Cell-free dihydropteroate synthetase activity: its use to investigate the relationship between structure and activity of p-aminobenzoic derivatives.
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4-Aminobenzoic Acid↗
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ADP-ribosyl transferase activity in Trypanosoma brucei.
Nuclear adenosine diphosphoribosyl transferase (ADPRT) catalyses the covalent modification of chromatin proteins by (ADP-ribose)n. This activity, which is entirely dependent on DNA containing strand breaks, is required for efficient DNA excision repair possibly because it regulates DNA ligation. ADPRT activity is also required for cytodifferentiation in a number of different cell types. We report here the presence of ADPRT activity in the blood-stream form of Trypanosoma brucei and its activation by DNA strand breaks formed by exposure to, either exogenously supplied deoxyribonuclease I, or treatment with the methylating agent, dimethylsulphate. 3-Aminobenzamide, but not its chemical analogue 3-aminobenzoic acid, is a competitive inhibitor of ADPRT activity in T. brucei. Intact trypanosomes are readily permeable to this competitive inhibitor of ADPRT activity.
Animals↗
Benzamide-DNA interactions: deductions from binding, enzyme kinetics and from X-ray structural analysis of a 9-ethyladenine-benzamide adduct.
The interaction of benzamide with the isolated components of calf thymus poly(ADP-ribose) polymerase and with liver nuclei has been investigated. A benzamide-agarose affinity gel matrix was prepared by coupling o-aminobenzoic acid with Affi-Gel 10, followed by amidation. The benzamide-agarose matrix bound the DNA that is coenzymic with poly(ADP-ribose) polymerase; the matrix, however, did not bind the purified poly(ADP-ribose) polymerase protein. A highly radioactive derivative of benzamide, the 125I-labelled adduct of o-aminobenzamide and the Bolton-Hunter reagent, was prepared and its binding to liver nuclear DNA, calf thymus DNA and specific coenzymic DNA of poly(ADP-ribose) polymerase was compared. The binding of labelled benzamide to coenzymic DNA was several-fold higher than its binding to unfractionated calf thymus DNA. A DNA-related enzyme inhibitory site of benzamide was demonstrated in a reconstructed poly(ADP-ribose) polymerase system, made up from purified enzyme protein and varying concentrations of a synthetic octadeoxynucleotide that serves as coenzyme. As a model for benzamide binding to DNA, a crystalline complex of 9-ethyladenine and benzamide was prepared and its X-ray crystallographic structure was determined; this indicated a specific hydrogen bond between an amide hydrogen atom and N-3 of adenine. The benzamide also formed a hydrogen bond to another benzamide molecule. The aromatic ring of benzamide does not intercalate between ethyladenine molecules, but lies nearly perpendicular to the planes of stacking ethyladenine molecules in a manner reminiscent of the binding of ethidium bromide to polynucleotides. Thus we have identified DNA as a site of binding of benzamide; this binding is critically dependent on the nature of the DNA and is high for coenzymic DNA that is isolated with the purified enzyme as a tightly associated species. A possible model for such binding has been suggested from the structural analysis of a benzamide-ethyladenine complex.
Adenine↗
Escherichia coli aminodeoxychorismate synthase: analysis of pabB mutations affecting catalysis and subunit association.
p-Aminobenzoic acid (PABA), an essential component of the vitamin folic acid, is derived from the aromatic branch-point precursor chorismate in two steps. 4-Amino-4-deoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate, and is composed of two subunits, PabA and PabB. While various experiments have suggested that PabA and PabB act as a complex, attempts to isolate the intact complex have failed. We report here the first successful copurification of PabA and PabB by gel filtration chromatography. The association of PabA and PabB is greatly enhanced by the presence of 5 mM glutamine, and by preincubation at 37 degrees C. Conversely, the association is greatly reduced at cold temperatures. We also report the isolation and characterization of both chemically induced and site-directed mutations in PabB. Mutated PabB enzymes fall into three categories according to their properties: deficiency of chorismate amination coupled with failure to associate with PabA, deficiency of chorismate amination coupled with retention of PabA association, and competency of chorismate amination with failure of PabA association.
Amination↗
Studies on age related changes in cytochrome P-450, cytochrome b-5 and mixed function oxidase activity in mouse liver microsomes, in relation to their phospholipid composition.
Liver microsomes were isolated from male mice of various defined ages, and their ability to metabolise ethylmorphine, p-nitroanisole, p-aminobenzoic acid and aniline was assessed as was their content of cytochrome P-450, cytochrome b-5 and protein, and their lipid composition. The rising capacity of the microsomes to metabolise these substrates in the first half of the lifespan was associated with a rise in the cytochrome P-450 and cytochrome b-5 content as well as an increase in the ratio of phosphatidylcholine to both phosphatidylethanolamine and sphingomyelin and, in early adult life, decreasing saturation of phospholipid fatty acids. The lipid changes would be expected to increase microsomal membrane fluidity. Declining mixed function oxidase activity in later life paralleled a decline in the ratio of phosphatidylcholine to phosphatidylethanolamine and sphingomyelin, and progressively reduced saturation of phospholipid fatty acids. Cytochrome content showed only a slight irregular decline with advancing age until the oldest animals were investigated, in which cytochrome b-5 was considerably reduced. Unfortunately, cytochrome P-450 was not measured in the oldest available group. Inducing the mixed function oxidase system with daily phenobarbitone injections for 4 days substantially increased enzyme activity in animals up to the middle of the age range tested. Older animals showed progressively reduced induction and none at all was seen in the oldest group. In contrast with the situation in non-induced mice, these changes were more closely associated with changes in cytochrome P-450 content than with alterations of lipid composition.
Aging↗
Long-term pancreatic duct occlusion impairs the entero-insular axis in the dog--failure of plasma VIP to respond as "incretin".
The response of VIP to either an oral glucose load (OGT) or intravenous glucose (IV glucose), aimed at reproducing the plasma glucose level after OGT, was studied in trained, conscious, sham-operated (Sham; n = 6) dogs, and dogs having initially (12 months before the glucose experiments) undergone occlusion of the pancreatic duct by the prolamine glue technique (Occ; n = 5). As a result, prior to glucose studies, the exocrine pancreas function was found subtotally reduced, as indirectly evaluated by the para-aminobenzoic acid (PABA) test, but no signs of diabetes were detected. The two studies with glucose administration designed to demonstrate the release of insulin, VIP, somatostatin into plasma as modified by enteric signals (represented by the difference of plasma peptide concentration during OGT minus peptide concentration during IV glucose) revealed the following: (1) basal plasma glucose, insulin, VIP, somatostatin did not differ between Sham and Occ dogs; (2) after OGT in Occ dogs the plasma glucose was elevated, whereas plasma insulin was markedly reduced, and VIP, somatostatin were largely unchanged; (3) the integrated output of insulin only was impaired when considering the so-called entero-insulin axis, while integrated VIP, somatostatin were unaltered. It was concluded (a) the Occ procedure in the dog has the capacity to subtotal destruction of the pancreatic acinar tissue, and of the entero-insular axis of insulin, the latter through yet unknown pathways, (b) the Occ technique may be a useful tool for investigation of the nature of "incretin," (c) VIP and somatostatin do not respond to elevated blood glucose and may have no role in the "incretin" concept of enteric modulation of the B-cell.
Animals↗
Genotypic and phenotypic characterization of an aroD deletion-attenuated Escherichia coli K12-Shigella flexneri hybrid vaccine expressing S. flexneri 2a somatic antigen.
The construction and characterization of EcSf2a-2, an aroD-deleted Escherichia coli-Shigella hybrid vaccine carrying chromosomal and plasmid genes from Shigella flexneri and expressing S. flexneri 2a somatic antigen in association with E. coli K12 core are described. Expression of hybrid lipopolysaccharide and deletion of aroD resulted in the attenuation of phenotypic characteristics associated with pathogenicity. The addition of an aroD deletion results in a requirement for an aromatic precursor of para-aminobenzoic acid (PABA), an essential bacterial metabolite not present in mammalian tissues. The biosynthesis of hybrid somatic antigen prevents expression of a Sereny-positive reaction by invasive bacteria capable of expressing a plaque-positive phenotype. A functional kcpA gene is required for expression of the plaque-positive phenotype. The presence of an aroD deletion does not interfere with expression of an invasive phenotype; however, in bacteria containing a functional kcpA gene, replication and spread by invading bacteria are limited, preventing development of the plaque-positive phenotype.
Animals↗
A predictive mouse ear-swelling model for investigating topical photoallergy.
The photoallergic potential of various compounds was assessed using a mouse ear-swelling model, which offers the advantage of being quantifiable and more objective than models based on subjective evaluation of erythematous skin reactions. Cyclophosphamide pretreated BALB/c mice were induced by topical treatment of the dorsal skin surface on 3 consecutive days and challenged on the ears 5 days after the last induction. For each induction and challenge treatment, mice were consecutively irradiated with ultraviolet (UV) A (10 J/cm2) and UVB (45 mJ/cm2) radiation 30 min to 1 hr after test material application. The photoallergic response to musk ambrette, a known human photoallergen, was significantly augmented when three consecutive induction exposures were used as compared with one or two inductions. The photoallergic potential of nine other known human photoallergens (tetrachlorosalicylanilide, bithionol, 6-methylcoumarin, chlorpromazine, sodium omadine, bisphenol A, sulphanilamide, fentichlor and p-aminobenzoic acid) was successfully detected using the mouse model. In each experiment, the ear thickness changes observed in the photoallergy test mice were significantly greater than the changes observed in the contact allergy, vehicle/radiation and phototoxicity control mice. Coumarin and homosalate, two agents not traditionally associated with causing photoallergy in humans or animals, did not demonstrate contact photoallergy using this model. With three of the photoallergens, musk ambrette, bithionol and tetrachlorosalicylanilide, the ear swelling response obtained was due to photoallergy alone and not due to the co-existence of both contact photoallergy and contact allergy. In addition, irradiating mice 24 hr, rather than 1 hr, following application of the test material during the induction phase resulted in a significantly reduced photoallergic response with both musk ambrette and tetrachlorosalicylanilide. These results indicate that the mouse ear-swelling model is a potentially useful model for investigative and predictive photoallergy testing.
Administration, Topical↗
Cetaben versus clofibrate: comparison of toxicity and peroxisome proliferation in rats.
Cetaben and clofibrate were each administered to groups of 6 rats (3 male and 3 female) by gavage at dosages of 50 mg/kg per day and 200 mg/kg per day for 2 weeks. Cetaben caused 1 death at 200 mg/kg per day and decreases in body weight gain and food consumption at 50 mg/kg per day or more. There were no effects on body weight or food consumption in the clofibrate-treated groups. At 200 mg/kg per day cetaben and clofibrate induced comparable increases in liver weight and in numbers of liver peroxisomes while cetaben caused increases in liver catalase activity as well.
4-Aminobenzoic Acid↗
Further evaluation of the local lymph node assay in the final phase of an international collaborative trial.
The local lymph node assay (LLNA) is a method used for the prospective identification in mice of chemicals that have the potential to cause skin sensitization. We report here the results of the second and final phase of an international trial in which the performance of the assay has been evaluated using seven test materials in five independent laboratories. The additional chemicals examined here included compounds which are considered less potent allergens than some of those tested in the first phase of the investigation, and includes hexylcinnamic aldehyde (HCA), a chemical recommended by the Organization for Economic Cooperation and Development (OECD) as a positive control for skin sensitization studies. In each laboratory all skin sensitizing chemicals examined (2,4-dinitrochlorobenzene {DNCB}, HCA, oxazolone, isoeugenal and eugenol) elicited positive responses of comparable magnitude as judged by the derived lowest concentration of test chemical required to elicit a 3-fold or greater increase in the proliferative activity of draining lymph node cells compared with vehicle-treated controls. We observed that sodium lauryl sulphate, considered to be a non-sensitizing skin irritant, also induced a positive response in the assay. Para-aminobenzoic acid (pABA), a nonsensitizing chemical, was negative at all test concentrations in each laboratory. Some laboratories incorporated minor modifications into the standard assay procedure, including the evaluation of lymph nodes pooled from individual mice rather than treatment groups and the use of statistical analyses. The use of statistics did not markedly change the determination of the lowest concentration yielding a positive response. These data confirm that the local lymph node assay is robust and yields equivalent results when performed independently.
Animals↗
In vitro metabolism of aniline by sheep intestine.
1. The major metabolite after incubating aniline with sheep intestine was acetanilide. 2. Other metabolites detected in smaller amounts were 2-aminophenol, 4-aminophenol, 2-acetamidophenol and 4-acetamidophenol. 3. The rumen, abomasum duodenum, jejunum, ileum and colon were all able to acetylate aniline. 4. 4-Aminophenol, 4-aminobenzoic acid, 4-anisidine and 4-nitroaniline were also acetylated.
Acetanilides↗
Cloning and characterization of a phosphate-regulated promoter involved in phosphate control of candicidin biosynthesis.
Phosphate strongly repressed the formation of p-aminobenzoic acid (PABA) synthase, an enzyme involved in candicidin biosynthesis. Expression in Streptomyces lividans of the pabS gene (encoding PABA synthase) of Streptomyces griseus is repressed by phosphate at concentrations above 0.1 mM. However, expression of the pabS gene in Escherichia coli is not regulated by phosphate. Phosphate control of the expression of the pabS gene was observed in all plasmids containing the original 4.5-kb BamHI fragment, whereas no phosphate regulation was found when an upstream 1-kb fragment that carries the pabS promoter was deleted. Using the promoter-probe plasmid pIJ424, a '114-bp' promoter was cloned. Expression of the promoterless kanamycin phosphotransferase gene when fused to the '114-bp' promoter was strongly reduced by phosphate (90% at 5 mM concentration). The '114-bp' promoter has been sequenced and the first transcribed nucleotide identified by S1 mapping. The '114-bp' fragment is A + T-rich (54%), as compared to the Streptomyces genome (70-73% GC). The presence of a phosphate control sequence (pcs) in the upstream region of the pabS gene is proposed.
Antifungal Agents↗
Toxicological studies on 4-(hexadecylamino)benzoate (PHB), an agent with anti-atherosclerotic properties, in the rat.
The subacute oral toxicity of sodium 4-(hexadecylamino)benzoate (PHB) was studied in male and female Sprague-Dawley rats. The animals were given PHB 0, 10, 30, 100 or 300 mg/kg body weight as a 3% gum arabic suspension for 13 weeks. During PHB administration all animals on the highest dose level died or were killed because of loss of weight: there were deaths also in the 100 mg/kg group. PHB caused a leukocytopenia which was significant only at the highest dose level. The hemoglobin decreased in the two lowest dose groups. In the highest dose group the histological liver pictures were pathological. Acidophilic bodies and karyorrhexis indicating severe liver cell damage occurred at this level. In the groups receiving 30, 100 and 300 mg/kg there were foci with poorly distinguishable liver cells, mononuclear cells and some neutrophils. PHB had no noticeable toxic effects on the other organs or parameters measured.
4-Aminobenzoic Acid↗
Arylamine N-acetyltransferase activities in cell lines of mouse, rat, hamster and man differing in their sensitivity to 1,6-dinitropyrene.
This study was aimed at monitoring N-acetyltransferase activities of continuous cell lines, which differ in their sensitivity to the toxic effects of nitroaromatic compounds. Transferase activities were measured toward the acetyl acceptors sulfamethazine and p-aminobenzoic acid in partially purified preparation of cytosols. Cell lines such as hamster V79, BHK, rat hepatoma H4IIEC3G- or fibroblast 208F, which are sensitive to 1,6-dinitropyrene (1,6-DNP), possess high transferase activities ranging from 120-270 nmol/min x mg protein. In contrast, human lung cells NCI-H322, mouse and rat hepatoma cells BW1J and H5, respectively, which are resistant to 1,6-DNP contain no or low transferase activity of less than 15 nmol/min x mg. There was no apparent correlation between 1,6-DNP sensitivity and acetyltransferase levels in a few cell lines, e.g. rat hepatoma HTC, 2sFou and 5L, which express intermediate transferase activities ranging from 25-50 nmol/min x mg protein. The results suggest that acetylation is an essential step in activating 1,6-DNP to toxic products in mammalian cells.
Animals↗
High-performance liquid chromatographic determination of primary aromatic amines in urine after derivatization to an azo dye with 2-aminoanthracene.
A sensitive HPLC method for the determination of primary aromatic amines (anilino compounds) is described. Samples were prepared by derivatization of the substrate to an azo dye with 2-aminoanthracene (2-AA). 2-AA was found to react with the diazonium salts prepared from substituted anilines such as 4-halo, -sulfonyl, -carboxyl, -nitro or -acetyl derivatives, but not 4-hydroxy or -alkyl derivatives. In this work, three model compounds [sulfanilamide, 4-aminobenzoyl-beta-alanine and 4-aminobenzoic acid (PABA)] were used to test the linearity and accuracy of the method. Chromatographic separation was carried out using a reversed-phase column (ODS) and ultraviolet detection at 279 nm. Good linearity for the three compounds was found within the range 50-2000 ng/ml. The intra-day coefficient of variation for the three compounds (at 100, 500, 1000 ng/ml) was below 10%. Using this method, the urinary excretion of PABA and its metabolites was studied after oral administration of PABA to rats.
Aniline Compounds↗
Analysis by surface enhanced Raman spectroscopy on silver hydrosols and silver coated filter papers.
Surface enhanced Raman spectrometry (SERS) is an analytical technique with a sensitivity comparable to that of conventional molecular absorption or fluorescence spectroscopy, with the additional major advantage of selectivity inherent in vibrational spectroscopies. The analytical application of flowing silver hydrosols is described. Under the controlled experimental conditions of flow injection analysis, it was possible to detect as low as 30 ng of p-aminobenzoic acid. The linear range was two orders of magnitude (1-100 microg ml(-1)) with a signal reproducibility of 3.2%. Silver coated filter paper is another SERS active substrate that is simple to prepare and handle. The SERS spectra of several nitrogen-containing molecules were obtained on these substrates. The effects of laser power and paper hydration are described. The relative advantages of both substrates are compared.
Journal Article↗
Construction of an auxotrophic Shigella flexneri strain for use as a live vaccine.
A virulent Shigella flexneri serotype Y strain, SFL1, was made auxotrophic for aromatic metabolites, including p-aminobenzoic acid, which is not available in mammalian tissues, by transduction of a Tn10-inactivated aroD gene from Escherichia coli K-12 NK5131. One transductant, SFL114, selected for further studies, had the same biochemical and serological characteristics as the parent strain and the O-antigen patterns of the two strains were identical in SDS-PAGE and Western blot experiments. SFL114 was as invasive for cultured epithelial cells as SFL1, and both strains could escape from the phagocytic vacuole into the cytoplasm of the infected cells. However, the ability of SFL114 to multiply intracellularly was considerably reduced. When applied to the conjunctival sac of guinea pigs, the parent strain gave rise to keratoconjunctivitis, i.e. was Serény-positive, in 13 of 16 animals. By contrast, SFL114 was Serény-negative in all 11 guinea pigs tested. These in vitro and in vivo results suggest that the aromatic-dependent transductant S. flexneri SFL114 is attenuated and possesses properties desirable for a live vaccine.
Animals↗
Evaluation of ultraviolet-A protection by sunscreen agents using a mouse model of contact photoallergy.
This study was conducted to investigate in vivo evaluation of protectiveness by sunscreens in the UVA range using a mouse model of contact photoallergy (CPS) to 3,3',4',5-tetrachlorosalicylanilide (TCSA). Mice were sensitized with TCSA painting plus UVA irradiation (TCSA/UVA) on the abdomen and, 5 days later, challenged with TCSA/UVA on the earlobe. Each of four sunscreen agents, benzophenone-3, Parsol 1789, p-aminobenzoic acid, and 2-ethyl-hexyl-p-methoxycinnamate, was applied to the earlobes before irradiation. Their protective efficacy was evaluated in the degree of inhibition of both ear swelling responses and TCSA-epidermal cell photoadduct formation. Two UVA-absorbing sunscreens, benzophenone-3 and Parsol 1789, dramatically inhibited the ear swelling response, while the two UVB-absorbers exhibited a much less suppressive effect. The UVA-absorbing agents functioned via inhibiting the formation of TCSA-epidermal cell photoadducts. This method is thought to be useful for in vivo estimation of UVA protection provided by sunscreens against the exquisite sensitivity involved in photoallergy.
Animals↗