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Estimation of biodegradation parameters of phenolic compounds on activated sludge by respirometry.

The bio-oxidation of phenol, catechol, resorcinol, m-cresol and 5-methylresorcinol on activated sludge was investigated by oxygen uptake measurements. In addition, the degradation of acetate with the same microbial population was studied. The substrate-dependent oxygen uptake data were analysed on the basis of the Michaelis-Menten kinetics. The extant kinetic parameters, the maximum rates of oxygen consumption and half-saturation constants for the processes with different substrates were determined. The simple respirometric approach also made it possible to determine the short-term oxygen demands of the substrates which formed 23-38% of the theoretical oxygen demand of the studied compounds.

Bacterial Physiological Phenomena↗

Formation of chloroform by aqueous chlorination of organic compounds.

Thirty organic compounds were selected to investigate their chloroform formation characteristics during chlorination with sodium hypochlorite at pH-values 7.0 and 8.0. These experiments were conducted under conditions similar to those applied on the chlorination of raw water. The results indicated that the chloroform concentrations occurred by the all tested compounds was in the ppm range. The maximum levels of chloroform (11-13 mg/l) were determined during the reaction of resorcinol and phloroglucinol at pH-value 8.0.

Chlorine Compounds↗

Detection of a tumour-associated ganglioside in plasma of patients with neuroblastoma.

An abnormal circulating ganglioside was found in patients with neuroblastoma. This ganglioside appeared as a single band by resorcinol-HCl staining of thin-layer chromatograms of purified total gangliosides isolated from as little as 1 ml of patient plasma. It is a major ganglioside of neuroblastoma tumour tissue and was present (250-1500 pmol lipid-bound sialic acid/ml) in the plasma of five patients with widespread neuroblastoma. In contrast, the ganglioside was not detected (less than 50 pmol/ml) in plasma samples of six patients in complete remission, nor in plasma samples of seventeen healthy children and adults. Measurement of this circulating tumour-associated ganglioside should be clinically useful in neuroblastoma, offering a new approach to the detection of tumour and the evaluation of therapy.

Adolescent↗

Development of non-electrophoretic assay method for DNA ligases and its application to screening of chemical inhibitors of DNA ligase I.

A new rapid assay method for DNA ligases has been developed, which allows direct quantification of enzyme activity without using the traditional polyacrylamide gel electrophoretic technique. In this method, the 5'-biotinylated nicked duplex was used as a substrate for the ligase reaction, in which the 5'-end of the first oligonucleotide (19-mer) on the nicked strand is biotinylated and the second oligonucleotide (20-mer) on the same strand is labeled with radioactive 32P at the 5'-end. After ligation of the biotinylated 19-mer oligonucleotide into the second oligonucleotide with the reaction of DNA ligases, the biotinylated 19-mer oligonucleotide is converted into the radioactive 39-mer oligonucleotide. The ligase reaction products were heat-denatured to release both ligated and unligated biotinylated oligonucleotides. The biotinylated oligonucleotides were then captured on a streptavidin-coated microtiter plate and counted. The results obtained using this method correlated very well with those from the standard assay method using electrophoresis. Using this assay method, we were able to screen a chemical library and identify new DNA ligase inhibitors structurally related to resorcinol, which has growth inhibitory effects on the human breast cancer cell, MCF-7. The method described here is anticipated to be very useful for screening DNA ligase inhibitors from chemical libraries.

Biotinylation↗

A new variant activator involved in the degradation of phenolic compounds from a strain of Pseudomonas putida.

A new variant type of regulatory activator and relevant promoters (designated capR, Pr and Po) involved in the metabolism of phenolic compounds were cloned from Pseudomonas putida KCTC1452 by using PCR. The deduced amino acid sequence of CapR revealed a difference in nine amino acids from the effector binding domain of DmpR. To measure effector specificity, plasmids were constructed in such a way that the expression of luc gene for firefly luciferase or lacZ for beta-galactosidase as a reporter was under the control of capR. When Escherichia coli transformed with the plasmids was exposed to phenol, dramatic increases in the activity of luciferase or beta-galactosidase were observed in a range of 0.01-1 mM. Among various phenolic compounds tested, other effective compounds included catechol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 2-chlorophenol, 4-chlorophenol, 2-nitrophenol, resorcinol, and 2, 5-dimethylphenol. The results indicate that CapR has effector specificity different from other related activators, CatR and DmpR. Waste water and soil potentially containing phenolic compounds were also tested by this system and the results were compared with chemical and GC data. The present results indicate that the biosensor consisting of capR and the promoters may be utilized for the development of a phenolic compounds-specific biosensor in monitoring the environmental pollutant.

Bacterial Proteins↗

Mutagenicity of commercial hair dyes in Salmonella typhimurium TA98.

Commercial permanent hair-dye formulations containing p-phenylenediamine, resorcinol and aminophenols were incubated with hydrogen peroxide and then tested for their ability to induce reverse mutations in Salmonella typhimurium TA98. Approximately half of the formulations (12 out of 25) gave positive results. The activity varied widely in degree and was observed only in the presence of an S-9 microsomal fraction from Aroclor-induced male rats. Five of the 12 positive formulations and one negative dye were administered topically to male rats; with one exception the urines of animals treated with the mutagenic hair dyes gave positive results in the presence of the S-9 mix.

Animals↗

Identification and sequence analysis of the genes encoding a polyketide synthase required for pyoluteorin biosynthesis in Pseudomonas fluorescens Pf-5.

Pyoluteorin is a chlorinated antifungal metabolite of mixed polyketide/amino-acid origin produced by certain strains of Pseudomonas spp., including the soil bacterium Pseudomonas fluorescens Pf-5. Sequence analysis of a gene cluster required for pyoluteorin biosynthesis by Pf-5 (Kraus, J., Loper, J., 1995. Appl. Environ. Microbiol. 61, 849-854) has identified two genes whose deduced peptide sequences exhibit characteristics of both fungal and bacterial Type I polyketide synthases (PKSs). The pyoluteorin PKS does not contain a loading domain that is typically present in bacterial Type I PKSs. Furthermore, this PKS possesses an acyltransferase domain that does not contain the conserved residues surrounding the active-site motif typically found in domains of similar function. Based on the organization of the functional domains within the pyoluteorin PKS, we propose a biosynthetic pathway analogous to non-aromatic polyketide biosynthesis within the Actinomycete bacteria that is responsible for the formation of the resorcinol moiety of pyoluteorin.

Acyl Carrier Protein↗

High-performance liquid chromatographic resolution of p-nitrobenzyloxyamine derivatives of brain gangliosides.

A new quantitative procedure for the high-performance liquid chromatographic (HPLC) resolution of human brain gangliosides employing reversed-phase chromatography is described. To provide a derivative which can be determined by UV absorption techniques, p-nitrobenzyloxyamine was coupled to the gangliosides. Derivatization involves ozonation and cleavage of the ceramide double bond followed by oxime formation to the nascent aldehyde. Individual gangliosides, as they were resolved by HPLC, were collected. These fractions were then identified by thin-layer chromatography (TLC) and by gas chromatography of their monosaccharides. Quantitative results were obtained along with a marked increase in sensitivity over conventional resorcinol-hydrochloric acid quantitation of TLC-resolved gangliosides.

Brain Chemistry↗

New spectrophotometric method for the determination of flutamide in pharmaceutical preparations.

A sensitive and simple spectrophotometric method for the determinations of reduction product of flutamide (FLA) is described. The method is based on the interaction of diazotized flutamide reduction product with N-(1-naphthyl) ethylenediamine dihydrochloride (NEDA) in neutral or resorcinol (RSL) in alkaline medium. Absorbance of the resulting chromophores is measured at 525 or 480 nm, respectively, and is stable for at least 7 days. The two coupling reagents are applied successfully for the determination of FLA in tablets. The common excipients used as additives in pharmaceutical preparations do not interfere with the determination. Results from the analysis of pure FLA and its commercial tablets by the proposed methods agree well with the reported method.

Androgen Antagonists↗

A new sensitive and selective spectrophotometric method for the determination of catechol derivatives and its pharmaceutical preparations.

A sensitive and simple spectrophotometric method for the estimation of certain catechol derivatives like pyrocatechol (PCL), dopamine hydrochloride (DPH), levodopa (LDP), methyl dopa (MDP) and adrenaline (ADH) in either pure form or in its pharmaceutical formulation is described. The method is based on the interaction of diazotised p-nitro aniline (DPNA) with catechol derivatives in presence of molybdate ions in acidic medium. Absorbance of the resulting red complex is measured at 500-510 nm, respectively, and is stable for 2-10 h. The method is highly reproducible and specific for these selected catechol derivatives. The common excipients used as additives in pharmaceuticals and phenol, hydroquinone, resorcinol, pyrogallol and phloroglucinol do not interfere in the proposed method. Analytical data for determination of the pure compound is presented together with the application of the proposed method to the analysis of some pharmaceutical formulations. The results compare favourably with those of official and reported methods.

Catechols↗

Spectrophotometric methods for the rapid determination of menadione and menadione sodium bisulphite and their application in pharmaceutical preparations.

Two simple, rapid and sensitive spectrophotometric determination of menadione and its sodium bisulphite derivative (MSB) have been carried out. The first method involves the reaction of menadione and its sodium bisulphite derivative with 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) is sodium hydroxide medium to give blue coloured product having maximum absorption at 625 nm and the coloured species is stable for more than 1 h. The Beer's law is obeyed in the range 0.4-16 microg ml(-1). The second method proposes the reaction of menadione and its sodium bisulphite derivative with resorcinol in concentrated sulphuric acid medium to give red coloured product having maximum absorption at 520 nm and is stable for 3 h. The Beer's law was obeyed in the range of 1-24 microg ml(-1). Molar absorptivity for the above two methods were found to be 7.6 x 10(3) and 4.5 x 10(3) l mol(-1) cm(-1), respectively. All the measurements were carried out at room temperature. These two methods have been successfully applied for menadione and its sodium bisulphite derivatives in injections and tablets of pharmaceutical formulations. The results compare favourably with official method.

Antifibrinolytic Agents↗

On-line preconcentration and determination of chromium in parenteral solutions by flow injection-flame atomic absorption spectrometry.

An on-line chromium preconcentration and determination system implemented with flame atomic absorption spectrometry (FAAS) associated to flow injection (FI) was studied. For the retention of chromium, 4-(2-Thiazolylazo)-resorcinol (TAR) and Amberlite XAD-16 were used, at pH 5.0. The Cr-TAR complex was removed from the micro-column with ethanol. An enrichment factor of 50 was obtained for the preconcentration of 50 ml of sample solution. The detection limit value for the preconcentration of 50 ml of aqueous solution of Cr was 20 ng l(-1). The precision for ten replicate determinations at the 5 microg l(-1) Cr levels was 2.9% relative standard deviation (RSD), calculated from the peak heights obtained. The calibration graph using the preconcentration system for chromium was linear with a correlation coefficient of 0.9997 at levels near the detection limits up to at least 100 microg l(-1). The method was successfully applied to the determination of chromium in parenteral solution samples.

Buffers↗

Structural aspects of the inhibitory effect of glabridin on LDL oxidation.

The inhibitory effects of glabridin, an isoflavan isolated from licorice (Glycyrrhiza glabra) root, and its derivatives on the oxidation of LDL induced by copper ions or mediated by macrophages were studied, in order to evaluate the contribution of the different parts of the isoflavan molecule to its antioxidant activity. The peak potential (E1/2) of the isoflavan derivatives, their radical scavenging capacity toward 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical and their ability to chelate heavy metals were also analyzed and compared to their inhibitory activity on LDL oxidation. In copper ion-induced LDL oxidation, glabridin (1), 4'-O-methylglabridin (2), hispaglabridin A (3), and hispaglabridin B (4), which have two hydroxyl groups at positions 2' and 4' or one hydroxyl at position 2' on ring B, successfully inhibited the formation of conjugated dienes, thiobarbituric acid reactive substances (TBARS) and lipid peroxides, and inhibited the electrophoretic mobility of LDL under oxidation. Compounds 1-3 exhibited similar activities, whereas compound 4 was less active. In macrophage-mediated LDL oxidation, the TBARS formation was also inhibited by these isoflavans (1-4) at a similar order of activity to that obtained in copper ion-induced LDL oxidation. On the other hand, 2'-O-methylglabridin (5), a synthesized compound, whose hydroxyl at 2'-position is protected and the hydroxyl at 4'-position is free, showed only minor inhibitory activity in both LDL oxidation systems. 2',4'-O-Dimethylglabridin (6), whose hydroxyls at 2'- and 4'-positions are both protected, was inactive. Resorcinol (7), which is identical to the phenolic B ring in glabridin, presented low activity in these oxidation systems. The isoflavene glabrene (8), which contains an additional double bond in the heterocyclic C ring, was the most active compound of the flavonoid derivatives tested in both oxidation systems. The peak potential of compounds 1-5 (300 microM), tested at pH 7.4, was similar (425-530 mV), and that for compound 6 and 8 was 1078 and 80 mV, respectively. Within 30 min of incubation, compounds 1, 2, 3, 4, 8 scavenged 31%, 16%, 74%, 51%, 86%, respectively, of DPPH radical, whereas compounds 5 and 6, which almost did not inhibit LDL oxidation, also failed to scavenge DPPH. None of the isoflavan derivatives nor the isoflavene compound were able to chelate iron, or copper ions. These results suggest that the antioxidant effect of glabridin on LDL oxidation appears to reside mainly in the 2' hydroxyl, and that the hydrophobic moiety of the isoflavan is essential to obtain this effect. It was also shown that the position of the hydroxyl group at B ring significantly affected the inhibitory efficiency of the isoflavan derivatives on LDL oxidation, but did not influence their ability to donate an electron to DPPH or their peak potential values.

Animals↗

Parallel solid-phase synthesis of vitronectin receptor (alphavbeta3) inhibitors.

A combinatorial approach for rapid optimization of a vitronectin receptor (alphavbeta3) inhibitor lead was accomplished by solid-phase synthesis. Orthogonally bis protected 2,3-diaminopropionic acid was used to immobilize the C-terminus of the molecule. Selective deprotection and functionalization of the alpha-amino group followed by acyl resorcinol scaffold attachment and N-terminus diversification was used to explore structure activity relationship (SAR).

Receptors, Vitronectin↗

Synthesis of a tetracyclic, conformationally constrained analogue of delta8-THC.

A tetracyclic, conformationally constrained analogue of delta8-THC (2) has been synthesized in which a two carbon bridge exists between C2 and C2'. Two conceptually related syntheses of 2 are described, both of which employ 5,7-dimethoxy-4-oxo-1,2,3,4-tetrahydronaphthoic acid (11) as starting material. This substrate was converted to 5,7dimethoxy-2-propyl-1,2,3,4-tetrahydronaphthalene (7) and its 4-keto derivative (18). Demethylation of 11 and 18 provided the corresponding resorcinols, which were condensed with trans-p-menthadienol to afford cannabinoid 2, and a keto derivative (20). LiA1H4/A1C1(3) reduction of 20 provided 2. Cannabinoid 2 has relatively low affinity for the cannabinoid brain receptor (Ki = 703+/-98 nM).

Cannabinoids↗

Spectroscopic studies on chromotropic mixed-ligand copper(II) complexes containing o-hydroxy benzoyl derivatives and dinitrogen bases.

Synthesis of chromotropic copper(II) mixed ligand complexes of o-hydroxy benzoyl derivatives (L1) and dinitrogen bases (L2), general formula Cun(L1)(L2)nXn; where n=1 or 2, L1=4,6-diacetyl-resorcinol (H2DACR), o-hydroxy benzaldhyde (HOHBZ) or o-hydroxy acetophenone (HOHAP), L2=N,N,N',N'-tetramethylethylenediamine (Me4en), 1,10-phenanthroline (phen) or bipyridine (bipy) and X=ClO4-, NO3- or Br- have been reported. Spectral, magnetic and molar conductance measurements as well as analytical data of these complexes show either mononuclear structure for OHBZ and OHAP or binuclear structure for DACR complexes. The IR stretching vibration frequencies of Cu-N and Cu-O are linearly correlated with the d-d absorption frequencies. The d-d absorption bands of Me4en-complexes in weak donor solvents suggest square-planar, distorted octahedral and distorted trigonal bipyramid geometries for the perchlorate, nitrate and bromide complexes, respectively. However, an octahedral structure is identified for the complexes in strong donor solvents. Perchlorate complexes show a remarkable color change from violet to green as the donor ability of the axial ligand increases, whereas bromide complexes are mainly affected by the Lewis acidity of the axial ligand. Specific and non-specific interactions of solvent molecules with the complexes are investigated using the unified solvation model. Calculations of the electronic transition probability (f) of the d-d band along with molecular orbital calculations of ligands have been carried out and correlated with the experimental data.

Chromogenic Compounds↗

Determination of phloroglucinol in human plasma by high-performance liquid chromatography-mass spectrometry.

A sensitive and selective liquid chromatographic method coupled with mass spectrometry (LC-MS) was developed for the quantification of phloroglucinol in human plasma. Resorcinol was used as internal standard, with plasma samples extracted using ethyl acetate. A centrifuged upper layer was then evaporated and reconstituted with mobile phase. The reconstituted samples were injected into a C(18) XTerra MS column (2.1 x 100 mm) with 3.5-microm particle size. The analytical column lasted for at least 500 injections. The mobile phase was 15% acetonitrile (pH 3.0), with flow-rate at 200 microl/min. The mass spectrometer was operated in negative ion mode with selective ion monitoring (SIM). Phloroglucinol was detected without severe interferences from plasma matrix when used negative ion mode. Phloroglucinol produced a parent molecule ([M-H](-)) at m/z 125 in negative ion mode. Detection of phloroglucinol in human plasma was accurate and precise, with quantification limit at 5 ng/ml. This method has been successfully applied to a study of phloroglucinol in human specimens.

Adult↗