Benzyl isothiocyanate is the chief or sole anthelmintic in papaya seed extracts.

Papaya (Carica papaya) seeds were extracted in an aqueous buffer or in organic solvents, fractionated by chromatography on silica and aliquots tested for anthelmintic activity by viability assays using Caenorhabditis elegans. For all preparations and fractions tested, anthelmintic activity and benzyl isothiocyanate content correlated positively. Aqueous extracts prepared from heat-treated seeds had no anthelmintic activity or benzyl isothiocyanate content although both appeared when these extracts were incubated with a myrosinase-containing fraction prepared from papaya seeds. A 10 h incubation of crude seed extracts at room temperature led to a decrease in anthelmintic activity and fractionated samples showed a lower benzyl isothiocyanate content relative to non-incubated controls. Benzyl thiocyanate, benzyl cyanide, and benzonitrile were not detected in any preparations and cyanogenic glucosides. which were present, could not account for the anthelmintic activity detected. Thus, our results are best explained if benzyl isothiocyanate is the predominant or sole anthelmintic agent in papaya seed extracts regardless of how seeds are extracted.

Isolation of a gene that down-regulates nitrate assimilation and influences another regulatory gene in the same system.

Glutamine is the preferred source of nitrogen of Neurospora crassa. In its presence and that of the gene product of MS5 (nmr-1), the fungus represses the assimilation of less preferred forms of nitrogen, such as nitrate. In the absence of glutamine and the presence of the product of gene nit-2, less preferred forms of nitrogen are assimilated as long as a specific pathway for their assimilation is induced. We report here the isolation, from a cosmid bank, of a gene that complements MS5 and can also complement nit-2. We speculate that this result suggests an interaction between the MS5 and nit-2 gene products and that this is important in the regulation of nitrate assimilation.

Physiological characterization of a Neurospora crassa mutant with impaired regulation of nitrate reductase.

This report describes the isolation and characterization of a Neurospora crassa mutant with an impaired regulation of nitrate reductase. Glutamine, which prevents the induction of nitrate reductase in N. crassa, did so relatively ineffectively in this mutant. The mutation did not affect the regulation of all enzymes regulated by "nitrogen metabolite regulation"; it did affect the regulation of nitrate reductase, nitrite reductase, histidase, and acetamidase, as well as that of thiourea sensitivity. The mutation was not allelic with nit-2, the gene controlling a general positive effector of nitrogen metabolite-regulated enzyme formation.

Repression of nitrate reductase in Neurospora studied by using L-methionine-DL-sulfoximine and glutamine auxotroph gln-1b.

The effect of L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase, on the formation of nitrate reductase in the wild-type strain of Neurospora in the presence of ammonium ions and of glutamine was studied. Under conditions in which glutamine synthetase was inactivated, it was found that only glutamine could repress nitrate reductase. In a mutant of Neurospora, gln-1b, which requires glutamine for growth, only glutamine could repress nitrate reductase. These results suggest a direct role for glutamine as corepressor of nitrate reductase in Neurospora.

Nitrogen metabolite repression of nitrate reductase in Neurospora crassa.

The effect of different nitrogen compounds on the induction of reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase was examined in Neurospora crassa. Whereas in the wild-type strain several amino acids and ammonia inhibit the formation of nitrate reductase, only glutamine, cysteine, and histidine are shown to inhibit the synthesis of nitrate reductase in a glutamine-requiring auxotroph. None of the amino acids inhibited nitrate reductase activity in vitro. The effects of cysteine and histidine are nonspecific, these amino acids being inhibitory of the growth of the organism. The effect of glutamine on the induction of nitrate reductase is not due to an inhibition of the uptake of the inducer nitrate. By the use of histidine-, pyrimidine-, and arginine-requiring auxotrophs, it was shown that glutamine appears to act per se and does not seem to be converted to another product in order to be effective in repression. The repression of nitrate reductase by ammonia appears, from the results described herein, to be indirect; ammonia has to be converted first to glutamine in order to be effective in repression.

Stability of messenger RNA for nitrate reductase in Neurospora crassa.

A method has been developed to study the synthesis and decay of the messenger RNA for nitrate reductase in Neurospora crassa. Glutamine prevents the synthesis of the mRNA which appears to have a half-life of approximately 8.5 min.

The regulation of the decay of nitrate reductase. Evidence for the existence of at least two mechanisms of decay.

There seem to be at least two different mechanisms of decay of nitrate reductase in Neurospora in vivo: one which is very sensitive to EDTA and cycloheximide, decreases with mycelial age and is not increased by an increase in temperature from 27 to 37 degrees C, the other which is relatively insensitive to EDTA and cycloheximide, increases with the age of the mycelium and with the above temperature shift.

Demonstration in vitro of two intracellular inactivators of nitrate reductase from Neurospora.

Two different inactivators of nitrate reductase have been found in cell free preparations of Neurospora. The first (Inactivator I) is very active at pH 9, is inhibited by disodium ethylene diamine tetraacetate (EDTA) and is present in all mycelia incubated under all conditions tested; the second (Inactivator II) is very active at pH 5, is repressed by ammonia or by a metabolic product of ammonia and derepressed by nitrogen starvation, cannot be derepressed by nitrogen starvation in strain nit-2, in which a number of "ammonia-represible" enzymes are permanently repressed, and is sensitive to phenyl methyl sulfonyl fluoride. Crude extracts of mycelia contain inhibitor(s) of both inactivators.

Effect of ammonium ions on the induction of nitrite reductase in Neurospora crassa.

Results with strain am-la, a glutamate dehydrogenaseless mutant, showed that ammonium ions must first be metabolized in order to repress nitrite reductase in Neurospora.

Enzymatic and non-enzymatic reduction of nitrite by extracts of Neurospora crassa.

Two activites causing nitrite disappearance are found in extracts of Neurospora; one, inducible by nitrate or nitrite and present only in nitrite-utilizing strains, catalyze the stoichiometric reduction of nitrite to ammonia; the other, present in all strains under all conditions, causes the disappearance of nitrite to something other than ammonia. The latter activity has a molecular weight of about 600 and may contain an oligopeptide, a metal, and an SH group(s). It has no known physiological function.

Effect of nitrate on the synthesis and decay of nitrate reductase of Neurospora.

1. A method was developed to examine the turnover of nitrate reductase by the use of tungstate. 2. Evidence is presented which suggests that the disappearance of nitrate reductase activity from Neurospora mycelia exposed to non-inducing conditions is due to the disappearance of the enzyme protein(s) from the mycelia, and not merely due to the disappearance of its (their) catalytic power. 3. The presence of NO(3) (-) in the culture medium slows down the rate of degradation of nitrate reductase in Neurospora in vivo.

Regulation of nitrate reductase of Neurospora at the level of transcription and translation.

The presence of nitrate is required for the induced synthesis of NADPH-nitrate reductase and its related partial activity Benzyl Viologen-nitrate reductase in a wild-type strain of Neurospora. In nit-3, a mutant lacking complete NADPH-nitrate reductase activity but retaining the partial activity Benzyl Viologen-nitrate reductase, the presence of nitrate ions is not required for the de-repressed synthesis of the latter enzyme. The accumulation of the capacity to synthesize nitrate reductase, and the related Benzyl Viologen-nitrate reductase, in the absence of protein synthesis does not require nitrate in the normal strain or in strain nit-3. Ammonia antagonizes the accumulation of this capacity in both strains. Nitrate is required for the synthesis of nitrate reductase and related activities from presumedly preformed mRNA in the wild-type strain. Nitrate is not required for the comparable function in strain nit-3. Ammonia appears to stop the synthesis of nitrate reductase and related activities from presumedly preformed mRNA in the wild-type strain and in strain nit-3. The effects of nitrate, or ammonia and of no nitrogen source on the induced synthesis of nitrate reductase cannot be explained on the basis of the effects of the different nitrogen sources on general synthesis of RNA or of protein.

Regulation of nitrate reductase in Neurospora crassa: stability in vivo.

Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-nitrate reductase and its related enzyme activities, NADPH-cytochrome c reductase and reduced benzyl viologen-nitrate reductase, are all induced following the transfer of ammonia-grown wild-type Neurospora mycelia to nitrate medium. After nitrate reductase is induced to the maximal level, the addition of an ammonium salt to, or the removal of nitrate from, the cultures results in a rapid inactivation of nitrate reductase and its two partial component activities. This rapid inactivation is slowed down by the protein synthesis inhibitor, cycloheximide. Experiments on the mixing of extracts in vitro rule out the presence of an inhibitor of nitrate reductase in free form in extracts containing inactivated nitrate reductase. Ammonia does not inhibit the uptake of nitrate by the mycelia. Inactivation of nitrate reductase in vivo by ammonia depends on the concentration of the ammonium salt and is not reversed by increasing the nitrate concentration of the medium. The nitrate-inducible NADPH-cytochrome c reductase activity and reduced benzyl viologen-nitrate reductase activity respectively of the nitrate-nonutilizing mutants nit-1 and nit-3 are not inactivated in vivo by the addition of an ammonium salt or the withdrawal of nitrate. This finding suggests that the integrity of the nitrate reductase complex is required for the in vivo inactivation of nitrate reductase and its associated activities.

Regulation of nitrate reductase in Neurospora crassa: regulation of transcription and translation.

A technique employing cycloheximide and actinomycin D has been used for the separation of transcription and translation during the induction of nitrate reductase in Neurospora crassa. Nitrate reductase is found to be synthesized in low efficiency when nitrate is not provided during both transcription and translation. Nitrate reductase synthesis is enhanced by nitrate. Nitrate is found to induce nitrate reductase by enhancing the increase of the capacity to synthesize nitrate reductase, and ammonia is found to repress nitrate reductase, by inhibiting the induced increase of the capacity to make the enzyme, or by making it unstable in vivo, or both. The effect of ammonia is partially reversed by nitrate. The addition of ammonium tartrate or the removal of nitrate during translation of the induced capacity to synthesize nitrate reductase is found to result in the inactivation of nitrate reductase in vivo. A low level of nitrate in the medium is found to be sufficient for enhancing the induced increase of the capacity to synthesize nitrate reductase, but a higher level of nitrate is required to stabilize the enzyme after its formation. The induced capacity to synthesize nitrate reductase is relatively stable in the presence or absence of nitrate, but not in the presence of ammonia.

Effect of nitrogenase components from mutant and wild-type strains of Azotobacter on the dilution effect of nitrogenase.

Nitrogenase activity exhibits a dilution effect. Evidence is presented that the reason for the dilution effect is that one of the component proteins of nitrogenase is limiting in preparations of this enzyme. The limiting component appears to be the non-haem-iron-containing protein (also called fraction II, iron protein, azoferredoxin), which is equivalent to the enhancement factor for nitrogenase activity present in extracts of nitrogenaseless mutant 22R1. A mathematical function of specific activity is described that is useful in describing nitrogenase. It takes into account the dilution effect and the exponential nature of the relationship between nitrogenase activity and enzyme protein concentration.

Nitrogenaseless mutants of Azotobacter vinelandii.

Mutants of Azotobacter which grow normally on excess ammonia under a variety of conditions and which grow slowly or not at all on atmospheric nitrogen have been isolated. Extracts of these strains have low or no detectable nitrogenase activity. There are three classes of mutants. Cell-free preparations of members of the first class possess an enhancement factor (EF+) which stimulates wild-type nitrogenase in vitro. Homogenates of members of the second class possess an enhanceable factor (EF-) which complements in vitro with extracts of the first class of mutants to give substantial nitrogenase activity. Preparations of members of the third class contain neither EF+ nor EF- activity. EF+ and EF- are repressed by the same conditions that repress nitrogenase. Molybdenum-deficient cells of the second class of mutants do not appear to contain EF- activity, but molybdenum deficient cells of the first class of mutants contain EF+. Because of these observations, EF+ is tentatively equated to azoferredoxin and EF- to molybdoferredoxin.

Regulation of nitrogen fixation in Azotobacter vinelandii OP: the role of nitrate reductase.

A number of chlorate-resistant mutants were selected, and one of these, clr68-5, was studied in detail. This mutant cannot utilize nitrate in vivo to overcome the effect of nonmetabolizable repressors of nitrogenase. The reason for this inability was that strain clr68-5 lacked nitrate reductase. Nitrate inhibited the activity of nitrogenase but did not act as a corepressor of nitrogenase in strain clr68-5 as it does in the wild type. Ammonia seemed to act as corepressor of nitrogenase in both strains.