Para-aminobenzoic acid inhibits a set of SOS functions in Escherichia coli K12

PABA - Vitamin H1 of group B, has obtained increasing fundamental interest as a very potent natural antimutagen after a series of our publications since 1979. In the first set of our experiments, we studied PABA in the assays with the alkylating agent N-methyl-N-nitrosourea (MNU). Mutagenic efficiency of this agent was suppressed up to 10-fold when PABA was administered into Escherichia coli cells concurrently with the mutagen or prior to the mutagenic treatment. NMR spectrometric and UV-spectrophotometric measurements did not reveal an interaction between the direct acting MNU and PABA, typical for some N-nitroso compounds and phenolics. PABA suppressed the error-prone DNA repair pathway induced by UV-irradiation. PABA decreased MNU-induced phage lambda lysogenic induction more than two orders of magnitude. PABA inhibited the thermal shift up to 400-fold in phage lambda from the permissive to non-permissive temperature in E. coli mutant tif-1 and decreased about two-fold W-reactivation of UV-damaged phage lambda. Chloramphenicol treatment of the cells just after the mutagenic treatment prevented the occurrence of PABA specific activity. The results suggest that PABA affects the SOS DNA repair pathway and the mutagenic response of E. coli. PABA appears to be an effective bioantimutagen reducing mutagenesis by modulating the error-prone DNA repair (SOS) response.     

Studies on the mechanism of action of sulfanilamide on mycelial felts of Rhizoctonia solani

Summary Sulfanilamide induced strong inhibitory effects on the growth, respiration and carbohydrate synthesis by mycelial felts of Rhizoctonia solani. The possible reasons for such inhibitory effects are discussed.The inhibitory effects of sulfanilamide were almost completely alleviated by the inclusion of p-aminobenzoic (PABA) acid in the culture medium of the fungal mats. R. solani normally produces certain amounts of PABA and its growth and metabolism is inhibited in presence of excess of it.     

Characterization and regulation of p-aminobenzoic acid synthase from Streptomyces griseus

p-Aminobenzoic acid synthase (PABA synthase) of Streptomyces griseus catalyses the conversion of chorismic acid to p-aminobenzoic acid (PABA), a precursor of the aromatic p-aminoacetophenone moiety of candicidin, a polyene macrolide antibiotic. This enzyme uses glutamine or ammonia as amino donors for PABA formation. Enzyme extracts converted [14C]chorismic acid to labelled PABA. PABA synthase was present in S. griseus IMRU 3570 only during the antibiotic producing phase. No detectable levels of the enzyme were found in cell-free extracts of nonproducing mutants of S. griseus obtained after UV mutagenesis. PABA synthase activity was found also in Streptomyces coelicolor var. aminophilus, producer of the polyene macrolide antibiotic fungimycin, but it was not present in extracts of several other streptomycetes that do not produce aromatic polyene macrolide antibiotics. PABA synthase (amidotransferase) activity was partially purified by DEAE-Bio-gel and Sephacryl S-200 filtrations. The estimated molecular weight was 50000. PABA synthase was repressed by aromatic amino acids and PABA but not by anthranilic acid. Inorganic phosphate strongly repressed but did not inhibit PABA synthase activity.     

Colorimetric plasma assay for the bentiromide test (BT-PABA) for exocrine pancreatic insufficiency

Bentiromide is a synthetic peptide, N-benzoyl-L-tyrosyl-p-aminobenzoic acid, which has been used as a test for exocrine pancreatic function. Following oral administration, bentiromide is hydrolyzed by chymotrypsin to yield free p-aminobenzoic acid (PABA) which is absorbed, conjugated and excreted in the urine. The PABA conjugates reach their peak levels in blood in 90-120 min. Healthy individuals have higher levels of PABA than patients with pancreatic insufficiency. A simple, accurate, and precise method for the determination of PABA in blood has been developed and validated. The plasma (1 ml) is deproteinized by perchloric acid. The conjugates are hydrolyzed and the total PABA is determined colorimetrically by the Bratton-Marshall test. The standard curve in plasma is linear up to 8 micrograms/ml of PABA. A similar semimicro method using 200 microliter of plasma suitable for pediatric samples shows comparable results. Average analytical recovery is 97% and precision studies of pooled within-run and total between-run showed CV% of 5.0 and 5.7%, respectively.     

Catalytic Properties of Tryptophanase from Proteus rettgeri

Formation of sclerotia in a strain of Sclerotinia libertiana Fuckel using Czapek-Dox agar medium was highest at pH 4.0~6.0 and at 22~25°C. The response was better under darkness than under light. However, when a potato-extract medium was used the optimum temperature range extended, and even at 15~27°C mature sclerotia formed. The addition of indole-3-acetic acid (IAA) to the Czapek-Dox medium containing riboflavine stimulated the formation of sclerotia under fluorescent light. Though iodoacetic acid, a ?SH reagent, also had a stimulatory effect, cysteine had little inhibitory effect on sclerotial formation. Formation was markedly inhibited by p-aminobenzoic acid (PABA), especially in the presence of tyrosine or tryptophan, and excess ammonium salts in the medium also produced an inhibitory effect. It was assumed that accumulation of an intermediary metabolite with high reactivity with ?SH groups was necessary for sclerotial formation, but PABA and excess ammonium salts inhibited the accumulation.     

Genetic Transformation in Methylococcus capsulatus

SUMMARY: Deoxyribonucleic acid (final concentration 100 μg/ml) extracted from wild-type cells of Methylococcus capsulatus was used to transform a p -amino benzoic acid (PABA) requiring strain (paba^-). Maximum levels of transformants were obtained towards the end of the exponential phase of growth in shake flask culture and the level of the population of transformants was not significantly altered by post-treatment incubation in enriched medium. The maximum transformation frequency was 0.17% of the initial population of PABA requiring cells and the back mutation frequency was 1 : 3.12 × 10⁸.     

Growth Inhibition Studies: II. SULPHANILAMIDE INHIBITION AND ITS REVERSAL

In a study of the inhibitory effect of sulphanilamide on thegrowth of flax seedlings and its reversal byp-aminobenzoic acid(PABA) and related compounds, two distinct types of reversalmechanism have been observed. The results, derived from growth-ratemeasurements, are interpreted as indicating that, in the flaxseedling, sulphanilamide interferes with the conversion of PABAto pteroyl-glutamic acid. None of the purine or pyrimidine compounds tested showed anyreversing action.     

The synthesis of folic acid-like compounds by Lactobacillus arabinosus

In the presence of p-aminobenzoic acid (PABA), Lactobacillus arabinosus synthesizes one or more compounds with folic acid (FA)-like activity during growth. The total FA activity formed is proportional to the amount of PABA added, up to 200 mμg./tube. Most of the FA activity is in a bound form which is present chiefly in the cells, and the remainder is present in free form which is mainly in the culture medium. Increasing levels of sulfanilamide in the medium competitively inhibit the utilization of PABA for growth of L. arabinosus and decrease the amount of free and combined FA compounds formed.Equimolecular amounts of p-aminobenzoylglutamic acid and PABA have approximately the same growth-promoting and antisulfanilamide activities for L. arabinosus under the conditions employed, and lead to the synthesis of similar amounts of the FA-like compound by L. arabinosus.Thymidine can replace PABA for growth of L. arabinosus and its activity is inhibited very little by sulfanilamide. Thymine and thymidylic acid are much less effective than thymidine. Crystalline vitamin B_c conjugate and vitamin B₁₂ cannot replace PABA for growth of L. arabinosus and do not augment the antisulfonamide activity of PABA.The growth-promoting and antisulfanilamide activities of synthetic folid acid (pteroylglutamic acid), citrovorum factor, and formylfolic acid preparations for L. arabinosus are much poorer than those of equimolecular amounts of PABA. The possibility that the compound(s) with FA-like activity produced by L. arabinosus may differ from pteroylglutamic acid, citrovorum factor, or formylfolic acid is discussed.     

Growth inhibition of Candida albicans by folate pathway inhibitors. Their potential in the selection of auxotrophs

Growth studies were conducted on C. albicans in a glucose — salts — biotin (GSB) medium in the presence of folate inhibitors. Sulfanilamide inhibited growth which was restored by PABA or tetrahydrofolate (THF). Aminopterin inhibited growth to about the same level as did sulfanilamide, but this inhibition was not reversed with PABA nor THF, singly or in combination. Inhibition by combined sulfanilamide and aminopterin was synergistic, reducing growth by more than 90% for 48 h. The sulfanilamide component of the combined inhibition was reversed by PABA or THF to the level of that of aminopterin alone. Cytochrome synthesis was not affected by the inhibitors, but marked increases occurred in -ketoglutarate, malate, isocitrate, and pyruvate dehydrogenases, especially in the presence of both inhibitors. The pyrimidines in combination with sulfanilamide were as inhibitory as was the combination of aminopterin and sulfanilamide, but they had no effect when added alone or in combination with aminopterin. Unlike the pyrimidines, the purines stimulated about a 50% recovery from inhibition by either of the inhibitors. Growth inhibition by combined sulfanilamide and aminopterin was overcome by about 50% by the addition of the THF-mediated end-produits: deoxythymidylate, adenine, histidine and methionine. The use of GSB medium containing adenine, histidine, methionine and the folate inhibitors but without deoxythymidylate resulted in thymineless death of prototrophic cells providing a method for the selection of auxotrophic mutants.     

Induced Resistance to Sulfonamides in Chilomonas paramecium

SUMMARY. Strains of Chilomonas paramecium differing in degree of resistance to sulfanilamide have been established through acclimatization to this sulfonamide at 50, 100 and 200 mg. %. Resistant strains differ from the normal stock in their enhanced sensitivity to p -aminobenzoic acid. In the normal stock, sulfanilamide inhibition is reversed at an SA/PABA ratio of 10,000 but not at 20,000; in the least resistant strain, at a ratio of 400,000 but not at 800,000. In resistant strains inhibition is reversed by folk acid, methionine, adenine, cytosine and thymine; in the normal stock, none of these metabolites produces reversal. In high concentrations of PABA (10–20 mg. %) growth of the normal stock is only retarded, whereas the strain least resistant to sulfanilamide fails to recover from exposure to 20 mg. % PABA. The strain most resistant to sulfanilamide is most susceptible to PABA in high concentrations. The data suggest that resistance to sulfanilamide in C. paramecium may depend mainly upon an accelerated synthesis of PABA.     

Inhibition of (Ca2+ + Mg2+)-ATPase by carbodiimides. A structure-activity study

Nine hydrophobic carbodiimides were synthesized and their chemical reactivities (towards acetic acid) and inhibitory capacities (towards the (Ca2+ + Mg2+)-ATPase were measured. No correlation between chemical reactivity and inhibitory efficacy emerges, but a significant effect of molecular bulk on reactivity towards the calcium-protectable carboxyl groups of the ATPase is noted: methyl-substituted compounds inhibit the enzyme in the presence of Ca2+, while aryl- or cyclohexyl-substituted compounds do not inactivate in the presence of Ca2+.     

Para-aminobenzoic acid--an interferon inducer]

Para-aminobenzoic acid (PABA) was shown to be an early type interferon inductor. PABA (10 micrograms/ml) induced interferon production in vitro in the cells of human peripheral blood and in vivo in albino mice (10 mg/kg). The results of the study suggested that PABA was able to induce production of interferon-alpha/beta in various immunocyte populations. By its interferonogenic activity PABA was comparable with the known interferon inductors. One of the mechanisms of the previously described in vivo antiherpes action of PABA can be attributed to its interferon inducing activity.     

Effect of L-ethionine on the expression of the SOS system in Escherichia coli

The effect of L-ethionine, the ethyl analog of the essential amino acid methionine, on the SOS system of Escherichia coli was studied. This compound does not induce either inhibition of cell division nor cessation of cell respiration in a RecA+ Met+ RelA+ strain, nor in RecA+ Met- RelA+ or RecA+ Met- RelA- mutants. Nevertheless, L-ethionine blocks the expression of both cited SOS functions in a recA441 mutant when it is growing at the restrictive temperature of 42 degrees C. Furthermore, the inhibitory effect of the L-ethionine on the induction of the SOS system in this mutant is increased when the cells are preincubated for several hours in the presence of the analog, before the temperature shift. Moreover, cultures of the recA441 mutant incubated at 42 degrees C in the presence of both L-ethionine and L-methionine present the same behaviour as the cultures of this mutant growing at the same temperature but without either amino acid. On the other hand, L-ethionine does not have any effect on the expression of the two mentioned SOS functions when these are induced by UV-irradiation in a RecA+ strain even if this compound is added to the cells several hours before irradiation.     

Antimutagenic effect of p-aminobenzoic acid on the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium

p-Aminobenzoic acid (PABA) exhibited antimutagenic activity toward N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)-induced mutagenicity in the Ames assay in Salmonella typhimurium. The antimutagenic effects were associated with an increased rate of decomposition of MNNG in the presence of PABA. The participation of other mechanisms, such as the alteration of cellular processes by PABA, however, cannot be excluded.     

Biochemical and genetic characterization of an early step in a novel pathway for the biosynthesis of aromatic amino acids and p-aminobenzoic acid in the archaeon Methanococcus maripaludis

Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon and facultative autotroph capable of biosynthesizing all the amino acids and vitamins required for growth. In this work, the novel 6-deoxy-5-ketofructose-1-phosphate (DKFP) pathway for the biosynthesis of aromatic amino acids (AroAAs) and p-aminobenzoic acid (PABA) was demonstrated in M. maripaludis. Moreover, PABA was shown to be derived from an early intermediate in AroAA biosynthesis and not from chorismate. Following metabolic labelling with [U-(13)C]-acetate, the expected enrichments for phenylalanine and arylamine derived from PABA were observed. DKFP pathway activity was reduced following growth with aryl acids, an alternative source of the AroAAs. Lastly, a deletion mutant of aroA', which encodes the first step in the DKFP pathway, required AroAAs and PABA for growth. Complementation of the mutants by an aroA' expression vector restored the wild-type phenotype. In contrast, a deletion of aroB', which encodes the second step in the DKFP pathway, did not require AroAAs or PABA for growth. Presumably, methanococci contain an alternative activity for this step. These results identify the initial reactions of a new pathway for the biosynthesis of PABA in methanococci.     

Laccase catalysed modification of lignin subunits and coupling to p-aminobenzoic acid

Laccase catalysed oxidation of syringyl and guaiacyl subunits of lignin and their modification with an aromatic amine, p-aminobenzoic acid (PABA) were investigated. Laccase from Galerina sp. HC1 isolated earlier by us was used as the main catalyst, and Trametes versicolor laccase was used for comparison. Among the syringyl compounds, syringic acid and syringaldehyde were oxidised to 2,6-dimethoxy-1,4-benzoquinone, and in the presence of PABA yielded a cross-coupling imine product. The reaction with methyl syringol resulted in several products whose structures were determined. The possible oxidative coupling pathways were proposed for the formation of the identified products. Oxidation of syringol and the guaiacyl compounds resulted mainly in homooligomers by free radical mechanism, with a negligible tendency of reaction with the nucleophilic group of PABA. Similar treatment of Eucalyptus Kraft lignin, which is rich in syringyl moieties, showed the presence of identical products obtained with syringic acid and syringaldehyde.     

Vitamin para-aminobenzoic acid inhibits development of SOS function in tif-1 mutants of Escherichia coli at nonpermissive temperatures

The development of "SOS" inducible functions in lysogenic and non-lysogenic strains of Escherichia coli tif-1 sfiA11 (lambda) at nonpermissive temperature of 42 degrees C was strongly suppressed by para-aminobenzoic acid (PABA). The rate of prophage lambda induction decreased 400 times, as compared to the control level; the efficiency of W-reactivation of UV-irradiated phage lambda decreased 37.5 to 16%. PABA also inhibited to some extent (1.5 times) the process of inducible recombination on the RecF pathway. The processes of spontaneous lambda induction and W-reactivation, as well as spontaneous recombination on RecBC and RecF pathways, were not influenced by PABA. The above data are in accordance with previous studies of PABA action when the manifestation of "SOS" functions was induced by chemical mutagens. The action of PABA has been tentatively interpreted on the basis of negative control of "SOS" repair pathway.     

Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli.

The metabolic fate of p-aminobenzoic acid (PABA) in Escherichia coli is its incorporation into the vitamin folic acid. PABA is derived from the aromatic branch point precursor chorismate in two steps. Aminodeoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate and is composed of two subunits, PabA and PabB. ADC lyase removes pyruvate from ADC, aromatizes the ring, and generates PABA. While there is much interest in the mechanism of chorismate aminations, there has been little work done on the ADC synthase reaction. We report that PabA requires a preincubation with dithiothreitol for maximal activity as measured by its ability to support the glutamine-dependent amination of chorismate by PabB. PabB glutamine enhances the protective effect of PabA. Incubation with fresh dithiothreitol reverses the inactivation of PabB. We conclude that both PabA and PabB have cysteine residues which are essential for catalytic function and/or for subunit interaction. Using conditions established for maximal activity of the proteins, we measured the Km values for the glutamine-dependent and ammonia-dependent aminations of chorismate, catalyzed by PabB alone and by the ADC synthase complex. Kinetic studies with substrates and the inhibitor 6-diazo-5-oxo-L-norleucine were consistent with an ordered bi-bi mechanism in which chorismate binds first. No inhibition of ADC synthase activity was observed when p-aminobenzoate, sulfanilamide, sulfathiazole, and several compounds requiring folate for their biosynthesis were used.     

Aldose-reductase- and protein-glycation-inhibitory principles from the whole plant of Duchesnea chrysantha

Ellagic acid (1), 3,3'-di-O-methylellagic acid (2), 3,3',4-tri-O-methylellagic acid (3), isovitexin (4), kaempferol 3-O-beta-D-glucuronide methyl ester (5), quercetin 3-O-alpha-L-arabinopyranosyl-(1-->6)-beta-D-galactopyranoside (6), ursolic acid, pomolic acid, tormentic acid, euscaphic acid, euscaphic acid 28-O-beta-D-glucopyranoside, and maslinic acid were isolated from the AcOEt- and BuOH-soluble MeOH extract of Duchesnea chrysantha (whole plant). The isolates were subjected to in vitro bioassays to evaluate their inhibitory activity on rat-lens aldose reductase (RLAR) and formation of advanced glycation end products (AGEs). The ellagic acids and flavonoids, compounds 1-6, exhibited moderate inhibitory effects on RLAR. However, compounds 1 and 4-6 showed excellent inhibitory activities towards the formation of AGEs. This is the first report that 4 and 6 exhibit inhibitory activity towards AR and AGEs formation.     

Mechanism of photoaffinity labeling of P2-purinergic receptors by arylazido aminopropionyl atp in isolated guinea-pig vas deferens

Following photolysis in the presence of the isolated guinea-pig vas deferens, arylazido aminopropionyl ATP (ANAPP₃), a photoaffinity label analog of ATP, produces an irreversible and specific pharmacological antagonism of contractile responses to adenine nucleotides. Experiments were performed to determine whether the antagonism follows the photolysis-dependent formation of nitrene intermediates at occupied receptors (true photoaffinity labeling) or if the reactive intermediate is photogenerated in solution prior to diffusion to the receptor and formation of covalent bonds (pseudo-photoaffinity labeling). When present during photolysis, para-aminobenzoic acid (PABA; 10 mM), a scavenger for nitrenes generated in solution, did not prevent the antagonism of ATP-induced responses by ANAPP₃. However, the absorption spectrum of ANAPP₃ photolyzed in the presence of PABA was different from that obtained when PABA was not present. This evidence for the formation of additional photolysis products suggests that ANAPP₃ had inserted into PABA during photolysis. Thus, covalent bonds arise from true photoaffinity labeling of the receptor. An analysis of the pharmacological effects of PABA indicated that responses to ATP, KCl and acetylcholine were unaffected either in the presence of, or after a 23 min incubation, with 10 mM PABA. In contrast, PABA produced a substantial, but reversible, antagonism of histamine- and norepinephrine-induced contractions. Irradiation of tissues in the presence of 10 mM PABA produced a slight potentiation of responses to ATP. Thus, information on the mechanisms of photoaffinity labeling may be obtained from functional studies on intact tissues. However, the pharmacological effects of agents used to define these mechanisms should be evaluated as well.