Heat-induced Resistance of Sugarcane to Helminthosporium sacchari and Helminthosporoside

Sugarcane clones susceptible to eye spot disease became resistant to the pathogen or helminthosporoside activity during warm summer months. Exposure of plants to 30 to 35 C temperatures, hot water treatment of excised leaves from 30 to 50 C, preincubation of leaves in an N₂ atmosphere, or preincubation in the presence of protein synthesis inhibitors also induced resistance. Hot water-treated leaves reverted to a susceptible condition. Five to 15 C temperatures, an N₂ atmosphere, or protein synthesis inhibitors prevented this recovery. Results indicate that temperature may influence host tissue sensitivity to helminthosporoside, thus governing disease development throughout the year.     

Transfer of toxin susceptibility to plant protoplasts via the hemlmintosporoside binding protein of sugarcane.

The eyespot disease of sugarcane is caused by $\text{Helminthosporium sacchari}$. Helminthosporoside, a host-specific toxin produced by $\text{H. sacchari}$, is essential for the pathogenicity of this fungus. The presence of the helminthosporoside-binding protein in sugarcane likewise appears to be essential for susceptibility to the toxin. The results of this report show that leaf cell protoplasts of tobacco and toxin resistant sugarcane effectively adsorbed the toxin-binding protein derived from membranes of susceptible sugarcane. These protoplasts then became susceptible to the helminthosporoside. They also functioned to takeup raffinose, a trisaccharide structurally related to the toxin. Tobacco protoplasts were treated with [¹⁴C] - binding protein, ruptured, and fractionated on a sucrose density gradient column. A peak of radioactivity was associated with the enriched plasma membrane fraction. The results support the hypothesis that the binding protein is the primary recognition site governing susceptibility of sugarcane to helminthosporoside.     

玉米大斑病菌HT—毒素与玉米细胞的膜脂过氧化研究

以土培玉米幼苗为材料,通过测定玉米大斑病菌HT－毒素处理后玉米叶片细胞膜透性和细胞内丙二醛（MDA）含量变化及其与细胞内过氧化物歧化酶（SOD）、过氧化物酶（POD）活性之间的关系来研究玉米细胞膜脂过氧化的程度。结果表明,HT－毒素胁迫后,亲和组合MDA含量上升,POD活性受到刺激,SOD活性受抑制较强;非亲和组合POD活性受到抑制,SOD活性受抑制较弱。HT－毒素对玉米叶片细胞膜有强烈的破坏作用,而且这种作用与毒素的浓度和毒素处理的时间呈正相关。试验结果初步推测在抗、感玉米的细胞膜上可能都有HT－毒素的结合位点,但毒素作用后幅地它们的活性氧代谢程度不同而导致了对HT－毒素敏感性的差异。     

Effect of the Specific Toxin in Helminthosporium victoriae on Host Cell Membranes

Helminthosporium victoriae toxin, which affects only hosts of the toxin-producing fungus, causes loss of electrolytes from roots, leaves, and coleoptiles of treated plants. Root hair cells lost the ability to plasmolyze after 20 minutes exposure to toxin in solution; comparable resistant cells retained plasmolytic ability during 3 hours exposure. Toxin stopped uptake of exogenous amino acids and Pi by susceptible but not by resistant tissue. Incorporation of ³²P into organic-P and ¹⁴C-amino acids into protein was blocked in susceptible but not in resistant tissue. Apparent free space increased in susceptible but not in resistant roots. The increase was evident within 30 minutes, and reached 80% free space after 2 hours exposure to toxin. When cell wall-free protoplasts were exposed to 0.16 μg toxin/ml, protoplasmic streaming stopped and all plasma membranes of susceptible protoplasts broke within 1 hour. Resistant protoplasts were not affected significantly. Data support the hypothesis of a primary lesion of toxin in the plasma membrane. Effects on synthesis could result from lack of transport of exogenous solutes to sites of synthesis. It is possible that all other observed effects of toxin are secondary to membrane damage.     

Kre1p, the plasma membrane receptor for the yeast K1 viral toxin

Saccharomyces cerevisiae K1 killer strains are infected by the M1 double-stranded RNA virus encoding a secreted protein toxin that kills sensitive cells by disrupting cytoplasmic membrane function. Toxin binding to spheroplasts is mediated by Kre1p, a cell wall protein initially attached to the plasma membrane by its C-terminal GPI anchor. Kre1p binds toxin directly. Both cells and spheroplasts of Deltakre1 mutants are completely toxin resistant; binding to cell walls and spheroplasts is reduced to 10% and < 0.5%, respectively. Expression of K28-Kre1p, an inactive C-terminal fragment of Kre1p retaining its toxin affinity and membrane anchor, fully restored toxin binding and sensitivity to spheroplasts, while intact cells remained resistant. Kre1p is apparently the toxin membrane receptor required for subsequent lethal ion channel formation.     

alpha-Galactoside Binding Proteins from Plant Membranes: Isolation, Characterization, and Relation to Helminthosporoside Binding Proteins of Sugarcane

α-Galactoside binding proteins were isolated from cellular membranes of mint and tobacco as well as two clones of sugarcane which differ in their sensitivity to helminthosporoside, a toxic galactoside. Sodium trichloroacetate was used to disrupt membranes after which the proteins were purified using a melibiose-Sepharose-6B affinity column. Proteins from mint, tobacco, and susceptible sugarcane had equal electrophoretic mobilities, whereas resistant sugarcane protein migrated more slowly. Pretreatment of the proteins with fluorescamine caused them to migrate with the tracking dye. Each of the proteins had molecular weights of about 100,000 and each was shown to be oligomeric. Gel filtration revealed that aqueous solutions of these membrane proteins contained a mixture of size species which included a high molecular weight multimer and lower molecular weight oligomers. The relative abundance of the oligomers was dependent upon protein concentration: the lower concentrations yielded higher relative amounts of oligomers (Kenfield and Strobel 1980 Biochim Biophys Acta 600: 705-712). Also, the binding activity of these receptors was inversely proportional to protein concentration. At low protein concentration (4 micrograms per milliliter), the K_d's of each of the proteins for galactinol, raffinose, and helminthosporoside was about 10 micromolar. At high protein concentrations (100 micrograms per milliliter), mint and resistant sugarcane proteins failed to bind α-galactosyl ligands, whereas proteins from tobacco and susceptible sugarcane exhibited a markedly decreased binding activity compared to that at lower protein concentrations. Binding proteins from susceptible sugarcane were mixed with receptors from either resistant sugarcane or mint at low protein concentrations, then assayed for binding activity. Such mixtures showed a concentration-dependent decrease in binding activity analogous to the activity of homogeneous protein solutions. Bovine serum albumin, a nonsubunit protein, had no effect on the binding activity of the protein from susceptible sugarcane. Thus, receptors from diverse plants can associate in vitro and form functional oligomers. The amino acid composition of each of the binding proteins was similar but not identical. The significance of these results is discussed in regard to regulation of carbohydrate transport and sensitivity to phytotoxins.     

Effect of mild heat treatment on the ATPase activity and proteolytic sensitivity of myosin subfragment-1

The K+-EDTA-activated ATPase activity of chymotryptic myosin subfragment-1 (S-1) decreased by 85-90% when S-1 was incubated over a 2-h period at 35 degrees C. Addition of F-actin, ATP, or ATP analogs, such as ADP or PPi, to S-1 before incubation at 35 degrees C prevented the loss of ATPase activity. The decrease in ATPase activity was also accompanied by changes in tryptic sensitivity. Instead of the normal peptide pattern--which is comprised of three heavy chain fragments (27K, 50K, and 20K)--only two fragments (27K and 20K) appeared on the sodium dodecyl sulfate-gel electrophoregram after limited tryptic digestion of thermally treated S-1. Addition of any ligand--e.g. ATP, ADP, pyrophosphate, or actin--which prevented the loss of ATPase activity during incubation at 35 degrees C also prevented the observed change in the tryptic peptide pattern of S-1. Tryptic digested S-1, whose heavy chain has been cleaved to 27K, 50K, and 20K fragments, also lost its ATPase activity upon mild heat treatment. The heat-treated trypsin-digested S-1 was subjected to a second tryptic digestion, which resulted in the disappearance of the 50K fragment, while the 50K fragment of tryptic S-1 not subjected to heat treatment was not susceptible to additional tryptic hydrolysis. The results indicate that the structural changes, that take place specifically in the 50K region of S-1 upon mild heat treatment, lead to both the loss of the ATPase activity and the changed tryptic sensitivity of S-1.     

Production of a Host-Specific Toxin by Germinating Spores of Alternaria tenuissima Causing Leaf Spot of Pigeon Pea

Production of a host-specific toxin by Alternaria tenuissima , the cause of pigeon pea leaf spot, was investigated in spore-germination fluids (SGF). The SGF selectively induced necrosis on pigeon pea leaves in a deteched leaf assay. Necrotic lesions were observed when a toxin from SGF was applied onto detached young leaves of the pigeon pea cultivar Bahar at concentration as low as 5 ng/ml. The resistant line Tanzania and nonhosts tolerated at least 20,000 times higher concentration of the toxin. The differential activity of the toxin on hosts and nonhosts of the fungus, as well as on susceptible and resistant cultivars or lines, suggested host-specific property of the toxin. At a concentration of 10 ng/ml, the toxin induced susceptibility of pigeon pea leaves to a non-pathogenic isolate of Alternaria alternata. The toxin possibly plays a role as a disease determinant of A. tenuissima , because the toxin was released from germinating spores as early as 3 h of incubation andthe, amount detected within 9 h was about 6 times of the concentration required for necrotic toxicity.     

Induction of a proteinase K inhibitor in a potato cultivar with a high degree of field resistance against Phytophthora infestans

A proteinase K inhibitor (PLPKI) was isolated from a potato cultivar with a high level of field resistance ( Solanum tuberosum L. cv. Pampeana INTA), after 24 h of infection with Phytophthora infestans , when inhibitory activity was markedly increased. Purification was performed by heat treatment, gel filtration chromatography and affinity chromatography. A size of 60 kDa was estimated by SDS-PAGE in partially denaturing conditions and by gel filtration. It is multimeric and the monomer has a molecular mass of 8.5–9.0 kDa. PLPKI is highly active against proteinase K (EC 3.4.21.14) but poorly inhibits two serine proteinases of animal origin, trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1). A differential expression (determined by activity and immunoblotting assays) of PLPKI was observed between two potato cultivars with different degrees of field resistance to P. infestans . In the resistant cultivar (cv. Pampeana INTA) PLPKI induction (19-fold with respect to healthy leaves) occurred 24 h after infection and remained over basal levels after 48 h infection. By contrast, in the susceptible cultivar (cv. Bintje), no induction was observed.     

用超微细胞化学定位技术揭示ATP酶在紫茎泽兰高温适应性中的作用

【背景】外来人侵植物紫茎泽兰自然演化出耐高温种群,其适应机制与各种生理代谢有关。【方法】本文从超微细胞化学水平,对紫茎泽兰抗高温种群、敏感种群ATP酶活性定位,明确其在高温适应性中的作用,试图阐明该草的生态适应机制。【结果】正常情况下,紫茎泽兰ATP酶主要定位于细胞壁及细胞间隙周围的细胞壁表面;经40℃高温处理后,在不同的处理时间下,抗性、敏感种群之间ATP酶的活性表现出明显差异,其中以处理12h时差异最大,具体表现为抗高温种群的ATP酶活性明显高于敏感种群,ATP酶的定位点除细胞壁外,在细胞膜上也呈现大量的分布,而敏感种群在处理12h时的酶活性明显降低,只在细胞壁上有零星的分布。处理24h时,敏感种群叶片已完全萎蔫,细胞结构毁坏,细胞膜破损;而抗高温种群叶片仍然完好,细胞膜上仍有ATP酶分布。【结论与意义】经40℃高温处理后,紫茎泽兰抗高温种群ATP酶活性明显高于敏感种群,初步认为紫茎泽兰对高温的适应性与ATP酶活性相关。本研究为进一步阐明与紫茎泽兰适应性相关的入侵机理提供了资料。     

Thermal Inactivation of Staphylococcal Enterotoxins B and C

Thermal inactivation profiles of staphylococcal enterotoxins B (SEB) and C (SEC) at 80, 100, and 121 C showed that SEC is more resistant than SEB to heat. After 24 h of incubation at 25 C, some reactivation (recovery of serological reactivity) occurred in toxins that had been inactivated by heat. If the toxin was stirred during heating, reactivation did not occur. An examination of the reactivation kinetics of heat-treated SEC showed that reactivation was temperature dependent. At 25 C, the incubation temperature of heat-treated crude SEC (80 C for 10 min), 100% reactivation occurred after 24 h, whereas at 4 C only slight reactivation was observed. We and others observed that heat-treated toxins initially lost more serological activity when heated at a low temperature (80 C) than at a higher temperature (100 C); in the present study we demonstrate that this is a reversible phenomenon.     

Temperature-specific inhibition of human red cell Na+/K+ ATPase by 2,450-MHz microwave radiation

The ATPase activity in human red blood cell membranes was investigated in vitro as a function of temperature and exposure to 2,450-MHz continuous wave microwave radiation to confirm and extend a report of Na+ transport inhibition under certain conditions of temperature and exposure. Assays were conducted spectrophotometrically during microwave exposure with a custom-made spectrophotometer-waveguide apparatus. Temperature profiles of total ATPase and Ca+2 ATPase (ouabain-inhibited) activity between 17 and 31 degrees C were graphed as an Arrhenius plot. Each data set was fitted to two straight lines which intersect between 23 and 24 degrees C. The difference between the total and Ca+2 ATPase activities, which represented the Na+/K+ ATPase activity, was also plotted and treated similarly to yield an intersection near 25 degrees C. Exposure of membrane suspensions to electromagnetic radiation, at a dose rate of 6 W/kg and at five temperatures between 23 and 27 degrees C, resulted in an activity change only for the Na+/K+ ATPase at 25 degrees C. The activity decreased by approximately 35% compared to sham-irradiated samples. A possible explanation for the unusual temperature/microwave interaction is proposed.     

Inhibitory effects of cations on the gastric H+, K+ -ATPase. A potential-sensitive step in the K+ limb of the pump cycle

The presence of a cation inhibitory site on the dephosphoform of the H+, K+ -ATPase was confirmed by comparing the effects of K+ and NH4+ on overall activity and on phosphorylation and dephosphorylation. Inhibition of ATPase activity was pronounced at high cation/ATP ratios, but NH4+ was much less effective. At 60 mM cation, although the ATPase activity was greater in the presence of NH4+ (17.1 mumol/mg.h) as compared to K+ (5.1 mumol/mg.h), dephosphorylation of preformed phosphoenzyme was faster with K+ (2101 min-1) than with NH4+ (1401 min-1). Increasing K+ concentrations at the cytosolic face of the enzyme, at constant ATP, decreased the rate of phosphorylation from 1343 to 360 min-1 at 25 mM K+. Increasing ATP concentrations in the presence of constant K+ concentrations accelerated ATPase activity and increased the steady-state phosphoenzyme level. Therefore, inhibition by cations was due to cation stabilization of a dephospho form of the enzyme at a cytosolically accessible cation-binding site. ATP promoted cation dissociation from this site. In ion-permeable vesicles, increasing K+ concentrations, at constant ATP, activated and then inhibited ATPase activity, with a K0.5(I) of 22 mM. In intact, ion-impermeable inside-out vesicles, in the presence of valinomycin, ATPase activity increased up to 175 mM K+. Collapse of this potential by the addition of the electrogenic protonophore 3,3',4', 5-tetrachlorosalicylanilide restored the K+ inhibition of ATPase activity. Thus, the cation inhibition of the ATPase activity appears to be voltage-sensitive; and hence, its connection to the voltage sensitivity of acid secretion demonstrated in intact gastric mucosa is discussed.     

黄瓜褐斑病菌毒素对抗、感黄瓜品种的作用

在黄瓜褐斑病菌毒素的作用下,黄瓜品种的根数、根长、芽长受到了抑制,根的电导值及叶片的苯丙氨酸解氨酶(PAL)活性升高。抗病品种的根数、根长、芽长受毒素的影响较感病品种小,即感病品种对毒素敏感,且抗、感品种的电导值差异达显著水平。抗病品种的PAL活性增加幅度较感病品种大。     

Effect of amiodarone on membrane fluidity and Na+/K+ ATPase activity in rat-brain synaptic membranes

In rat-brain synaptic membranes at a fixed temperature (37 degrees C), amiodarone dose-dependently inhibits the Na+/K+ ATPase activity (IC50 approximately equal to 2.10(-5)M) and produces a linear increase in the degree of fluorescence depolarization (P) of 1,6-diphenylhexatriene embedded in the lipid matrix. Amiodarone has no effect on Mg++ ATPase and K+PNPase activity up to 3.10(-4)M. Studies carried out at different temperatures indicate that 10(-5)M amiodarone inhibits the Na+/K+ ATPase and decreases the lipid fluidity at all the temperatures studied (9 - 40 degrees C). The compound significantly displaces the temperature of transition observed around 20 degrees C in both Na+/K+ ATPase activity and lipid fluidity to 24 degrees C with no changes in slopes. The results suggest that part of the selective inhibition of Na+/K+ ATPase activity by amiodarone could be due to the effects of the drug on lipid dynamics.     

Endovesiculation of human erythrocytes exposed to sphingomyelinase C: a possible explanation for the enzyme-resistant pool of sphingomyelin

When human erythrocytes are treated with Staphylococcus aureus sphingomyelinase C at 37 degrees C they become susceptible to cold lysis and appear to endovesiculate. Endovesiculation has been confirmed by showing that in parallel with sphingomyelin breakdown, the cells accumulate [3H]inulin or [14C]sucrose (without losing intracellular K+) and also experience a loss of cell-surface acetylcholinesterase activity into a latent intracellular pool which can be revealed by treatment with detergent. On the basis of these observations it can be calculated that endovesicles account for about 2-4% of cell volume and about 25% of total cell surface. Pretreatment of cells with bee venom phospholipase A2 completely blocked sphingomyelinase-induced endovesiculation but this effect was related to a concomitant decrease in sphingomyelin breakdown which was reduced by about 90%. These results indicate that the pool of sphingomyelin which is not susceptible to attack by sphingomyelinase C (about 15% of total sphingomyelin) may be resistant because of membrane internalisation and not because it originally resides in the inner leaflet of the plasma membrane.     

The enhanced ATPase activity of glutathione-substituted actin provides a quantitative approach to filament stabilization

[Cys374]glutathionyl-actin was prepared by isolation of the reaction product of G-actin with Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid], followed by reaction with glutathione. Filaments of this actin disulfide are susceptible to even weak shearing stress as exerted, for example, by heating to 37 degrees C. This treatment produces a 25-fold enhanced steady-state ATPase activity as compared to unsubstituted F-actin at room temperature. Monitoring the reduction of this enhanced ATPase activity is a reliable method for quantifying the effectiveness of filament-stabilizing agents and for determining their apparent dissociation constants. A detailed comparative study of filament-stabilizing agents was performed, and some hitherto unknown filament-protecting effects were revealed. Inorganic phosphate provides stabilization only to a maximum of 45% ATPase inhibition, but reaches this effect already at cytoplasmic Pi concentrations (approximately 4 mM). Arsenate seems to bind with similar affinity, but with distinctly less protective activity (maximum of 16%). High concentrations of alkali ions provide a more effective protection (maximum of 95%), Li+ being more efficient than Na+ and K+. Divalent cations (Ca2+, Mg2+) had a strong stabilizing effect on KCl-polymerized actin; we confirmed the presence of two distinct classes of binding sites for divalent metal ions with moderate and low affinities, apparent in a strong stabilizing effect on KCl-polymerized actin. The stabilizing effects of KCl and Pi are independent and additive. Correspondingly, at K2HPO4 concentrations greater than 4 mM, K+ ions contribute considerably to stabilization. In the presence of 100 mM KCl plus 4 mM Pi, conditions which mimic the physiological environment, filament protection is nearly as effective as with the mushroom toxin phalloidin. The strong stabilizing effect of phalloidin occurred at concentrations far below stoichiometric, suggesting a very high degree of cooperativity in its interaction with actin filaments.     

Effect of mild heat treatment on actin and nucleotide binding of myosin subfragment 1

Chymotryptic subfragment 1 (S-1) prepared from rabbit skeletal myosin has lost its ATPase activity upon incubation at 35 degrees C for 3 h. The loss in ATPase activity was accompanied by the perturbation of the structure of the 50K domain as indicated by a dramatic increase in the tryptic susceptibility of this domain without any change in the susceptibility of the other domains of S-1. The perturbation starts at the C-terminal region of the domain as suggested by the appearance of a 29K intermediate protein band in the tryptic peptide pattern of the heat-treated S-1. The heat-treated molecule essentially retained its actin and polyphosphate binding ability, and the actin binding was still sensitive to the presence of ATP or pyrophosphate. However, as opposed to native S-1, in heat-treated S-1 the addition of ATP does not induce an increase in tryptophan fluorescence, and, in the case of the treated species, the fluorescence of 1,N6-ethenoadenosine 5'-diphosphate added to the mixture is quenchable by acrylamide. This latter observation suggests that the binding of the adenine ring of the nucleotide has been altered following the heat treatment. The results indicate that the actin and polyphosphate binding sites of S-1 are distinct and that they are relatively independent of the adenine ring binding site.     

Beneficial effects of gamma linolenic acid supplementation on nerve conduction velocity, Na+, K+ ATPase activity, and membrane fatty acid composition in sciatic nerve of diabetic rats

Metabolic and vascular abnormalities are implicated in the pathogenesis of diabetic neuropathy. Two principal metabolic defects are altered lipid metabolism resulting from the impairment of delta-6-desaturase, which converts linoleic acid (LA) into gamma linolenic acid (GLA), and reduced nerve Na+, K+ ATPase activity. This reduction may be caused by a lack of incorporation of (n-6) fatty acids in membrane phospholipids. Because this ubiquitous enzyme maintains the membrane electrical potential and allows repolarization, disturbances in its activity can alter the process of nerve conduction velocity (NCV). We studied the effects of supplementation with GLA (260 mg per day) on NCV, fatty acid phospholipid composition, and Na+, K+ ATPase activity in streptozotocin-diabetic rats. Six groups of 10 rats were studied. Two groups served as controls supplemented with GLA or sunflower oil (GLA free). Two groups with different durations of diabetes were studied: 6 weeks with no supplementation and 12 weeks supplemented with sunflower oil. To test the ability of GLA to prevent or reverse the effects of diabetes, two groups of diabetic rats were supplemented with GLA, one group for 12 weeks and one group for 6 weeks, starting 6 weeks after diabetes induction. Diabetes resulted in a 25% decrease in NCV (P < 0.0001), a 45% decrease in Na+, K+ ATPase activity (P < 0.0001), and an abnormal phospholipid fatty acid composition. GLA restored NCV both in the prevention and reversal studies and partially restored Na+, K+ ATPase activity in the preventive treatment group (P < 0.0001). These effects were accompanied by a modification of phospholipid fatty acid composition in nerve membranes. Overall, the results suggest that membrane fatty acid composition plays a direct role in NCV and confirm the beneficial effect of GLA supplementation in diabetic neuropathy.     

Changes in the Plasma Membrane Distribution of Rice Phospholipase D during Resistant Interactions with Xanthomonas oryzae pv oryzae

Phospholipase D (PLD; EC 3.1.4.4), which hydrolyzes phospholipids to generate phosphatidic acid, was examined in rice leaves undergoing susceptible or resistant interactions with Xanthomonas oryzae pv oryzae. RNA analysis of leaves undergoing resistant interactions revealed different expression patterns for PLD over 5 days relative to control plants or those undergoing susceptible interactions. By using an activity assay and immunoblot analysis, we identified three forms of PLD (1, 2, and 3). PLD 1 was observed only at 1 day after tissue infiltration. PLDs 2 and 3 were detected up to 3 days in all interactions. Immunoelectron microscopy studies revealed PLD to be associated predominantly with the plasma membrane. In cells undergoing a susceptible response, PLD was uniformly distributed along the plasma membrane at 3, 6, 12, and 24 hr after inoculation. However, within 12 hr after bacterial challenge in resistant interactions, PLD was clustered preferentially in membranes adjacent to bacterial cells.