Chemical Constitution of the Host-Specific Toxin of Helminthosporium carbonum

The host-specific toxin of Helminthosporium carbonum Ullstrup has a molecular formula approximating C(32)H(50)N(6)O(10). The compound has been crystallized and a crystalline hydrochloride derivative has been produced. The molecular weight, as determined by chromatography on Sephadex G-10, is slightly less than 700. The toxin appears to be a cyclic peptide, since, although it does not react with ninhydrin or dinitrofluorobenzene, it yields, on hydrolysis, compounds which react to these reagents. It is unstable in dilute acids, yielding ninhydrin-reacting products. Complete acid hydrolysis yields alanine, proline, and three other ninhydrin-reacting components. The infrared spectrum of the toxin reveals an ester band in addition to amide absorption. Its ultraviolet spectrum reveals the presence of unsaturation in the molecule. The toxin is relatively unstable and loses its specific toxicity. This loss of activity appears to be associated with loss of nitrogen and with decreased solubility in water.     

Effects of Helminthosporium carbonum Toxin on Absorption of Solutes by Corn Roots

Susceptible corn roots exposed to the host-selective toxin of Helminthosporium carbonum took up and retained more NO₃⁻, Na⁺, Cl⁻, 3-o-methylglucose, and leucine than did control roots. Stimulatory effects on uptake were more pronounced with freshly cut roots than with roots that were washed and aged. Solutes were accumulated against a concentration gradient, and toxin-treated tissues developed a steeper gradient than did control tissues. Toxin affected both the low and high affinity uptake systems for Na⁺ and Cl⁻. Toxin did not affect uptake of Na₂⁻, K⁺, Ca²⁺, phosphate ion (H₂PO₄⁻ and HPO₄⁻), SO₄⁻, and glutamic acid. No toxin-induced leakage of any solute tested was detected within 5 to 6 hr after initial exposure to toxin. The data suggest that toxin from H. carbonum does not cause the general plasma membrane derangement caused by other host-selective toxins. Instead, H. carbonum toxin may cause specific changes in characteristics of the plasmalemma, which result in increased uptake of certain solutes.     

Effects of Helminthosporium carbonum Toxin on Nitrate Uptake and Reduction by Corn Tissues

Susceptible corn tissues exposed to the host-specific toxin of Helminthosporium carbonum race 1 reduced more nitrate to nitrite than did control tissues, as measured by an in vivo method. There were no differences in nitrate reductase activities extracted from treated and control tissues and assayed by an in vitro method. Toxin-treated susceptible roots removed nitrate from solution and accumulated it in the tissues twice as fast as did control roots. Uptake by resistant roots was stimulated also, provided approximately 100 times higher concentrations of toxin were used. Toxin-stimulated nitrate uptake occurred in the presence of tungstate, which eliminates nitrate reductase activity. Toxin did not cause leakage of nitrate from roots under these conditions. Thus, toxin-enhanced nitrate accumulation was caused by increased nitrate uptake rather than by decreased nitrate metabolism or decreased nitrate leakage. The data indicate that toxin increases the rate of nitrate reduction in vivo by increasing the availability of substrate, not by stimulation of enzyme synthesis.     

Abolishment of Specific Toxicity of Host-Specific Toxin of Helminthosporium carbonum by Electrolytic Reduction

The host-specific toxin produced by the plant pathogen, Helminthosporium carbonum Ullstrup is normally about 300 times as toxic for corn plants susceptible to the disease as it is for disease-resistant plants. Electrolytic reduction of the toxin destroys this specificity, rendering the material no more toxic for disease susceptible plants than for disease resistant ones.     

615小鼠血红蛋白α链的氨基酸组成及个别肽段的氨基酸序列

用CM-Cellulose-23柱层析分离纯化了615小鼠珠蛋白α链,测定其N端氨基酸残基为缬氨酸.615小鼠珠蛋白α链含有141个氨基酸残基,其中19个亮氨酸残基,10个组氨酸残基,9个缬氨酸残基,上述氨基酸残基的数目与文献中其亲本C₅₇BL不同.用胰蛋白酶水解615小鼠珠蛋白α链,发现有不溶性的‘核心’和可溶性的酶解片段.其中一个酶解肽段从N端数第8位氨基酸残基发生了突变,由亲本的缬氨酸变为亮氨酸.     

Purification and structure of the host-specific toxin from Helminthosporium carbonum race 1

The host-specific toxin from Helminthosporiumcarbonum race 1 was purified from culture filtrates by solvent extraction, gel filtration, and high pressure liquid chromatography. High resolution mass spectrometry of the purified toxin gave a MW of 436.2318 and an elemental composition C₂₁H₃₂N₄O₆. Amino acid analysis and proton and¹³C-NMR indicated a peptide containing four amino acids. Their sequence was determined by gas chromatography mass spectrometry. Finally, digestion of the amino acids with D- and L-amino acid oxidases gave the complete structure cyclo[(L-2-amino-9, 10-epoxy-8-oxodecanoyl)-D-prolyl-L-alanyl-L-alanyl].     

Isolation and Biological Activities of Four Selective Toxins from Helminthosporium carbonum

A new and simpler purification procedure was developed for host selective toxins from Helminthosporium carbonum race 1. Four analogs or forms of toxin with the same selectivity as the fungus were isolated from culture fluids; two forms (HC toxins III and IV) have not been reported by other workers. Crystals of the major form of toxin (HC toxin I) were recovered in high yields (>80 milligrams per liter of culture fluid) without the use of high performance or preparative thin layer liquid chromatography. ED₅₀ values, based on inhibition of root growth of susceptible seedlings, for HC toxins I, II, III, and IV were 0.2, 0.4, 2.0, and 20 micrograms per milliliter, respectively. The specific activity of crystalline HC toxin I matched the most active preparation reported previously; the preparation of HC toxin II was more active than that reported previously. Resistant seedlings tolerated 100-fold higher concentrations of each form of toxin than did susceptible seedlings. Hydrolysis of the epoxide group of HC toxin I to a diol destroyed toxicity to susceptible and resistant seedlings. The data suggest that the same mechanisms are affected in resistant and susceptible plants.     

Conversion of Helminthosporium sacchari Toxin to Toxoids by beta-Galactofuranosidase from Helminthosporium

Helminthosporium sacchari produces a host-selective toxin and structurally related nontoxic compounds, here referred to as `toxoids.' Toxin and the three toxoids were each isolated to a high level of purity and were hydrolyzed under acidic conditions. The released galactose was measured by a galactose oxidase/peroxidase assay. Toxin was found to contain four units of galactose per molecule, as previously reported. Toxoids I, II, and III contained one, two, and three units of galactose, respectively. In cultures of the fungus, toxin concentration peaked at 3 weeks, followed by a rapid decline; as toxin levels fell, the total amount of toxoids increased. An enzyme with β-galactofuranosidase activity was found in small amounts in the cultures of H. sacchari; the enzyme converted toxin to the toxoids in vitro. β-Galactofuranosidase was previously known from very few micro-organisms; therefore, several pathogenic Helminthosporia and other fungi were tested for production. β-Galactofuranosidase activity in culture filtrates and mycelia of H. victoriae, H. maydis, H. carbonum, and H. turcicum was much greater than in filtrates and mycelium of H. sacchari. More work is needed to determine the significance of enzyme production by these fungi. No β-galactofuranosidase was evident from Fusarium oxysporum and Cladosporium cucumerinum.     

Amino Acid Composition of Elm Seeds

In the biosynthetic pathway of aromatic amino acids of Brevibacterium flavum, ratios of each biosynthetic flow at the chorismate branch point were calculated from the reaction velocities of anthranilate synthetase for tryptophan and chorismate mutase for phenylalanine and tyrosine at steady state concentrations of chorismate. When these aromatic amino acids were absent, the ratio was 61, showing an extremely preferential synthesis of tryptophan. The presence of tryptophan at 0.01 mM decreased the ratio to 0.07, showing a diversion of the preferential synthesis to phenylalanine and tyrosine. Complete recovery by glutamate of the ability to synthesize the Millon-positive substance in dialyzed cell extracts confirmed that tyrosine was synthesized via pretyrosine in this organism. Partially purified prephenate aminotransferase, the first enzyme in the tyrosine-specific branch, had a pH optimum of 8.0 and Km’s of 0.45 and 22 mM for prephenate and glutamate, respectively, and its activity was increased 15-fold by pyridoxal-5-phosphate. Neither its activity nor its synthesis was affected at all by the presence of the end product tyrosine or other aromatic amino acids. The ratio of each biosynthetic flow for tyrosine and phenylalanine at the prephenate branch point was calculated from the kinetic equations of prephenate aminotransferase and prephenate dehydratase, the first enzyme in the phenylalanine-specific branch. It showed that tyrosine was synthesized in preference to phenylalanine when phenylalanine and tyrosine were absent. Furthermore, this preferential synthesis was diverted to a balanced synthesis of phenylalanine and tyrosine through activation of prephenate dehydratase by the tyrosine thus synthesized. The feedback inhibition of prephenate dehydratase by phenylalanine was proposed to play a role in maintaining a balanced synthesis when supply of prephenate was decreased by feedback inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP*) synthetase, the common key enzyme. Overproduction of the end products in various regulatory mutants was also explained by these results.     

Isolation and purification of canadaphore, a siderophore produced by Helminthosporium carbonum

A new siderophore was isolated and purified from the spent growth medium of the fungus Helminthosporium carbonum by solvent extraction and reverse phase high pressure liquid chromatography. This new molecule has been assigned the name Canadaphore. Canadaphore was detected in culture filtrates after 15 days of growth and production was maximal after growth of H. carbonum to maximal stationary phase in modified Fries basal medium. Production of Canadaphore was completely suppressed when the organism was grown in medium supplemented with iron. Mass spectral analysis yielded a molecular mass of 680 Daltons for the iron-Canadaphore complex and 627 Daltons for the iron-free molecule. Spectroscopic analysis indicates that Canadaphore is a siderophore of the hydroxamate type.     

Inhibition of Dark CO(2) Fixation and Photosynthesis in Leaf Discs of Corn Susceptible to the Host-specific Toxin Produced by Helminthosporium maydis, Race T

The host-specific toxin produced by Helminthosporium maydis, race T, causes 50% inhibition of dark fixation of ¹⁴CO₂ by leaf discs of susceptible (Texas male sterile) corn when it is diluted to approximately 1/10,000 of the volume of the original fungus culture filtrate. Dilutions of 1/10 or less are required for equivalent inhibition of discs prepared from resistant (N) corn. Root growth and photosynthesis were considerably less sensitive (dilution values 1/3000 and 1/1200, respectively), as was leakage of ¹⁴C induced by toxin from preloaded discs. Based on literature values for dilutions causing ion leakage or inhibition of mitochondrial oxidation, toxin dilutions several orders of magnitude greater bring about inhibition of dark CO₂ fixation. Preincubation of discs in light increased sensitivity of dark fixation to toxin and an effect of light on symptom development was shown. Phosphoenolypruvate carboxylase activity in extracts of roots or leaves was not affected by toxin nor was the enzyme level altered in excised leaves treated with toxin. Inhibition of dark fixation of CO₂ provides a bioassaay for race T toxin which is both reliable and rapid.     

氨基酸组成对蛋白质耐热性的影响

为了研究一级结构对蛋白质耐热性的影响,利用软件DNAMAN对16个家族32种蛋白质序列进行了氨基酸含量分析,并统计分析了氨基酸组成对蛋白质耐热性的影响。通过比较同一家族的高低温蛋白质序列及16个家族中所有高温和低温蛋白质序列中氨基酸含量的变化可以推断(从低温到高温)：Ser、Cys．含量降低显著,Arg、Ile、Pro含量升高显著。由此可知高温蛋白质倾向于含有疏水性氨基酸而避免亲水性氨基酸。     

Amino Acid Composition of Certain Bacterial Cell-Wall Proteins

Analyses were made to determine the amino acid composition of the cell-wall proteins of Salmonella pullorum, S. senftenberg 775W, S. derby, and Escherichia coli. These proteins consist of the usual 18 amino acids found in most proteins with diaminopimelic acid in addition. Quantitative determination of these amino acids showed that their amounts were similar.     

圈养大熊猫乳中氨基酸组成的研究

为了认识大熊猫乳中氨基酸的组成特点,将大熊猫乳经6 mol/L盐酸水解后,用高效液相色谱测定了不同泌乳阶段的20个乳样的氨基酸组成.结果显示,大熊猫初乳中含量最高的氨基酸是谷氨酸,约为5.2 g/L;常乳和干乳期乳腺分泌物中含量最高的是脯氨酸;总氨基酸含量在泌乳2～10 d内变化不明显,平均值约为34 g/L;干乳期乳中氨基酸含量变化显著.