Degradation of p-toluidine by Pseudomonas testosteroni

Abstract A bacterium, which utilizes p -toluidine as sole source of carbon and energy was isolated from soil. The bacterium was identified as Pseudomonas testosteroni .
From enzymatic studies we propose the following pathway for the degradation of p -toluidine: p -toluidine is oxidatively converted to 4-methyl-catechol, which is then cleaved by a meta -pyrocatechase to 2-hydroxy-5-methyl- cis-cis -muconate semialdehyde.     

Glucose measurement errors in enzymic starch hydrolysates at high enzyme-glucose weight ratios

The 3,5-dinitrosalicylic acid (DNS), o -toluidine, and glucose oxidase methods accurately measured concentrations of standard glucose solutions in the absence of the starch hydrolyzing enzymes Diazyme (amyloglucosidase) and Clarase (α-amylase). In the presence of high enzyme concentrations, particularly at low glucose concentrations, glucose oxidase and o -toluidine somewhat underestimated standard glucose concentrations while DNS overestimated the glucose concentration by 100%. DNS also overestimated glucose in hydrolysates of standard potato starch. Glucose recovery was estimated at almost 200% of that given by glucose oxidase when enzyme starch weight ratios were 9:1 or more. Glucose was underestimated by o -toluidine in starch hydrolysates in the presence of Diazyme at high enzyme-starch weight ratios. DNS similarly overestimated glucose in starch hydrolysates from white spruce ( Picea glauca (Moench.) Voss) and some other species, as enzyme-starch weight ratios increased. The o -toluidine and glucose oxidase reactions were more reliable. Overestimation of the DNS reaction was not improved by treating the glucose-enzyme solutions with anion or cation exchange resins or by removing the enzyme prior to measurement.     

Anaerobic Degradation and Transformation of p-Toluidine by the Sulfate-Reducing Bacterium Desulfobacula toluolica

The ability of the strictly anaerobic sulfate-reducing bacterium Desulfobacula toluolica (strain Tol2) to cometabolically degrade p-toluidine (p-methylaniline) while using toluene as the primary source of carbon and energy has been studied. This organism has been shown to modify and degrade toluidine in dense cell suspensions when no other source of carbon and energy is added. The metabolism led to the formation of a variety of metabolites. From these metabolites a biphenyl-like compound as well as phenylacetic acid have been identified by means of HPLC/MS techniques. The probable conversion of p-toluidine to p-aminophenylacetic acid and phenylacetic acid as dead end products suggested that this organism initiates p-toluidine degradation by the carboxylation of the methyl group. If this could be validated in further experiments, it would be the first time that a toluidine was carboxylated at the methyl moiety by an anaerobic, sulfate-reducing bacterium. Received: 6 March 1998 / Accepted: 3 April 1998     

Conversion ofN-acetyl-o-toluidine into 4′-hydroxy-N-acetyl-o-toluidine byFusarium verticillioides

Summary Approximately 850 fungus and yeast strains were tested for their ability to hydroxylateN-acetyl-o-toluidine in the 4-position. The strain Y-1 selected as the best producer, was identified asFusarium verticilliides, and accumulated 1.5 mg of 4-hydroxy-N-acetyl-o-toluidine per ml of culture broth.     

Induction of mitotic crossing over in Saccharomyces by p-Toluidine.

Summary p-Toluidine, a carcinogen for rats, does not cause genetic damage when tested directly in Saccharomyces cerevisiae; however, certain chemical derivatives of p-toluidine do induce gene conversion when tested directly. It may be suspected by analogy with other aromatic amines that p-toluidine, a monocyclic aromatic amine, requires conversion to breakdown products which are then the genetically active and carcinogenic entities. The Udenfriend hydroxylation medium, which has been used previously to show the genetic activity of certain other aromatic amines and nitrosamines, was used in the incubation of p-toluidine with Saccharomyces cerevisiae. The resulting breakdown products, but not the parent compound, induced reciprocal mitotic recombination in a diploid strain D-3. Recombination was monitored by using induced homozygosity of the red ade2 marker, and the reciprocal nature of the event was confirmed by observing the simultaneous homozygosity of two peripheral markers.     

Anaerobic Metabolism of 1-Amino-2-Naphthol-Based Azo Dyes (Sudan Dyes) by Human Intestinal Microflora

The rates of metabolism of Sudan I and II and Para Red by human intestinal microflora were high compared to those of Sudan III and IV under anaerobic conditions. Metabolites of the dyes were identified as aniline, 2,4-dimethylaniline, o-toluidine, and 4-nitroaniline through high-performance liquid chromatography and liquid chromatography electrospray ionization tandem mass spectrometry analyses. These data indicate that human intestinal bacteria are able to reduce Sudan dyes to form potentially carcinogenic aromatic amines.     

Chlorination of harman and norharman with sodium hypochlorite and co-mutagenicity of the chlorinated products

Harman and norharman are widely distributed in the environment and consequently contaminate in domestic waste-water. It has been reported that they have co-mutagenic activity in the presence of non- mutagenic aromatic amines such as aniline and o-toluidine with S9 mix. When these β-carbolines were treated with sodium hypochiorite under mild conditions, chlorinated derivatives were produced. Among them, 6-chloroharman and 6-chloronorharman showed much more potent co-mutagenic activities than harman and norharman in the presence of o-toluidine toward Salmonella typhimurium TA98 with S9 mix. These results suggest that the chlorination of harman and norharman occurs during disinfection at the sewage plant to produce potent co-mutagens that contaminate river water.     

Nitrite and Nitrous Oxide Accumulation During Denitrification in the Presence of Pesticide Derivatives

Temporary accumulation of nitrite and nitrous oxide was observed in soil incubated under anaerobic conditions when derivatives of the insecticide chlordimeform [(N-4-chloro-o-tolyl)-N′,N′ -dimethylformamidine] were added. Chlordimeform did not affect the denitrification process, but N-formyl-4-chloro-o-toluidine and 4-chloro-o-toluidine caused an inhibition as determined by the accumulation of nitrite and nitrous oxide. A simultaneous application of the insecticide and its derivatives resulted in a stronger inhibitory effect than the application of each compound separately. Aniline intermediates of other pesticides also inhibited denitrification in soil, and they proved to be more effective than their parent compound.     

Conversion of Intrasporal Trehalose into Reducing Sugars During Germination of Nosema algerae (Protista: Microspora) Spores: A Quantitative Study

ABSTRACT. Carbohydrates were extracted from dormant, stimulated and germinated spores of Nosema algerae . Concentrations of total sugars were measured by the Anthrone test. Non-reducing sugars were quantified by NaOH hydrolysis followed by the Anthrone reaction, and reducing sugars by the Nelson's test. Glucose was measured by the o -toluidine test and a glucose oxidase assay. The concentrations of trehalose in the cytoplasm of the dormant, ungerminated spore was estimated to be in excess of 1.0 M. Trehalose decreased by 70% during the five-minute course of germination. All of the lost trehalose was converted to reducing sugar of which 70–78% was glucose. The osmotic potential increase caused by catabolism of trehalose appears to be sufficient for germination.     

Studies on the oxidation products from 2,4-diaminotoluene by hydrogen peroxide and their mutagenicities. II.

2,4-Diaminotoluene (DAT) was reacted with hydrogen peroxide at room temperature for 2 days, and the resulting red precipitates were separated into 5 fractions on silica gel column chromatography. On the gas chromatographic (GC) study, the first fraction (Fr. 1), which is mutagenic (1425 and 1391 revertants/μg in the absence and presence of S9 respectively) in Salmonella typhimurium TA98, contained several peaks. Fr. 1 was further separated into 4 subfractions (Fr. 1-I-Fr. 1-IV) by silica gel column chromatography. The red crystals were separated from Fr. 1-III and the structure of the compound was determined to be 1,8-diamino-2,7-dimethylphenazine from physicochemical and chemical evidence.Further, o-nitro-p-toluidine, p-nitro-o-toluidine, 3,3′-diamino-4,4′-dimethylazobenzene and 3,3′ diamino-4,4′-dimethylazoxybenzene were identified with authentic and synthesized samples by gas chromatography/mass spectrometry. These compounds without nitrotoluidines were mutagenic, and phenazine, azo and azoxy compounds induced 49, 301 and 245 revertants/nmole in Salmonella typhimurium TA98 with 25 μl S9 per plate, respectively.     

Purification,Cloning and Immuno-Biochemical Characterization of a Fungal Aspartic Protease Allergen Rhi o 1 from the Airborne Mold Rhizopus oryzae

Background

Fungal allergy is considered as serious health problem worldwide and is increasing at an alarming rate in the industrialized areas. Rhizopus oyzae is a ubiquitously present airborne pathogenic mold and an important source of inhalant allergens for the atopic population of India. Here, we report the biochemical and immunological features of its 44 kDa sero-reactive aspartic protease allergen, which is given the official designation ‘Rhi o 1’.

Method

The natural Rhi o 1 was purified by sequential column chromatography and its amino acid sequence was determined by mass spectrometry and N-terminal sequencing. Based on its amino acid sequence, the cDNA sequence was identified, cloned and expressed to produce recombinant Rhi o 1. The allergenic activity of rRhi o 1 was assessed by means of its IgE reactivity and histamine release ability. The biochemical property of Rhi o 1 was studied by enzyme assay. IgE-inhibition experiments were performed to identify its cross-reactivity with the German cockroach aspartic protease allergen Bla g 2. For precise characterization of the cross-reactive epitope, we used anti-Bla g 2 monoclonal antibodies for their antigenic specificity towards Rhi o 1. A homology based model of Rhi o 1 was built and mapping of the cross-reactive conformational epitope was done using certain in silico structural studies.

Results

The purified natural nRhi o 1 was identified as an endopeptidase. The full length allergen cDNA was expressed and purified as recombinant rRhi o 1. Purified rRhi o 1 displayed complete allergenicity similar to the native nRhi o 1. It was recognized by the serum IgE of the selected mold allergy patients and efficiently induced histamine release from the sensitized PBMC cells. This allergen was identified as an active aspartic protease functional in low pH. The Rhi o 1 showed cross reactivity with the cockroach allergen Bla g 2, as it can inhibit IgE binding to rBla g 2 up to certain level. The rBla g 2 was also found to cross-stimulate histamine release from the effector cells sensitized with anti-Rhi o 1 serum IgE. This cross-reactivity was found to be mediated by a common mAb4C3 recognizable conformational epitope. Bioinformatic studies revealed high degree of structural resemblances between the 4C3 binding sites of both the allergens.

Conclusion/Significance

The present study reports for the first time anew fungal aspartic protease allergen designated as Rhi o 1, which triggers IgE-mediated sensitization leading to various allergic diseases. Here we have characterized the recombinant Rhi o 1 and its immunological features including cross-reactive epitope information that will facilitate the component-resolved diagnosis of mold allergy.     

DNA adducts of ortho-toluidine in human bladder

Background: 4-Aminobiphenyl (4-ABP) and o-toluidine are known human bladder carcinogens, but only 4-ABP-releasing DNA adducts are known.

Methods: Determination of 4-ABP and o-toluidine-releasing DNA adducts in epithelial and submucosal bladder tissues of sudden death victims (SDV: n?=?46), and bladder tumours (n?=?12) by gas chromatography/mass spectrometry.

Results: Above background, 4 and 11 of 12 tumour samples contained adducts of 4-ABP (0.057?±?0.125?fmol/µg DNA) and o-toluidine (8.72?±?4.49?fmol/µg DNA), respectively. Lower adduct levels were present in both epithelial and submucosal bladder tissues of SDV (4-ABP: 0.011?±?0.022 and 0.019?±?0.047?fmol/µg DNA; o-toluidine: 0.24?±?0.63 and 0.27?±?0.70?fmol/µg DNA).

Conclusion: Detection of o-toluidine-releasing DNA adducts support the carcinogenicity of o-toluidine in the human bladder.     

V-Type H+-ATPase/synthase from a thermophilic eubacterium, Thermus thermophilus. Subunit structure and operon

V-type ATPase (V(o)V(1)) capable of ATP-driven H(+) pumping and of H(+) gradient driven ATP synthesis was isolated from a thermophilic eubacterium, Thermus thermophilus. When the enzyme was analyzed by gel electrophoresis in the presence of sodium dodecyl sulfate, it showed eight polypeptide bands of which four were subunits of V(1). We also isolated the V(o)V(1) operon, containing nine genes in the order of atpG-I-L-E-X-F-A-B-D, which encoded proteins with molecular sizes of 13, 43, 10, 20, 35, 11, 64, 53, and 25 kDa, respectively. The last four genes were identified as those for V(1) subunits; atpA, B, D, and F encoded the A, B, gamma, and delta subunits, respectively. The first five genes, atpG-atpX, were identified as genes for the V(o) subunits. The product of atpL, the proteolipid subunit, lacked a 19-amino acid presequence and, unlike V-type ATPases, contained two membrane-spanning domains rather than four. The hydrophobic 43-kDa product of atpI is the smallest member so far found of the eukaryotic 100-kDa subunit family. Its electrophoretic band overlapped with the band of the A subunit. Therefore, all the gene products were found in our purified V(o)V(1). We isolated the A(3)B(3) subcomplex reconstituted from the isolated subunits and the A(3)B(3)gamma subcomplex from subunit-expressing Escherichia coli. Electron microscopic observation of these subcomplexes revealed that the gamma subunit of V(1) filled the central cavity of A(3)B(3) and might be central subunit, similar to the gamma subunit of F(1)-ATPase.     

The role of renal aromatic N-deacetylase in selective toxicity of avicide 3-chloro-p-toluidine in birds

1.

1. Several species of birds were studied to determine the amount of N-deacetylase activity present in the kidneys. This information was desired to learn if there was any apparent relation between the presence of the enzyme and the selective nephrotoxicity of the avicide 3-chloro-p-toluidine.     

Application of p-Toluidine in Chromogenic Detection of Catechol and Protocatechuate, Diphenolic Intermediates in Catabolism of Aromatic Compounds

In the presence of p-toluidine and iron, protocatechuate and catechols yield color. Inclusion of p-toluidine in media facilitates the screening of microbial strains for alterations affecting aromatic catabolism. Such strains include mutants affected in the expression of oxygenases and Escherichia coli colonies carrying cloned or subcloned aromatic catabolic genes which encode enzymes giving rise to protocatechuate or catechol. The diphenolic detection system can also be applied to the creation of vectors relying on insertion of cloned DNA into one of the latter marker genes.     

Phosphoinositide 3-kinases and membrane traffic

Phosphoinositide 3-kinases (PI 3-kinases) and their 3-phosphoinositide products were identified initially as components of intracellular signalling pathways emanating from cell surface receptors. A new role for 3-phosphoinositides in the constitutive movement o f proteins from one intracellular compartment to another was proposed with the discovery of homology between the product of a yeast gene important for vacuolar sorting, Vps34p, and a mammalian PI 3-kinase. Recent studies have implicated PI 3-kinase as an essential component in membrane traffic at specific steps o f the trans-Golgi-network-endosomal pre-lysosomal system. Evidence largely emerging from the insulin-stimulated glucose transport system suggests that PI 3-kinase may also mediate the effects o f growth factors on membrane traffic events. These studies suggest a possible link between growth-factor-stimulated and constitutive membrane traffic in the endosomal system.     

Synthesis of 3-deoxy-4-O-β-D-galactopyranosyl-D-erythro-hexos-2-ulose and of some of its derivatives from lactose

3-Deoxy-4-O-β-D-galactopyranosyl-D-erythro-hexos-2-ulose (1) was obtained from lactose by reaction with benzoylhydrazine in the presence of a slightly acidic solution of p-toluidine, followed by hydrazinolysis of the product, 3-deoxy-4-O-β-D-galactopyranosyl-D-erythro-hexos-2-ulose bis(benzoylhydrazone) (3), with benzaldehyde. A variety of derivatives of 1 and 3 was prepared. Lactose aroylhydrazones were also prepared. Quantitative determination of the oxidant during the periodate oxidation of 3 was studied. Periodate oxidation of monosaccharide arylhydrazones gave glyoxal mono(arylhydrazones) which afforded the corresponding, mixed bis(hydrazones).     

Discovery of novel 2-aminopyridine-3-carboxamides as c-Met kinase inhibitors

A series of 2-aminopyridine-3-carboxamide derivatives against c-Met were designed and synthesized by employing bioisosteric replacement of heterocyclic moieties with the amide bond. The structure-activity relationship (SAR) at various positions of the scaffold was explored. In this study, a promising compound (S)-24o with a c-Met IC(50) of 0.022μM was identified. The compound exhibited dose-dependent inhibition of the phosphorylation of c-Met as well as downstream signaling in EBC-1 cells. Furthermore, the interactive binding model of (S)-24o with c-Met was elucidated by virtue of a molecular modeling study.     

Regulation of AE2-mediated Cl- transport by intracellular or by extracellular pH requires highly conserved amino acid residues of the AE2 NH2-terminal cytoplasmic domain

We reported recently that regulation by intracellular pH (pH(i)) of the murine Cl-/HCO(3)(-) exchanger AE2 requires amino acid residues 310-347 of the polypeptide's NH(2)-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. 36Cl- efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pH(o)) with unclamped or clamped pH(i), or during variation of pH(i) at constant pH(o). Wild-type AE2-mediated 36Cl- efflux was profoundly inhibited by acid pH(o), with a value of pH(o50) = 6.87 +/- 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)6]bloc substitutions between aa 312-347 identified the greatest acid shift in pH(o(50)) value, approximately 0.8 pH units in the mutant (A)6 342-347, but only a modest acid-shift in the mutant (A)6 336-341. Two of the six (A)6 mutants retained normal pH(i) sensitivity of 36Cl- efflux, whereas the (A)6 mutants 318-323, 336-341, and 342-347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336-347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pH(o) and to pH(i) were found independently and in concert. The E346A mutation acid-shifted the pH(o(0) value to the same extent whether pH(i) was unclamped or held constant during variation of pH(o). Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pH(o) as well as pH(i).     

Microbial transformation of nitroaromatic compounds in sewage effluent

The transformation of mono- and dinitroaromatic compounds was measured in sewage effluent maintained under aerobic or anaerobic conditions. Most of the nitrobenzene, 3- and 4-nitrobenzoic acids, and 3- and 4-nitrotoluenes and much of the 1,2- and 1,3-dinitrobenzenes disappeared both in the presence and absence of oxygen. Under anaerobiosis, 2,6-dinitrotoluene and 3,5-dinitrobenzoic acid disappeared slowly, but no loss was evident in 28 days in aerated sewage. Aromatic amines did not accumulate during the aerobic decomposition of the mononitro compounds. They did appear in nonsterile, but not in sterile, sewage incubated aerobically with the dinitro compounds and anaerobically with all the chemicals. Analysis by gas chromatography and combined gas chromatography-mass spectrometry showed that aniline was formed from nitrobenzene, toluidine was formed from 3- and 4-nitrotoluenes, and aminobenzoic acid was formed from 3- and 4-nitrobenzoic acids under anaerobiosis, and that nitroaniline was formed from 1,2- and 1,3-dinitrobenzenes, aminonitrotoluene resulted from 2,6-dinitrotoluene, and aminonitrobenzoic acid was a product of 3,5-dinitrobenzoic acid under both conditions. The isomeric forms of the metabolites were not established. Aniline, 4-toluidine, and 4-aminobenzoic acid added to sewage disappeared from aerated nonsterile, but not from sterile, sewage or sewage in the absence of oxygen. 2-Nitroaniline, 2-amino-3-nitrotoluene, and 2-amino-5-nitrobenzoic acid added to sewage persisted for at least 60 days in aerobic or anaerobic conditions. Gas chromatographic and gas chromatographic-mass spectrometric analyses demonstrated that acetanilide and 2-methylquinoline were formed from aniline, 4-methylformanilide and 4-methylacetanilide were formed from 4-toluidine, 2-methylbenzimidazole was a product of 2-nitroaniline, and unidentified benzimidazoles were formed from 2-amino-3-nitrotoluene in the absence of oxygen, and that 2-nitroacetanilide and 2-methyl-6-nitroacetanilide were formed from 2-nitroaniline and 2-amino-3-nitrotoluene, respectively, in the presence or absence of oxygen. It is suggested that the transformations of widely used nitroaromatic compounds should be further studied because of the persistence and possible toxicity of products of their metabolism.