assay and almost completely reactivated by incubation with thiosulfate, lauryl maltoside, and 2-mercaptoethanol. These results are similar to those observed when rhodanese is inactivated by hydrogen peroxide. These observations, as well as the cyanide-dependent, oxidative inactivation by phenylglyoxal, are explained by invoking the formation of reactive oxygen species such as superoxide or hydrogen peroxide from autooxidation of alpha-hydroxy carbonyl compounds, which can be facilitated by cyanide.     

Proteome analysis of tobacco leaves under salt stress

The mechanisms responsible for the effects of salt stress on tobacco plants were examined by means of proteomic analysis. Tobacco plants were exposed to 0, 150, 250, 300, or 400 mM NaCl. At 150 mM NaCl or above, the plants showed a reduction in fresh weight and an increase in proline levels. Proteins extracted from the leaves of tobacco plants exposed to 150 mM NaCl were separated by 2-DE. Of 205 protein spots that were detected reproducibly in each gel, 18 were differentially expressed under NaCl treatment. Up-regulated proteins belonged to the photosynthesis category, whereas down-regulated proteins correspond to defense-related functions. Dose- and time-dependent studies showed that a stromal 70-kDa heat shock-related protein was markedly down-regulated by NaCl. Thus, down-regulation of the stromal 70-kDa heat shock protein in response to salt stress is likely the cause of failure to protect cells against salt stress of tobacco plants.