Cell-wall lectins during winter wheat cold hardening

The polypeptide composition and functional activity of cell-wall lectins from roots of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) seedlings during cold hardening were studied. Several phases of lectin activity changes were observed, which indicates their involvement in the development of general adaptation syndrome of the cell. After 0.5-h low-temperature treatment, marked alterations occurred in the profile of protein elution: lectins with mol wts of 78 and 42.5 kD disappeared and new ones with mol wts of 72, 69, 37, and 34.5 kD appeared. It was established that 17.5-and 69-kD lectins and most lectins eluted with glucose were arabinogalactan proteins (AGP), which permitted a supposition that these lectins were involved in the interaction between the cell wall and cytoskeleton. After 7-day-long hardening, total protein content reduced and lectins with mol wts of 69 and 37 kD disappeared, which corresponded to reduced lectin activity by the end of hardening. A transient appearance of 37-and 69-kD lectins, which are AGP, might indicate their involvement in the triggering the development of plant-cell defense responses.     

Lectins of the <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> pollen grain walls stimulating in vitro pollen germination

Proteins diffusing from tobacco pollen grains into external medium, being inactivated by low temperature (0°C), were shown to stimulate pollen germination in vitro. Fractionation of these proteins by affinity chromatography using α-D-methylmannopyranoside (MMP) immobilized on agarose resulted in the isolation of lectins stimulating germination. The mol wts of these lectins were estimated by SDS-PAGE as 58, 69, and 74 kD. A stimulatory effect of these lectins was determined by their specific interaction with carbohydrate determinants because a competitive sugar (0.3 M MMP) suppressed completely lectin effect on germination. Polyvalent lectins capable of erythrocyte agglutination were also found among diffused proteins. These lectins are glycoproteins with Glu/Man carbohydrate determinants. MMP did not affect their capability of agglutination. This finding permits a conclusion that pollen grain wall contains lectins differing in their carbohydrate specificity.     

DNA-Binding Proteins and Structural Characteristics of Chloroplast Nucleoids

A comparative analysis of proteins from chloroplast nucleoids was performed in two higher-plant species (Pisum sativumL. andArabidopsis thalianaL.) and a green alga Chlamydomonas reinhardtiiDang. In the nucleoids of the higher plants and the alga, 26–27 proteins were detected with their mol wts ranging from 10 to more than 94 kD. In all the species tested, the distribution of nucleoid proteins by their mol wts was similar, especially between the predominant proteins with mol wts of 10 to 40 kD. Six DNA-binding proteins (12–18 kD) were detected in nucleoids fromCh. reinhardtiichloroplasts after in vivocovalent cross-linking between chloroplast DNA and proteins. Under an electron miscroscope, some regular structures resembling nucleosome-like particles of bacterial cells were revealed.     

Human non-secretory ribonucleases. I. Purification, peptide mapping and lectin blotting analysis of the kidney, liver and spleen enzymes

Human non-secretory neutral ribonucleases (RNases) from kidney,liver and spleen have been purified and characterized. SDS—PAGEindicates that all three RNases are highly purified and haveapparent mol. wts of 17–18 kDa. Kinetic analysis indicatesthat all three RNases have a broad pH optimum centred around6.5, and all three have similar substrate specificities withsignificant preference for RNA and poly(U) when compared topoly(C), poly (A) and poly(G). All of the above data, as wellas immunoblotting data using three polyclonal antibodies (anti-humanliver RNase, anti-human pancreatic RNase, anti-human eosino-phil-derivedneurotoxin), indicate that the three proteins are highly purifiedand are non-secretory RNases (IIN). Further characterizationby cyanogen bromide peptide mapping and extensive lectin blottingindicated no significant differences between the three humanRNases. All three RNases appear to have very similar, if notidentical, protein backbones and all three are glycoproteinswhich are recognized by lectins with specificity for GlcNAc,Fuc and, to a lesser extent, with specificity for Galß(1–4)GlcNAc.No significant tissuespecific differences were found among thethree human non-secretory RNases. lectin blotting non-secretory RNases peptide mapping     

Prediction and comparison of the secondary structure of legume lectins

Secondary structure prediction for the 4 legume lectins: Concanavalin A, soybean agglutinin, favabean lectin and lentil lectin, was done by the method of Chou and Fasman. This prediction shows that these four lectins fall into a structurally distinct class of proteins, containing high amounts of β-sheet and β-turns. There is a notable similarity in the gross structure of these proteins; all four of them contain about 40–50% of β-sheet, 35–45 % β-turn and 0–10% of α-helix. When the secondary structure of corresponding residues in each pair of these lectins was compared, there was a striking similarity in the Concanavalin A-soybean agglutinin and favabean lectin-lentil lectin pairs, and considerably less similarity in the other pairs, suggesting that these legume lectins have probably evolved in a divergent manner from a common ancestor. A comparison of the predicted potential β-turn sites also supports the hypothesis of divergent evolution in this class of lectins.     

Lectin binding characterstics of mouse epididymal fluid and sperm extracts

Glycoproteins from luminal fluid of the mouse cauda epiciidymidis have been compared with glycoproteins from Triton X-100 extracts of mouse spermatozoa from varying regions of the epididymis, using lectins with specific affinity for different sugar residues. Concanavalin A recognizes 11 glycocomponents on Western blots of fractionated caudal fluid; wheat germ agglutinin (WGA) binds 12 proteins; Ulex europaeus agglutinin (UEA) binds seven; and Dolichos biflorus agglutinin (DBA) recognizes nine. Several of these glycoproteins display an affinity for more than one lectin, indicating a diversity in their exposed carbohydrate residues; whereas other proteins bind only one of the four lectins used. The results also show that some glycoproteins exhibit a higher affinity for particular lectins. Eight glycoproteins of similar mobility and lectin-binding characteristics are detected in Triton X-100 extracts of spermatozoa from different regions of the epididymis and in caudal fluid. The lectin affinity of some proteins appears or increases in spermatozoa from distal epididymal regions (54 kD, 32 kD), whereas the lectin affinity of others decreases (29 kD, 40 kD). There are differences in lectin affinities between proteins in sperm extracts and in caudal fluid. Some proteins show an affinity for three or four lectins in caudal fluid, but proteins of similar electrophoretic mobility in sperm extracts bind only one or two of the lectins. These data show that glycoproteins of similar mobility are present in caudal fluid and in Triton-X-100 sperm extracts, implying a potential interaction between caudal fluid components and epididymal sperm.     

Proteins of cotyledons of mature horse chestnut seeds

This is the first characterization of proteins from storage parenchyma of cotyledons of mature dormant recalcitrant horse chestnut (Aesculus hippocastanum L.) seeds and evaluation the cell protein-synthesizing capacity. It was established that the content of protein in cotyledons did not exceed 0.5% of tissue fresh weight. Soluble proteins (the proteins of the postmitochondrial supernatant or cytosol) comprised the bulk (up to 90%) of total proteins. Protein of subcellular structures (20000 g-pellet) comprised 5–7% of total protein. Cotyledon proteins were heterogenous in their charges and molecular weights of subunits. Cotyledon protein was easily extracted with a salt (1 M NaCl); they comprised 90% of water-soluble albumin-like proteins. The proportion of globulins was insignificant; it did not exceed 5%. Most water-soluble proteins (more than 80%) were tolerant to heat denaturing. Among these heat-stable proteins, two major groups of polypeptides dominated: an electrophoretically homogeneous component with a mol wt of 24–25 kD and a complex group from three to five polypeptides with mol wts in the range between 6 and 12 kD. Native heat-stable proteins had disulfide bonds. Four fractions of heat-stable proteins were obtained by ammonium sulfate fractionation; three of them were alike in their polypeptide composition and contained major components with mol wts of 24–25 and 5–12 kD. It was established that the active translational machinery functioned in the cells of storage parenchyma in cotyledons of mature dormant horse chestnut seeds. During each stage of stratification, cotyledon fragments incorporated ³⁵S-methionine into TCA-insoluble material more actively than axial organs. We discuss cotyledon protein composition, their function as a storage organ, and a possible role of heat-stable proteins.     

Toxicity of lectins and processing of ingested proteins in the pea aphidAcyrthosiphon pisum

Acute toxicity of thirty lectins was tested against the pea aphidAcyrthosiphon pisum (Harris) (Homoptera, Aphididae: Macrosiphini). Activity was measured on artificial diets containing moderate concentrations of lectins (10–250 μg/ml) by scoring mortality and growth inhibition over the whole nymphal period (7 days at 20°C). Most of the proteins tested exhibited low toxicity, but some induced significant mortality; these included the lectins from jackbean (Concanavalin A), amaranth, lentil and snowdrop. There was no direct correlation between toxicity and sugar specificity of the lectin; however, many mannose-binding lectins were toxic towardsA. pisum. Concanavalin A was also tested on five other aphid species (Aphis gossypii, Aulacortum solani, Macrosiphum euphorbiae, Macrosiphum albifrons andMyzus persicae) at concentrations between 10–1500 μg/ml. Mortality was very variable from one species to another. Strong growth inhibition invariably occurred within this concentration range, although dose-response curves differed substantially between aphid species. The peptidase complement ofA. pisum’s digestive tract was also investigated, as well as the oral toxicity of some protease inhibitors (PIs) to this aphid. Most protein PIs were inactive, and no part of the digestive tract contained detectable amounts of endo-protease activity. This is in contrast to the strong amino-peptidase activity which was shown to occur predominantly in the midgut and crop portions of the digestive tract. The potential of lectins in transgenic crops to confer Host-Plant Resistance to aphids is discussed.     

Association of dehydrins with wheat mitochondria during low-temperature adaptation

In mitochondria from the crowns of field-grown winter wheat plants or their seedlings hardened in the laboratory, thermostable proteins immunologically related to dehydrins were detected. It was found that two dehydrins with mol wts of 63 and 52 kD bound with the outer mitochondrial membrane during autumnal hardening or during adaptation to low temperature in the laboratory. Dehydrins of similar mol wts were detected among proteins in the total membrane fraction from low-temperature-adapted wheat plants. In addition, dehydrins with mol wts of 209 and 196 kD were present in this fraction as well. Dehydrins of similar mol wts were bound with mitochondria from seedlings adapted to low temperature and those from the crowns of plants after autumnal hardening. In spring, the amount of dehydrins associated with mitochondria from the crowns declined to the level characteristic of early autumn. Dehydrin association with mitochondria is evidently an important defense mechanism of frost-resistant plants.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 221–226.Original Russian Text Copyright © 2005 by Borovskii, Stupnikova, Antipina, Anuchina, Voinikov.This revised version was published online in April 2005 with a corrected cover date.     

Spatial Distribution of Lectin Activity in Perilesional Areas of Tobacco Leaves Inoculated with Tobacco Mosaic Virus

The activities of lectins and peroxidase and lignin content were studied in the perilesional area of leaves in two tobacco species (Nicotiana tabacumL., cultivar Samsun NN, and N. glutinosa L.) inoculated with tobacco mosaic virus. The development of hypersensitivity response proved to be accompanied by a complex spatial and temporal distribution of lectin activity. The area 5–9 mm away from the lesion center was characterized by the highest activity of loosely bound membrane lectins eluted with 0.05% Triton X-100. In the fraction of tightly bound membrane lectins (eluted with 0.5% detergent), lectin activity decreased during the first two days but increased on day 4 after inoculation. The activity of loosely bound membrane lectins increased in the leaf areas distant from the lesion. Two-phase dynamics in the interlesional area were also observed for lectin activity in the tightly bound membrane fraction (decrease on day 2 days and increase on day 4 after inoculation) and for peroxidase activity (increase on day 2 days and decrease on day 4). The relationship between the dynamics and spatial distribution of lectins in the perilesional area and the possible involvement of these proteins in pathogen-induced changes in photosynthesis are discussed.     

Voltage-dependent calcium channels in cultivated neurons of the rat hippocampus

Calcium currents through the somatic membrane of cultivated (a low-density culture) hippocampal neurons of rats were studied with the use of a patch-clamp technique in the whole-cell configuration. Low- and high-threshold components of calcium currents were found in the somata of all studied cells. Low-threshold currents were activated at a membrane potential of about−75 mV and reached the maximum amplitude at −45±4 mV, while the maximum amplitude of high-threshold currents was observed at 17±6 mV. Low-threshold calcium currents differed from high-threshold current in weak suppression by low Cd²⁺ concentration (10–20 μM), while Ni²⁺ inhibited both types of calcium currents to an equal extent. Experiments with organic channel blockers showed that in most neurons at least four channel types were expressed: these were L, N, P, and channels insensitive to the used blockers (presumably, R-type). A blocker of L-type calcium channels, nifedipine (10 μM), blocked, on the average, 22.7±5.2%; a blocker of N-type channels, ω-CTx-GVIA (1.0 μM), blocked 30.0±5.0% and a blocker of P/Q channels, ω-Aga-IVA (200 nM), blocked 37.2±13.3% of the integral high-threshold current. A resistive component equalled 15.7±5.1% of the latter current. It is concluded that hippocampal neurons cultivated with a low density express a pharmacologically heterogeneous population of calcium channels, and the relative proportions of different type channels are close to the earlier described channel type composition in rat hippocampal slices. Our study shows that the low-density culture can be used as an adequate model for studying calcium channels in the somatic membrane of hippocampal neurons.     

The major tuber storage protein of araceae species is a lectin. Characterization and molecular cloning of the lectin from Arum maculatum L.

A new lectin was purified from tubers of Arum maculatum L. by affinity chromatography on immobilized asialofetuin. Although this lectin is also retained on mannose-Sepharose 4B, under the appropriate conditions free mannose is a poor inhibitor of its agglutination activity. Pure preparations of the Arum lectin apparently yielded a single polypeptide band of approximately 12 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, N-terminal sequencing of the purified protein combined with molecular cloning of the lectin have shown that the lectin is composed of two different 12-kD lectin subunits that are synthesized on a single large precursor translated from an mRNA of approximately 1400 nucleotides. Lectins with similar properties were also isolated from the Araceae species Colocasia esculenta (L.) Schott, Xanthosoma sagittifolium (L.) Schott, and Dieffenbachia sequina Schott. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration of the different Araceae lectins have shown that they are tetrameric proteins composed of lectin subunits of 12 to 14 kD. Interestingly, these lectins are the most prominent proteins in the tuber tissue. Evidence is presented that a previously described major storage protein of Colocasia tubers corresponds to the lectin.     

Effect of Azospirillum Lectins on the Activities of Wheat-root Hydrolytic Enzymes

This work studied the effect of two cell-surface lectins isolated from the nitrogen-fixing soil bacterium Azospirillum brasilense Sp7 and from its mutant defective in hemagglutinating activity, A. brasilense Sp7.2.3, on the activities of α-glucosidase, β-glucosidase and β-galactosidase in the exocomponent, membrane and apoplast fractions of wheat-seedling roots. Lectin (40 μg mL⁻¹) incubation for 1 h of the plant fractions increased the enzymes’ activities; both wild-type and mutant lectins were most stimulatory to the activities of all the exocomponent-fraction enzymes studied and to the apoplast-fraction β-glucosidase. Pretreatment of the lectins with their carbohydrate hapten, L-fucose, lowered the effect. The observed differences in the lectins’ ability to influence enzyme catalytic activity are explained by change in the antigenic properties of the mutant lectin.     

Garlic compounds induced calpain and intrinsic caspase cascade for apoptosis in human malignant neuroblastoma SH-SY5Y cells

Malignant (N-type) neuroblastoma continues to defy current chemotherapeutic regimens. We tested the garlic compounds diallyl sulfide (DAS) and diallyl disulfide (DADS) for induction of apoptosis in human malignant neuroblastoma SH-SY5Y cells. Viability of human primary neurons was unaffected after 24 h treatment with 50 and 100 μM DAS and 50 μM DADS but slightly affected with 100 μM DADS. Treatment with 50 and 100 μM DAS or DADS significantly decreased viability in SH-SY5Y cells. Wright staining showed morphological features of apoptosis in SH-SY5Y cells treated with 50 and 100 μM DAS or DADS for 24 h. ApopTag assay demonstrated DNA fragmentation in apoptotic cells. Apoptosis was associated with an increase in [Ca²⁺]_i, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, increase in cytosolic Smac/Diablo, and down regulation of inhibitor-of-apoptosis proteins and nuclear factor kappa B (NFκB). Activation of caspase-9 and caspase-3 indicated involvement of intrinsic pathway of apoptosis. Calpain and caspase-3 activities produced 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Also, caspase-3 activity cleaved inhibitor of caspase-activated DNase (ICAD). Results strongly suggested that the garlic compounds DAS and DADS suppressed anti-apoptotic factors and activated calpain and intrinsic caspase cascade for apoptosis in SH-SY5Y cells.     

Woody plants of Yakutia and low-temperature stress

Physiological and biochemical features of woody plants (Pinus sylvestris L. and Betula platyphylla Sukacz.) during transition from vegetative to frost-resistant state under conditions of extremely severe climate of Yakutia were studied. In P. sylvestris such transition was accompanied by a decrease in the content of chlorophylls long before first frosts and by an increase in the proportion of Xanth and simultaneous decrease in the content of β-carotene in needles during the first and second phases of hardening. In the period of preparation to dormancy, overwintering organs of both P. sylvestris (needles) and B. platyphylla (buds) accumulated the two groups of major dehydrins, with low mol wts of 15–21 kD and middle mol wts of 66–141 kD. Simultaneously, low temperature led to a great increase in the content of polyunsaturated fatty acids (FAs) in lipids of P. sylvestris needles and B. platyphylla buds, primarily linoleic acid and also eicosenoic FAs differing in the extent of desaturation. Observed qualitative and quantitative changes in pigments, total proteins, dehydrins, and FAs during autumn hardening of P. sylvestris and B. platyphylla plants presume their important role in the development of resistance of these tree species to low-temperature stress (down to −60°C) in the cryolithic zone of Yakutia.     

A mannose-specific tetrameric lectin with mitogenic and antibacterial activities from the ovary of a teleost,the cobia (<Emphasis Type="Italic">Rachycentron canadum</Emphasis>)

A tetrameric lectin, with hemagglutinating activity toward rabbit erythrocytes and with specificity toward d-mannosamine and d(+)-mannose, was isolated from the ovaries of a teleost, the cobia Rachycentron canadum. The isolation protocol comprised ion exchange chromatography on CM-cellulose and Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q, and finally gel filtration by FPLC on Superose 12. The lectin was adsorbed on all ion exchangers used. It exhibited a molecular mass of 180 kDa in gel filtration on Superose 12 and a single 45-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is a tetrameric protein. The hemagglutinating activity of the lectin was stable up to 40°C and between pH 4 and pH 10. All hemagglutinating activity disappeared at 60°C and at pH 1 and pH 13. The hemagglutinating activity was doubled in the presence of 0.1 μM FeCl₃. The lectin exerted antibacterial activity against Escherichia coli with 50% inhibition at 250 μg. There was no antifungal activity toward Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, and Rhizoctonia solani at a dose of 300 μg. The lectin exhibited maximal mitogenic response from mouse splenocytes at a concentration of 14 μM.     

Effect of Ca<Superscript>2+</Superscript> and cAMP on protein phosphorylation in mitochondria of maize seedlings

The effect of common intracellular signals (Ca²⁺ and cAMP) on the activity of protein phosphorylation in mitochondria was investigated in coleoptiles of maize (Zea mays L.). Treatment of isolated mitochondria with 2 mM CaCl₂ brought about an increase in the level of phosphorylation of proteins with mol ws of 74, 60, and 33 kD but considerably reduced phosphorylation of the protein with a mol wt of 51.5 kD. In the presence of Ca²⁺, phosphorylation of polypeptides with mol wts of 59 and 66 kD was also detected. cAMP considerably reduced phosphorylation of essentially all the investigated proteins in isolated mitochondria, which could be explained by activation of their dephosphorylation. Phosphorylation of mitochondrial proteins involves a polypeptide of about 94 kD showing kinase activity, which may be proper protein kinase or one of the subunits of a compound structure. In maize mitochondria, PP1A phosphatases were found. A hypothesis was advanced that redox-dependent phosphorylation/dephosphorylation of mitochondrial proteins plays an important role in mitochondrial signaling in higher plants.     

Alpha-Ketoisocaproic Acid Increases Phosphorylation of Intermediate Filament Proteins from Rat Cerebral Cortex by Mechanisms Involving Ca<Superscript>2+</Superscript> and cAMP

We have previously described that α-ketoisocaproic acid (KIC), the main metabolite accumulating in maple syrup urine disease (MSUD), increased the in vitro phosphorylation of cytoskeletal proteins in cerebral cortex of 17- and 21-day-old rats through NMDA glutamatergic receptors. In the present study we investigated the protein kinases involved in the effects of KIC on the phosphorylating system associated with the cytoskeletal fraction and provided an insight on the mechanisms involved in such effects. Results showed that 1 mM KIC increased the in vitro incorporation of ³²P into intermediate filament (IF) proteins in slices of 21-day-old rats at shorter incubation times (5 min) than previously reported. Furthermore, this effect was prevented by 10 μM KN-93 and 10 μM H-89, indicating that KIC treatment increased Ca²⁺/calmodulin- (PKCaMII) and cAMP- (PKA) dependent protein kinases activities, respectively. Nifedipine (100 μM), a blocker of voltage-dependent calcium channels (VDCC), DL-AP5 (100 μM), a NMDA glutamate receptor antagonist and BAPTA-AM (50 μM), a potent intracellular Ca²⁺ chelator, were also able to prevent KIC-induced increase of in vitro phosphorylation of IF proteins. In addition, KIC treatment was able to significantly increase the intracellular cAMP levels. This data support the view that KIC increased the activity of the second messenger-dependent protein kinases PKCaMII and PKA through intracellular Ca²⁺ levels. Considering that hyperphosphorylation of cytoskeletal proteins is related to neurodegeneration it is presumed that the Ca²⁺-dependent hyperphosphorylation of IF proteins caused by KIC may be involved to the neuropathology of MSUD patients.     

Isolation and some properties of mammalian hepatic membrane lectins

Membrane lectins were isolated from sheep, goat, and buffalo liver by chromatography on an asialofetuin (ASF)-Sepharose 4B column. The lectins moved as a single protein band in SDS-PAGE with molecular masses of 42, 54 and 50 kDa, respectively, for sheep, goat and buffalo lectins. The molecular masses remained unchanged in 0.2 M 2-mercaptoethanol. As judged from the inhibition of binding of the lectin to ASF gel, the three lectins were beta-galactoside-specific. Sheep, goat and buffalo lectins were found to be sialoglycoproteins containing 18.6, 27 and 38.8 mol/mol lectin of neutral hexose, respectively; the corresponding values for the sialic acid content being 5.3, 8.7 and 11.8 mol/mol lectin. Thus goat and buffalo lectins are physico-chemically different from many mammalian hepatic lectins described so far.     

Rat lung 29 kD beta-galactoside-binding lectin is secreted by bronchiolar Clara cells into airways

We isolated a mixture of beta-galactoside-binding lectins from rat lung and raised polyclonal antibody against 14 kD lectin purified from the mixture of lectins. Immunoblotting of the mixture of lectins, which was separated with SDS-PAGE under reducing condition and transferred onto a NC paper, showed that the antibody reacted with two bands at 14 and 29 kD, indicating that these two lectins have common antigenic determinants(s). Immunohistochemically, the antibody recognized only bronchiolar Clara cells with intense immunofluorescence in their apical cytoplasmic protrusions where the secretory granules of the cells are known to be stored. Thus, to determine if the lectin(s) might be secreted into airways, we next raised antibody against airway secretions free from serum as well as surfactant proteins. By immunoblot analysis, the resulting antibody stained 29,45 and 55 kD bands, but not 14 kD band, on a NC paper transferred with the mixture of lectins. These findings suggest that at least 29 kD lung lectin is located in bronchiolar Clara cells and secreted by these cells into airways.