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.     

para-Aminobenzoic acid as a sole source of carbon and energy for Pseudomonas desmoliticum]

A strain of Psuedomonas desmoliticum has been isolated from soil. It utilizes, as a source of carbon and energy, p-aminobenozic (PABA), p-fluorobenzoic acids, and some other aromatic compounds. The strain has been isolated by inoculating soil suspensions onto Petri plates with a solid mineral medium containing 0.1% PABA as a carbon source. The preparatory metabolism of PABA was studied in this work; p-hydroxybenzoic and protocatechuic acids were found to be its intermediate products. Enzyme systems catalysing oxidation of aromatic compounds and glucose are inducible.     

Production of para-aminobenzoate by genetically engineered Corynebacterium glutamicum and non-biological formation of an N-glucosyl byproduct

para-Aminobenzoate (PABA), a valuable chemical raw material, can be synthesized by most microorganisms. This aromatic compound is currently manufactured from petroleum-derived materials by chemical synthesis. To produce PABA from renewable resources, its production by fermentation was investigated. The evaluation of the sensitivity to PABA toxicity revealed that Corynebacterium glutamicum had better tolerance to PABA than several other microorganisms. To produce PABA from glucose, genetically engineered C. glutamicum was constructed by introducing both pabAB and pabC. The generated strain produced 20 mM of PABA in a test-tube scale culture; however, during the investigation, an unidentified major byproduct was detected in the culture supernatant. Unexpectedly, the byproduct was also detected after the incubation of PABA with glucose in a buffer solution without bacterial cells. To elucidate the mechanism underlying the formation of this byproduct, PABA analogues and several kinds of sugars were mixed and analyzed. New chemical compounds were detected when incubating aniline with glucose as well as PABA with reducing sugars (mannose, xylose, or arabinose), indicating that an amino group of PABA reacted non-enzymatically with an aldehyde group of glucose. The molecular mass of the byproduct determined by LC-MS suggested that the molecule was generated from PABA and glucose with releasing a water molecule, generally known as a glycation product. Because the glycation reaction was reversible, the byproduct was easily converted to PABA by acid treatment (around pH 2-3) with HCl. Then, pab genes were screened to improve PABA production. The highest PABA concentration was achieved by a strain expressing the pabAB of Corynebacterium callunae and a strain expressing the pabC of Xenorhabdus bovienii, respectively. A plasmid harboring both the pabAB of C. callunae and the pabC of X. bovienii, the best gene combination, was introduced into a strain overexpressing the genes of the shikimate pathway. The resultant strain produced 45 mM of PABA in a test-tube scale culture. Under a fermenter-controlled condition, the strain produced up to 314 mM (43 g/L) of PABA at 48 h, with a 20% yield. To our knowledge, this is the highest concentration of PABA produced by a genetically modified microorganism ever reported.     

Protective effect of two sunscreens against lethal and genotoxic effects of UVB in V79 Chinese hamster cells and Saccharomyces cerevisiae strains XV185-14C and D5.

Effects of p-aminobenzoic acid (PABA) and of 4-[(2-oxo-3-bornylidene)methyl]-phenyl trimethylammonium methylsulfate (OMM), two components used in sunscreen formulations, on the mutagenicity of UVB irradiation are compared in three genetic assay systems. A haploid strain of Saccharomyces cerevisiae XV185-14C was used to measure reverse mutations at three loci. The diploid strain D5 of Saccharomyces cerevisiae was used to screen for reciprocal mitotic recombination. The induction of forward mutations was measured in Chinese hamster V79 cells. Our results indicate that UVB irradiation induced HGPRT- mutants in V79 cells, reverse mutations in Saccharomyces cerevisiae strain XV185-14C, and mitotic crossing over and other genetic alterations in Saccharomyces cerevisiae strain D5. V79 Chinese hamster lung cells were the most sensitive to UVB irradiation, followed by Saccharomyces cerevisiae haploid strain XV185-14C and the diploid strain D5. PABA and OMM were both capable of protecting all three types of cells from UVB irradiation regarding both lethality and induction of various types of genetic alterations. At higher concentrations (above 10(-5) M), OMM was more effective in its photoprotective effect toward UVB irradiation than PABA.     

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-β-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.     

Anti-mycobacterial activity of biocides

The effects of different biocides on the growth and viability of Mycobacterium tuberculosis and other mycobacteria were studied. Mycobacterium phlei was the most sensitive of the test strains with a strain of the M. avium intracellulare (MAI) complex the most resistant. Chlorhexidine diacetate, quaternary ammonium compounds, a phenolic and esters of para (4)-hydroxybenzoic acid were inhibitory but not lethal to MAI, whereas 2% glutaraldehyde was bactericidal against all strains.     

Adaptation of Chilomonas paramecium to Isonicotonic Acid Hydrazide (Isoniazid)

SYNOPSIS. Chilomonas paramecium developed significant resistance to INH at concentrations up to 150 mg/100 ml. Once established, the resistant strains grew subnormally in drug-free medium and were more susceptible than the normal strain to sulfanilamide, but were much more resistant to PABA (10-20 mg/100 ml). In addition, INH-adapted strains were less responsive than the controls in PABA reversal of SA-inhibition. Strains adapted to sulfanilamide, sulfapyridine, and nicotinamide were less resistant to INH than the normal strain.     

Spectrofluorimetric analysis of ethopabate in veterinary formulations with application to residue determination in chicken muscles and liver

Ethopabate is a veterinary drug used in the prophylaxis and treatment of coccidiosis in chickens. The presence of drug residues in edible tissues can be dangerous to human consumers. It may cause direct toxic effects, allergic reactions and increased bacterial resistance. A highly sensitive, simple and rapid spectrofluorimetric method was developed for the determination of ethopabate in its veterinary formulations. The proposed method is based on measuring the native fluorescence of ethopabate in water at 364 nm after excitation at 270 nm. The fluorescence–concentration plot was rectilinear over the range of 2–100 ng/mL, with a limit of detection of 2.9 ng/g and a limit of quantification of 9.8 ng/g for ethopabate. The method was successfully applied to the analysis of ethopabate in its commercial veterinary formulations and the results were in good agreement with those obtained with the reference method. The method was extended to the determination of ethopabate residues in chicken muscles and liver, and the results were satisfactory. The recoveries obtained were in the 108.36–113.42% range. No organic solvents are used in the procedure, so it can be considered a type of ‘green’ chemistry. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis and in vitro antibacterial activity of novel 5'-O-analog derivatives of zidovudine as potential prodrugs

An efficient, short synthesis of four potential prodrugs of 3'-azido-3'-deoxythymidine (AZT) and their antibacterial activity are reported. The 5'-OH group of AZT was functionalized with oxalyl chloride obtaining an acyl chloride derivative (AZT-Ox), which by further transformation with leucine, isoleucine and valine amino acids led to the corresponding AZT analogs, namely AZT-Leu, AZT-iLeu and AZT-Val. A carboxyl acid derivative (AZT-Ac) was also obtained by hydrolysis of AZT-Ox. These compounds, which exhibit anti HIV activity, have killed collection and clinical strains of some opportunistic infectious agents in AIDS-related complex. Thus, the clinical strains, K. oxytoca, S. typhi and K. pneumoniae, and collection strain K. pneumoniae ATCC 10031 showed sensitivity to antibiotics. The activity order for the studied compounds against the most sensitive strain (K. pneumoniae ATCC 10031) was AZT-Leu > AZT-iLeu > AZT-Val > AZT-Ac > AZT. On the other hand, the activity order for the second most sensitive strain (K. oxytoca) was AZT-Leu > AZT-Val = AZT-Ac > AZT-iLeu > AZT. The most effective antibacterial drug AZT-Leu, M.I.C.=0.125 microgmL(-1)) was 16 times more active than AZT (AZT, M.I.C.=2 microg mL(-1)) against K.     

Relationship between Sulfaguanidine Resistance and Increased Cellulose Production in Acetobacter xylinum BPR3001E

The mechanism of the increased cell growth and cellulose production of Acetobacter xylinum subsp. sucrofermentans BPR3001E, a sulfaguanidine (SG)-resistant mutant, was investigated. We found that adding p-aminobenzoic acid (PABA) to cultures of the parent strain, BPR2001, led to increased levels of intracellular adenosine-related purine compounds and increased cellulose production. Furthermore, adding ATP increased the cellulose production by permeabilized BPR2001 cells. On the other hand, the intracellular levels of PABA and adenosine-related purine compounds in BPR3001E cells were higher than those in BPR2001 cells. These results suggest that SG resistance increases enhance cellulose production through increased levels of intracellular high-energy compounds caused by increased PABA biosynthesis, reflecting the promoted supply of cellulose precursors.     

The effects of genetic variation in N-acetyltransferases on 4-aminobiphenyl genotoxicity in mouse liver

Inbred, congenic and transgenic strains of mice were characterized for acetylation of p-aminobenzoic (PABA) and the carcinogen 4-aminobiphenyl (4ABP). C57Bl/6 mice have the NAT2*8 allele, A/J mice have NAT2*9 and congenic B6.A mice have NAT2*9 on the C57Bl/6 background. The first transgenic strain with human NAT1, the functional equivalent of murine NAT2, was also tested. The murine NAT2*9 allele correlated with a slow phenotype measured with the murine NAT2 selective substrate PABA. The two strains having this allele also had a lower capacity to acetylate 4ABP. A line with five copies of the human NAT1 transgene was bred for at least five generations with either C57Bl/6 or A/J mice. There was no significant change in PABA NAT activity on the C57Bl/6 background but a 2.5-fold increase was seen in hNAT1:A/J compared with A/J. The effect of variation in NATs on 4ABP genotoxicity was assessed in these strains. Twenty-four hours after exposure to a single oral dose of 120 mg 4ABP/kg, hepatic 4ABP-DNA adducts were detected by immunofluoresence in all strains. Nuclear fluorescence intensities (mean+/-S.D.) were 41.1+/-3.6 for C57Bl/6, 37.9+/-1.11 for A/J and 36.3+/-2.44 for B6.A. There was no correlation between murine NAT2 alleles and 4ABP-DNA adduct levels. Similar results were seen with the transgenic strains. The data indicate that the range of variation present in these strains of mice was insufficient to alter susceptibility to 4ABP genotoxicity. The impact of these relatively modest differences in the acetylation of the activation of 4ABP may be masked by other competing biotransformation reactions since 4ABP is a substrate for both NAT1 and NAT2. Mouse models with variation in both isoforms are needed to adequately assess the role of variation in NATs in susceptibility to 4ABP genotoxicity.     

Chemotaxis of Bdellovibrio bacteriovorus toward pure compounds.

Positive chemotaxis by Bdellovibrio bacteriovorus strain UKi2 was measured for 139 compounds. Twenty-one compounds were attractants; sensitive attraction was elicited by acetate, propionate, thioacetate, malonate, cis-oxalacetate, D-glucose-6-phosphate, acetyl coenzyme A, ammonium ion, barium ion, manganous ion, and potassium ion. Several of the attractants for B. bacteriovorus strain UKi2 also were attractants to strains 6-5-S and 114; however, strains 109D and 109J were not attracted by the compounds tested. Of 33 compounds tested, 8 were repellents for B. bacteriovorus strain UKi2: n-caproate, alanine, isoleucine, leucine, phenylalanine, tyrosine, cobaltous chloride, and hydronium ion. None of the organic repellents for strain UKi2 elicited repulson from strains 114 or 109D. However, all three strains of Bdellovibrio show aerotaxis. Several compounds were tested for their effects on viability and predacious growth of B. bacteriovorus strain UKi2. No simple correlation was found between attraction or repulsion and benefit or harm to bdellovibrios. The data are consistent with the view that in nature, the greatest survival value of chemotaxis for bdellovibros may be in aerotaxis, attraction to certain inorganic ions and acetate, and repulsion by hydronium ion.     

Nutritional Requirements of Methanosarcina sp. Strain TM-1

Methanosarcina sp. strain TM-1, an acetotrophic, thermophilic methanogen isolated from an anaerobic sludge digestor, was originally reported to require an anaerobic sludge supernatant for growth. It was found that the sludge supernatant could be replaced with yeast extract (1 g/liter), 6 mM bicarbonate-30% CO₂, and trace metals, with a doubling time on methanol of 14 h. For growth on either methanol or acetate, yeast extract could be replaced with CaCl₂ · 2H₂O (13.6 μM minimum) and the vitamin p-aminobenzoic acid (PABA, ca. 3 nM minimum), with a doubling time on methanol of 8 to 9 h. Filter-sterilized folic acid at 0.3 μM could not replace PABA. The antimetabolite sulfanilamide (20 mM) inhibited growth of and methanogenesis by Methanosarcina sp. strain TM-1, and this inhibition was reversed by the addition of 0.3 μM PABA. When a defined medium buffered with 20 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid was used, it was shown that Methanosarcina sp. strain TM-1 required 6 mM bicarbonate-30% CO₂ for optimal growth and methanogenesis from methanol. Cells growing on acetate were less dependent on bicarbonate-CO₂. When we used a defined medium in which the only organic compounds present were methanol or acetate, nitrilotriacetic acid (0.2 mM), and PABA, it was possible to limit batch cultures of Methanosarcina sp. strain TM-1 for nitrogen at NH₄⁺ concentrations at or below 2.0 mM, in marked contrast with Methanosarcina barkeri 227, which fixes dinitrogen when grown under NH₄⁺ limitation.     

Role of killer effect in fermentations conducted by mixed cultures of Saccharomyces cerevisiae

Abstract The present work reports on population dynamics in musts (pH 3.2) inoculated with pairs of Saccharomyces cerevisiae wild strains. Two assays determined the growth of both killer and sensitive strains; the latter were not totally eliminated from the must and non-proliferating populations were detected. Another two were carried out with two killer or two sensitive strains, respectively; the exponential growth of the two populations was observed in both cases. The succession of Saccharomyces cerevisiae strains was seen to be common in the four assays; only one strain proved to have the ability to complete fermentation, whereas the other disappeared after 28 days of fermentation. The most important fermentation compounds were estimated at the end of fermentations.     

Biosynthesis and phosphate control of candicidin byStreptomyces acrimycini JI2236: effect of amplification of thepabAB gene

Summary Biosynthesis of candicidin byStreptomyces acrimycini JI2236 was strongly inhibited by phosphate.p-Aminobenzoic acid (PABA) synthase activity, required for the synthesis of PABA, a candicindin precursor, was reduced by 72% in cells grown in medium supplemented with 7.5 mM phosphate. Hybridization studies showed that the DNA region ofS. acrimycini carrying thepabAB gene (encoding PABA synthase) is very similar to the homologous region ofS. griseus 3570.S. acrimycini was easily transformed with plasmids containing thepabAB gene ofS. griseus. Four transformants were studied in detail; three of the transformants synthesized higher levels of PABA synthase and two transformants produced more candicidin than control cultures transformed with pIJ699. The fourth transformant was unable to synthesize the antibiotic. Formation of PABA synthase and candicidin production was equally sensitive to phosphate regulation in transformants with thepabAB than in the untransformedS. acrimycini strain.     

Specificity of the sensitivity of inbred mouse strains TPS, WR and CBA/LacY to the action of chemical mutagens

The cytogenetic effect of thioTEPA, ethyleneimine, mitomycin C, cyclophosphamide and phtorafurum in bone marrow cells of mouse strains TPS, WR, CBA/LacY was studied. Mice of the TPS strains were most sensitive to the action of all mutagens. Mice of the WR strain were sensitive to thioTEPA and phtorafurum but relatively resistant to mutagens which require metabolic preactivation, i. e. mitomycin C and cyclophosphamide. It was suggested to carry out the study of the cytogenetic activity of chemical compounds using the panel of TPS, WR, 101/H, C57BL/6Y, CBA/LacY strains.     

Duration and Reversibility of Sulfonamide-Resistance in Chilomonas Paramecium

Resistance to sulfanilamide has persisted in a strain of Chilomonas Paramecium for 255 transfers (63 months) in a drug-free medium. In attempts to modify resistance, stocks derived from sulfonamide-resistant and normal strains have been acclimatized to and then maintained in media containing p -aminobenzoic acid at 5.0, 10.0, 15.0 and 20.0 mgJ100 ml. Each PABA-acclimatized strain was more susceptible to sulfanilamide than its parent stock. In other words, sulfanil-amide-resistant strains lost their resistance and normal strains became hypersensitive. One strain, adapted first to sulfanilamide, subsequently to PABA (15 mgJ100 ml) and again to sulfanilamide, showed a loss of and finally a restoration of sui-fonamide-resistance (but to a degree somewhat lower than the original level).     

Ca2+-ions and pattern control in Hydra

The fresh water polyp Hydraforms buds which develop a foot at their base and separate from the parent. In the strain H. vulgaris (Zürich), various compounds including phorbolesters, diacylglycerols, cantharidin and Li+-ions were found to prevent foot formation at the bud's base. Therewith, the bud transforms into a branch which persists at the parent. Other strains were found to be unaffected by such treatments. Here we show that a reduced Ca2+-ion concentration of the culture medium causes branch formation in the H. vulgaris (Zürich) strain but not in the other strains tested. However, all strains tested transformed their buds into branches when the medium was enriched with Ba2+ and Sr2+ ions. We suggest that the various treatments either reduce the internal concentration of Ca2+-ions by stimulating Ca2+-ion export or compete with Ca2+-ions at theirtarget. H. vulgaris (Zürich) isthe most sensitive strain tested and appears to have the most efficient Ca2+-pumps. This appears to be necessary forthese animals derived from a lake which is extremely rich in Ca2+ ions.     

The use of rec-bacteria for testing of carcinogenic substances.

A series of rec-Escherichia coli strains were tested for their sensitivity to four known carcinogenic compounds by examination of a zone of inhibited bacterial growth around a central well containing the test chemical. The mutants recA-, recB-, recC-, and recA- recB- recC- were all more sensitive to the mutagens than the parental strain AB1157. The recB- recC- strain was examined with a larger series of compounds and was found to respond to many of the substances in a similar way as the Salmonella typhimurium strains of Ames but with some notable exceptions. Nitrosamines, with rat liver microsomal activation, could be detected at lower levels and a group of aromatic amino compounds failed to react with these rec-E. coli. An unusual feature of these rec-mutants is their sensitivity to mixtures of nitrosamines and 2-acetyl amino-fluorene in the absence of microsomal activation.     

Development of gallic acid formazans as novel enoyl acyl carrier protein reductase inhibitors for the treatment of tuberculosis

The enoyl acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis (MTB) is an attractive target for developing novel antitubercular agents. A series of gallic acid formazans, were computationally designed and docked into the active site of InhA to understand their binding mode and potential to inhibit InhA. Nine compounds from the designed series were identified as potential InhA inhibitors, on the basis of good Glide score. These compounds were synthesized in the laboratory and evaluated for in vitro antitubercular activity against drug-sensitive and multi-drug resistant strains of MTB. Out of nine compounds, three compounds exhibited the most promising MIC of <2 μM against the sensitive strain of MTB, H37Rv. The compounds were evaluated against five resistant strains of MTB. Most of the compounds exhibited activity superior to the standard, linezolid, against all these resistant strains. The mechanism of action of these compounds was concluded to be InhA inhibition, through InhA enzyme inhibition study. Insignificant cytotoxicity of these compounds was observed on RAW 264.7 cell line. Inactivity of all these compounds against gram positive and gram negative bacteria indicated their specificity against MTB. The compounds were further analyzed for ADME properties and showed potential as good oral drug candidates. The results clearly identified some novel, selective and specific InhA inhibitors against sensitive and resistant strains of MTB.