Cytokinin Reversal of Abscisic Acid Inhibition of Growth and α-Amylase Synthesis in Barley Seed

The effect of cytokinin, kinetin, on abscisic acid (dormin) inhibition of α-amylase synthesis and growth in intact barley seed was investigated. Abscisic acid at 5 × 10^?5M nearly completely inhibited growth response and α-amylase synthesis in barley seed. Kinetin reversed to a large extent abscisic acid inhibition of α-aniylase synthesis and coleoptile growth. The response curves of α-amylase synthesis and coleoptile growth in presence of a fixed amount of abscisic acid (6 × l0^?6M) and increasing concentrations of kinetin (from 5 × l0^?7M to 5 × 10^?5 M) showed remarkable similarity. Kinetin and abscisic acid caused synergistic inhibition of root growth. Gibberellic acid was far less effective than kinetin in reversing abscisic acid inhibition of α-amylase synthesis and coleoptile growth. A combination of kinetin and gibberellic acid caused nearly complete reversal of abscisic acid inhibition of α-amylase synthesis but not the abscisic acid inhibition of growth. The results suggest that factors controlling α-amylase synthesis may not have a dominant role in all growth responses of the seed. Kinetin possibly acts by removing the abscisic acid inhibition of enzyme specific sites thereby allowing gibberellic acid to function to produce α-amylase.     

Peroxidase and α-Amylase Activities in Relation to Germination of Dormant and Nondormant Wheat

Germination capacity, and α-amylase production in relation to the peroxidase and isoperoxidase activities in the grains of three varieties of wheat have been analysed and compared. A high percentage of germination and α-amylase producation at 25°C are associated with low peroxidase activity of the isolated embryo. This correlation is lacking when the intact grain is considered. A 2-day treatment at 4°C which further increases the percentage germination and enhances α-amylase synthesis, lowers the activity of peroxidase in the embryos. A general decrease in activity of all the isoenzymes is observed. Based on the above data and on differences in the activity of the most cathodic isoperoxidasic bands, a hypothesis is put forward which suggests that a sufficiently low peroxidase activity and a minimum auxin level of the embryo are responsible for the onset of germination.     

Effect of Growth Retardants on α-Amylase Production in Germinating Barley Seed

The effect of growth retarding compounds, (2-chloroethyl)trimethylammonium chloride (CCC), 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride (AMU-1618), tributyl-2,4-dichlorobenzylphosphonium chloride (Phosfon D) and N-dimethylamino succinamic acid (B-995) on α-amylase production in germinating barley seed was studied. Seeds were germinated in growth retardants in presence and absence of gibberellic acid (GA₃). CCC, AMO-1618 and Phosfon D inhibitedα-amylase production in germinating seed and the effect was reversed by GA₃ Phosfon D and AMO-1618 were stronger inhibitors of α-amylase production than CCC. CCC was by far the strongest inhibitor of all the other analogs tested. B-995 was comparatively only slightly inhibitory. The results reported here, when viewed in light of the results of other workers, provide good evidence that CCC, AMO-1618 and Phosfon D inhibit α-amylase production by inhibiting the synthesis of gibberellin or gibberellin-like hormone(s) during germination of barley seed. Consistent with other reports, B-995 possibly acts by other mechanism (s).     

Effects of AMO-1618 on Growth, Morphology, and Gibberellin Content of Phaseolus Cocineus Seedlings

A study was made of the effects of AMO-1618 on the endogenousgibberellin (GA) levels and stem, leaf, and root growth of Phaseoluscoccineus seedlings. The data establishes that some of the effectsof AMO-1618 on the growth of Phaseolus seedlings are mediatedby factors other than an inhibition of GA biosynthesis.     

Abscisic Acid Inhibition of Gibberellic Acid and Cyclic 3',5'-Adenosine Monophosphate Induced α-Amylase Synthesis

The induction of α-amylase synthesis in barley aleurone by cyclic 3′,5′-adenosine monophosphate or GA₃ was inhibited by abscisic acid. The concentration of ABA required to inhibit α-amylase induction by the cyclic nucleotide in the extract was one-fiftieth to one hundredth of that required for GA₃-induced α-amylase. It is concluded that the effects of ABA on GA₃ and cyclic nucleotide induced α-amylase synthesis in the aleurone are independent and indirect.     

Inhibition of Abscisic Acid Biosynthesis in Cercospora rosicola by Inhibitors of Gibberellin Biosynthesis and Plant Growth Retardants

The fungus Cercospora rosicola produces abscisic acid (ABA) as a secondary metabolite. We developed a convenient system using this fungus to determine the effects of compounds on the biosynthesis of ABA. Inasmuch as ABA and the gibberellins (GAs) both arise via the isoprenoid pathway, it was of interest to determine if inhibitors of GA biosynthesis affect ABA biosynthesis. All five putative inhibitors of GA biosynthesis tested inhibited ABA biosynthesis. Several plant growth retardants with poorly understood actions in plants were also tested; of these, six inhibited ABA biosynthesis to varying degrees and two had no effect. Effects of plant growth retardants on various branches of the isoprenoid biosynthetic pathway may help to explain some of the diverse and unexpected results reported for these compounds. Knowledge that certain inhibitors of GA biosynthesis also have the ability to inhibit ABA biosynthesis in C. rosicola indicates the need for further studies in plants on the mode of action of these compounds.     

Effect of Gibberellin A3 on the Elongation, α-Amylase Activity, Reducing Sugar Content and Oxygen-uptake of the Leaf Sheath of Dwarf Maize Seedlings

GA₃-treatment of dwarf maize seedlings resulted in the elongation of the leaf sheath and also an increase in α-amylase activity. Excised leaf sheaths did not respond to GA₃ in leaf shealh length and α-amylase activity. Increase in the enzyme activity is always accompanied by an increase in the length of the leaf sheath. α-Amylase activity gradually increased as the growth of the first leaf proceeded, and a parallelism was found between the length of the leaf sheath and the enzyme activity, suggesting that the degree, of the enzyme activity depends on the length of the leaf sheath. On the other hand, IAA did not affect α-amylase activity while it promoted leaf sheath elongation. This suggests that elongation per se is not associated with the increase in α-amylase activity and that the enzyme-promoting effect is specific to gibberellin. Higher α-amylase activity and lower content of reducing sugars were detected in the older tissue of the leaf sheath, that is, in the upper half. This was the same for GAlrealed seedlings. The amount of reducing sugars was less in GA₃-trealcd seedlings. Oxygen-uptake of the leaf sheath was higher in the upper half in both controls and GA₃-treated seedlings. It was slightly higher in the latter than in the former. From these results it was discussed 1o conclude that the processes of the GA₃-induced elongation and increase in α-amylase activity of the leaf sheath are independent of one another.     

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the ααααααα/βββββββ-type protect spores against this DNA damage

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α⁻β⁻) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and α⁻β⁻ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and α⁻β⁻ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.     

Effects of AMO-1618 and B-995 on Growth and Endogenous Gibberellin Content of Cupressus arizonica seedlings

Soil drenches of 250, 500 or 1000 mg/l of the growth retardants AMO-1618 or B-995 effectively reduced dry matter production and stem elongation in young seedlings of Cupressus arizonica Greene. In seedlings treated with AMO-1618, the acidic, ethyl acetate-soluble gibberellin-like substances (GAs), as detected. by bioassay, were reduced to almost undetectable levels. However, the endogenous GA content in seedlings treated with B-995 were at least 11-fold greater than in control seedlings and differed as well in chromatographic characteristics, being of a more polar nature than the endogenous GAs of control seedlings. It was concluded that while AMO-1618 probably acts through interference with GA biosynthesis, B-995 may act through the interconversion of GAs.     

Abscisic Acid as Inhibitor of α-Amylase from Aspergillus and Bacillus subtilis

Abscisic acid (ABA) inhibited the activity of α-amylase from both Aspergillus and Bacillus subtilis in vitro if ABA and enzyme solutions were allowed to react with each other before adding to the starch solution. If the ABA solution was put to starch before adding the enzyme, no inhibition occurred. The inhibition increased with increasing time between mixing ABA and enzyme solutions and adding the mixture to starch. It was not the absolute amounts of enzyme and ABA which were of importance for the inhibition, but the concentrations of ABA and enzyme in the ABA + enzyme mixture. Within certain limits the inhibition was proportional to the concentration of ABA, so that it should be possible to use the inhibition in quantitative tests for inhibitors. Dialysis of a mixture of ABA and enzyme showed that ABA is bound to the enzyme. The enzyme was still inhibited after dialysis for 25 h. On the other hand, partitioning with diethylether from acid water solution could free the enzyme from all ABA. Supposedly ABA acts as an allosteric inhibitor. The results may offer the foundation for one possible way to explain why inhibitors in plants sometimes inhibit growth and sometimes do not. If inhibitor, enzyme and substrate are compartmentalized, the degree of reaction should depend upon the sequence in which the three components meet each other.     

Inhibition of Astrocyte Glutamine Production by α-Ketoisocaproic Acid

Abstract: We have evaluated the effect of α-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used ¹⁵NH₄Cl as a metabolic tracer to measure the production of both [5-¹⁵N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-¹⁵N]glutamine, representing the reductive amination of 2-oxoglutarate via glutamate dehydrogenase and subsequent conversion of [¹⁵N]-glutamate to [2-¹⁵N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-¹⁵N]glutamine and stimulated the formation of [2-¹⁵N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5-¹⁵N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-¹⁵N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2-¹⁵N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of ¹⁴CO₂ from [U-¹⁴C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic α-ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate. The data indicate that KIC diminishes flux through glutamine synthetase by lowering the intraastrocytic glutamate concentration below the K_m of glutamine synthetase for glutamate, which we determined to be ～7 mM.     

The Effect of the Plant Growth Retardants Amo 1618 and CCC on Gibberellin Production in Fusarium monili-forme: Light Stimulated Biosynthesis of Gibberellins

Through the use of a single gene dwarf mutant of Zea mays L., dwarf-1, the interaction of growth retardants with gibberellin biosynthesis was studied in Fusarium monitiforme. It was demonstrated that the growth retardants 2-isopropyl-4-dimcthylamine-5-methyphenyl-1-piperidine-cai'boxylate methyl chloride (Amo 1618) and (2-chloroethyl) trimethylammonium chloride (CCC) are more effective inhibitors of gibberellin biosynthesis in cultures maintained under continuous illumination. Light grown cultures produced significantly more biologically active gibberellin-like materials than dark grown cultures. Stock cultures exposed to light also promoted the subsequent biosynthesis of gibberellins in the dark. Chromatographical analysis of the soluble gibberellins extracted from the culture medium revealed that large amounts of chromatographically detectable A₃ and A₇ were produced in light cultures with only A₇ produced in the dark. Light also induced a greater incorporation of acelate-2-¹⁴C into the gibberellins A₇, A₃ and an unidentified gibberellin. Growth returdants occasionally caused a complete disappearance of chromatographically detectable gibberellins in the dark; however, in the light at no concentration tested was it possible to detect the complete disappearance of gibberellin-like material. A₃ was always detectable. Like higher plants, different strains of F. moniliforme exhibit variation which makes them more or less sensitive to the growth retardants. This variation is interpreted to mean that there may be more than one pathway leading to the synthesis of the gibberellins.     

Purification and partial amino acid sequence of plantaricin 1.25ααα and 1.25βββ, two bacteriocins produced by Lactobacillus plantarum TMW1.25

Two bacteriocins produced by Lactobacillus plantarum TMW1.25 have been purified by a four-step purification procedure, including ammonium sulphate precipitation and cation-exchange chromatography followed by hydrophobic-interaction chromatography on octyl sepharose. The final purification was performed by repeated reversed-phase chromatography steps which yielded two bacteriocin fractions designated plantaricin 1.25 alpha and plantaricin 1.25 beta. The molecular masses of the peptides in these fractions were 5979 and 5203 Da, respectively. Combination of the fractions did not have any synergistic effects on bacteriocin activity, indicating that they each contain a one-peptide bacteriocin. The major peptide in the alpha fraction was blocked at its N-terminus, and a partial sequence (25 residues) could only be obtained after cleavage with CNBr. This sequence did not show clear homologies with known bacteriocins. The beta peptide has been sequenced almost completely and consists, presumably, of 53 residues. This peptide displayed strong homology to the known N-terminal part of brevicin 27 produced by Lactobacillus brevis SB27. The results showed that the beta peptide contains as many as six consecutive lysine residues at the N-terminus.     

The Formation, General Characteristics and Production of α-Amylase in Heterocaryons and Diploids of Aspergillus oryzae

S ummary . Heterocaryons and diploids from Aspergillus oryzae were investigated with respect to nuclear number/conidium and to conidial size. Heterocaryons usually had larger conidia and more nuclei/conidium than diploids and the haploid parent mutants. Diploids contained significantly fewer nuclei/conidium than haploids. However, they could not be distinguished from haploids by measurement of conidial size. The strains were examined for the production of α-amylase. All auxotrophic mutants produced less α-amylase than the prototrophic wild type. Heterocaryons gave yields which were intermediate between that of their parent mutants or the same as the best producing parent. Diploids which produced more α-amylase than the best producing parent strain were synthesized. The highest yield from a diploid was of the same order of magnitude as the yield from the wild type.     

Inhibition of Gibberellin Biosynthesis by Geraniol Derivatives and 17-Nor-16-azakauranes

Four analogues of the intermediates of gibberellin biosynthesis, 1-geranylimidazole (V) and three 17-nor-16-azakauranes (VIII), (IX) and (X) were found to inhibit gibberellin production of Gibberella fujikuroi by the procedure of bioassay which the author established. These compounds showed also growth retarding effects on rice seedlings.     

Inhibition of Tyrosine Aminotransferase by β-N-Oxalyl-l-α,β-Diaminopropionic Acid, the Lathyrus sativus Neurotoxin

Abstract: Species differences in susceptibility are a unique feature associated with the neurotoxicity of β-N-oxalyl-l -α,β-diaminopropionic acid (l -ODAP), the Lathyrus sativus neurotoxin, and the excitotoxic mechanism proposed for its mechanism of toxicity does not account for this feature. The present study examines whether neurotoxicity of l -ODAP is the result of an interference in the metabolism of any amino acid and if it could form the basis to explain the species differences in susceptibility. Thus, Wistar rats and BALB/c (white) mice, which are normally resistant to l -ODAP, became susceptible to it following pretreatment with tyrosine (or phenylalanine), exhibiting typical neurotoxic symptoms. C57BL/6J (black) mice were, however, normally susceptible to l -ODAP without any pretreatment with tyrosine. Among the various enzymes associated with tyrosine metabolism examined, the activity of only tyrosine aminotransferase (TAT) was inhibited specifically by l -ODAP. The inhibition was noncompetitive with respect to tyrosine (K_i = 2.0 ± 0.1 mM) and uncompetitive with respect to α-ketoglutarate (K_i = 8.4 ± 1.5 mM). The inhibition of TAT was also reflected in a marked decrease in the rate of oxidation of tyrosine by liver slices, an increase in tyrosine levels of liver, and also a twofold increase in the dopa and dopamine contents of brain in l -ODAP-injected black mice. The dopa and dopamine contents in the brain of only l -ODAP-injected white mice did not show any change, whereas levels of these compounds were much higher in tyrosine-pretreated animals. Also, the radioactivity associated with tyrosine, dopa, and dopamine arising from [¹⁴C]tyrosine was twofold higher in both liver and brain of l -ODAP-treated black mice. Thus, a transient increase in tyrosine levels following the inhibition of hepatic TAT by l -ODAP and its increased availability for the enhanced synthesis of dopa and dopamine and other likely metabolites (toxic?) resulting therefrom could be the mechanism of neurotoxicity and may even underlie the species differences in susceptibility to this neurotoxin.     

New Strains of Bacillus licheniformis and Bacillus coagulans producing Thermostable α-Amylase Active at Alkaline pH

Two species of Bacillus producing thermostable α-amylase with activity optima at alkaline pH are reported here. These organisms were isolated from soil and have been designated as Bacillus licheniformis CUMC 305 and B. coagulans CUMC 512. The enzymes released by these two species were partially purified up to about 81- and 72-fold respectively of the initial activity. The enzyme from B. licheniformis showed a wide temperature-range of activity, with optimum at 91°C. At this temperature it remained stable for 1 h. It retained 40–50% activity at 110°C and showed only 60% of its activity at 30°C. The enzyme showed a broad pH range of activity (4–10) retaining substantial activity on the alkaline side. The optimum pH was 9·5. The enzyme of B. coagulans showed activity up to 90°C, with optimum at 85°C and had a wide pH range with optimum at 7·5–8·5. The hydrolysis pattern of the substrate starch by these enzymes indicated that glucose, maltose, maltotriose and maltotetraose are the principal products rather than higher oligosaccharides.     

The Influence of the Synthetic Growth Regulator α-Chloro-(β-(3-chloro-o-tolyl) propionitrile on Some Phytochrome-mediated Processes

In intact Sinapis alba seedlings the synthetic growth regulator α-chloro-β-(3-chloro-o-tolyl)propionitrile (PRB-8) inhibits the phytochrome-mediated anthocyanin synthesis, carotenoid synthesis and L-phenylalanine ammonia-lyase activity. This action was compared with that of other auxins and with that of α-chloro-β-(3-chloro-o-tolyl)propionitrile-homologues. From the results it appeared that PRB-8 and some other o-tolyl derivatives are more inhibitive than other known auxins.