Search

Lactofen is a chiral herbicide and widely used against broadleaf weeds in agriculture. As a pesticide, it is directly released to the environment, and easily caused contamination in soil and aquatic ecosystem. The enantioselective degradation of lactofen in the environment has been reported, but the molecular biological mechanism of this phenomenon is still unclear. In this study, strain Edaphocola flava HME-24 could degrade 96.7% of 50 mg L -1 lactofen within 72 h. Lactofen was initially hydrolyzed to desethyl lactofen and subsequently acifluorfen by strain HME-24. A novel gene lanE, involved in lactofen transformation, was obtained from Edaphocola flava HME-24. Gene lanE encoded a protein of 471 amino acids that contained the conserved GXSXG esterase motif and clustered into esterase subfamily V. LanE shared the highest identity with esterase EstD (Q9WYH1) from Thermotoga maritima MSB8 (29.14%). This esterase was also able to transform p-nitrophenyl esters (C4-C8), and the activity decreased when the carbon chain length increased. LanE showed enantioselectivity during the degradation of lactofen, diclofop-methyl, and quizalofop-ethyl, with a higher degradation efficiency of (S)-enantiomers than (R)-enantiomers. The three-dimensional structure of LanE was simulated, and molecular docking revealed that when the (S)-enantiomers of lactofen occupied the active sites, the distance between the ligand molecule and the coordination atom was shorter than that when the (R)-enantiomers occupied the active sites, which facilitated the formation of the transition state complex. The results in this study enhanced our understanding of the preferential catabolism of the (S)-enantiomers of lactofen on the molecular level and could illustrate the reported enantioselective degradation of lactofen in the environment., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In the fumarylacetoacetate hydrolase deficient (Fah -/- ) mouse, massive liver repopulation can be easily obtained after transplanted hepatocytes. Understanding the mechanisms of complete liver repopulation in Fah -/- mice will be useful for future clinical application. Here, we found that the endogenous hepatocytes in liver of Fah -/- mice undertook senescence during the time of tyrosinemia symptoms. Increase of senescent hepatocytes in Fah -/- mice provided proliferative advantage to the transplanted hepatocytes. Importantly, senescent hepatocytes upregulated the expression of extracellular matrix enzyme, contributing to degradation of extracellular matrix components and weakness of cell adhesion and connection. The liver exhibiting a loose architecture provided the space for the engraftment and expansion of transplanted hepatocytes. These findings underscore the underlying mechanisms of completed liver repopulation in Fah -/- mice. Senescence followed by loose hepatic parenchyma is a preconditioning for liver repopulation, which would be a promising strategy to achieve therapeutic liver repopulation in clinical settings., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The influence of salinity, pH, temperature, and dissolved organic matter on the photolysis rate of 2,4,6-trinitrotoluene (TNT) in marine, estuary, and laboratory-prepared waters was studied using a Suntest CPS+® solar simulator equipped with optical filters. TNT degradation rates were determined using HPLC analysis, and products were identified using LC/MS. Minimal or no TNT photolysis occurred under a 395-nm long pass filter, but under a 295-nm filter, first-order TNT degradation rate constants and apparent quantum yields increased with increasing salinity in both natural and artificial seawater. TNT rate constants increased slightly with increasing temperature (10 to 32°C) but did not change significantly with pH (6.4 to 8.1). The addition of dissolved organic matter (up to 5mg/L) to ultrapure water, artificial seawater, and natural seawater increased the TNT photolysis rate constant. Products formed by TNT photolysis in natural seawater were determined to be 2,4,6-trinitrobenzaldehyde, 1,3,5-trinitrobenzene, 2,4,6-trinitrobenzoic acid, and 2-amino-4,6-dinitrobenzoic acid., (Published by Elsevier Ltd.)

Purpose: Combining the results of presumptive cord formation in smear and MPT64 Antigen Immunochromatographic Assay has been suggested to reduce the false negative and positive rates for identification of Mycobacterium tuberculosis (MTB) complex in liquid culture. This study was done to evaluate the clinical utility of combining the results of the two tests for rapid identification MTB complex in mycobacterial isolates., Methods: 484 isolates of mycobacteria obtained in MGIT culture were identified using presumptive cord formation in smear and further by MPT64 Antigen ICT assay. Result obtained were analyzed taking PNB inhibition test as the reference standard., Results: Combining the results of the two tests, 464 (95.9%) isolates were correctly identified while discrepant results were obtained in 20 (4.1%) isolates. When the results of the two tests were intersected, the specificity and PPV was 100%, but the sensitivity decreased to 96.4% and the NPV to 68.6%. On the other hand, when the results of the two methods were combined, the sensitivity and NPV was 100%, but the specificity decreased to 88.6% and the PPV to 99.1%., Conclusion: Presumptive cord formation and MPT64 antigen ICT assay can be used in combination for identification of MTB complex. When both the test are positive, the culture can be reported to contain MTB complex. If both the tests are negative, the culture should be reported to contain NTM. Only when discrepant results are obtained by the two tests, further evaluation is necessary to ensure an accurate diagnosis., (Copyright © 2015 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.)

Background: Microscopically observed drug susceptibility (MODS) assay relies on visualisation of characteristic serpentine cord for identification of Mycobacterium tuberculosis (MTB) complex. Since a few Non-tuberculous mycobacteria (NTM) also form cords or pseudocords, additional tests are required to obviate false positive result. Revised MODS platform by including p-nitro benzoic acid (PNB) in one of the wells is suggested to differentiate MTB from NTM., Methods: 464 cultures positive by both MODS assay and reference solid LJ/liquid MGIT were included in the study. MODS protocol was modified by addition of a PNB well. Identification of MTB in reference culture methods was done by standard methods. Results obtained were compared with that of modified MODS assay., Results: Overall concordance for species identification between MODS assay and reference solid LJ/liquid MGIT culture on the basis of visual detection of serpentine cords was 94.8% (95% Confidence interval- 92.3%-96.5%). When results of visual detection of serpentine cords were combined with PNB inhibition test, concordance was 100% (95% CI 98.9%-100%)., Conclusion: Based on results of our study, we recommend modification of MODS assay protocol by adding PNB to one well to differentiate MTB complex from NTM species.

Chloride channels belong to diverse group of anion selective channels involved in different signaling processes. The present study was planned to investigate the involvement of chloride channels in crush injury-induced neuropathic pain in rats by using ivermectin, a ligand gated chloride channel opener and NPPB, a CaCC blocker. The effect of ivermectin (5, 10, 20 mg/kg i.p. or 50, 100 and 200 microg/rat by i.c.v. route) and NPPB (10, 20 and 40 mg/kg i.p.) was investigated on pain behavioural thresholds in crush injury-induced neuropathic pain rat model. Reduction in pain threshold by mechanical, thermal and cold stimuli confirmed the development of neuropathic pain in rats after crush injury. Ivermectin administered either by i.p. or i.c.v. route did not alter the pain threshold in mechanical, thermal and, cold allodynia tests in rats. NPPB (20 and 40 mg/kg i.p.) significantly reduced the pain threshold crush injury neuropathic pain model suggesting its hyperalgesic effect. The results showed that NPPB increased significantly the mechanical and thermal hyperalgesia in crush injury-induced neuropathic pain rat model, whereas ivermectin, either by i.p. or i.c.v. route of administration, has no effect on pain symptoms in this model. NPPB hyperalgesic effect is independent of CaCCs inhibition and may be due to blockade of Ca2+-activated K+ channel.

This work reports the development of a simple, robust, automated sequential injection analysis (SIA) system for the enzymatic determination of total (tGSH) and oxidized (GSSG) glutathione in human whole blood. The reduced (GSH) glutathione concentration is then obtained as the difference between the tGSH and GSSG concentrations. The determination was based on the DTNB-GSSG reductase recycling assay, which couples the specificity of the GSSG reductase (GR) with an amplification of the response to glutathione, followed by spectrophotometric detection of the 2-nitro-5-thiobenzoic acid (TNB) formed (lambda=412 nm). The implementation of this reaction in a SIA flow system with an in-line dilution strategy permitted the necessary distinct application ranges for tGSH and for GSSG. It also guaranteed the exact timing of fluidic manipulations and precise control of the reaction conditions. The influence of parameters such as reagents concentration, temperature, pH, flow rate of the carrier buffer solution, as well as reaction coil length, etc., on the sensitivity and performance of the SIA system were studied and the optimum reaction conditions subsequently selected. Linear calibration plots were obtained for GSH and GSSG concentrations up to 3.00 and 1.50 microM, with detection limits of 0.031 and 0.014 microM, respectively. The developed methodology showed good precision, with a relative standard deviation (R.S.D.)<5.0% (n=10) for determination of both glutathione forms. Statistical evaluation showed good compliance, for a 95% confidence level, between the results obtained with the SIA system and those furnished by the comparison batch procedure.

Since protein kinase C (PKC) serves as a receptor for phorbol ester type tumor promoters and oxidants and has unique redox-active cysteine-rich regions, we have determined whether various chemopreventive selenocompounds could affect this enzyme. At lower concentrations, selenite decreased the kinase activity (IC50 = 0.5 microM), while at higher concentrations it decreased phorbol ester binding. However, when the catalytic and regulatory domains of PKC were separated by proteolysis, the catalytic domain retained its sensitivity to selenite, while the regulatory domain lost its sensitivity. Cysteine residues were quantitated in PKC modified with selenite by using 5,5'-dithiobis(2-nitrobenzoic acid) and also by using 2-nitro-5-thiosulfobenzoic acid after sulfitolysis. At lower concentrations, selenite induced a modification of four cysteine residues resulting in the formation of two disulfides, while at higher concentrations it induced a modification of seven to eight cysteine residues resulting in the formation of three to four disulfides. Contrary to selenite, selenocystine and selenodiglutathione (GSSeSG) readily inactivated the kinase activity, but not the phorbol ester binding. These two agents induced a two-stage modification of PKC; a limited modification at low concentrations leads to a loss of affinity for ATP, while an excessive modification at high concentrations leads to a loss of Vmax. Selenocystine and GSSeSG were 100,000-fold more potent than GSSG in inactivating PKC. The isoenzymes alpha, beta, and gamma exhibited an identical susceptibility to these selenocompounds. These results suggested that the cysteine residues present within the catalytic domain of these isoenzymes, although apart in the sequence, may be clustered in the tertiary structure to react with selenite, as well as may be in close proximity to some of the cysteines in the regulatory domain. Selenite did not affect protein kinase A, whereas GSSeSG and selenocystine inactivated the catalytic subunit after dissociation from the regulatory subunit at concentrations 100- and 800-fold, respectively, higher than that required for PKC inactivation. All three selenocompounds did not affect the activities of phosphorylase kinase and protein phosphatase 2A. Taken together, these results suggest that the accessible redox-active cysteine residues present in the PKC catalytic domain can react with certain specificity with redox-active selenocompounds such as selenite, selenocystine, and GSSeSG relative to other protein kinases tested.

MPL immunostimulant, a 4'-monophosphoryl lipid A (MLA) preparation obtained from the lipopolysaccharide of Salmonella minnesota R595, is being developed for several clinical indications. MPL comprises a mixture of MLA congeners that contain 4, 5, and 6 fatty acids. In this paper, we report a new high-performance liquid chromatography (HPLC) method for analyzing the congener composition and purity of MPL. MPL is first derivatized with dinitrobenzyloxyamine (DNBA), resulting in incorporation of the dinitrobenzyl chromophore at the reducing end of all MLA congeners. DNBA-MPL is then analyzed by reversed-phase HPLC on a Waters NovaPak C18, 4 microns particle size, 300 mm x 3.9 mm column. Optimal separation of DNBA-MLA species is obtained using a linear gradient of 10% to 80% isopropanol-water (95:5, v/v), 5 mM tetrabutylammonium dihydrogenphosphate (TBAP), in acetonitrile-water (95:5, v/v), 5 mM.TBAP, over 45 min. A synthetic compound, corresponding to a hexaacyl MLA congener, is used for determination of the detector response factor, allowing the MLA content of MPL (i.e., purity) to be determined. Overall, this method provides better separation, higher sensitivity, and is faster and saler than previous methods used for the analysis of MPL.

n-Octyl-5-dithio-2-nitrobenzoic acid (ODNB), the reaction product of n-octane-1-thiol and Ellman's reagent, can be isolated in crystalline form. Like Ellman's reagent, ODNB can be used for the titration of SH groups in proteins, but with considerable advantages: Due to the absence of one negative charge and the presence of a lipophilic hydrocarbon chain, titrations of protein thiols are significantly faster. Normally, endpoints are reached after 5-30 min, while the corresponding reaction with Ellman's reagent may take 1-2 h. Another advantage of ODNB is that it finds access to thiol groups which normally remain undetected. With myosin S1, for example, ODNB reacts with a thiol group which is not reactive with Ellman's reagent. Although ODNB has detergent properties, reaction of this thiol group as a consequence of denaturation seems unlikely, because ODNB was used below its critical micelle concentration and all other thiols of myosin S1 remained buried. The n-octylthiol derivatives of proteins formed by reaction with ODNB can be used for transient protection of thiols, for example, during purification procedures, or for a reversible blocking of SH groups involved in biochemical reactions.

A novel method for detecting microorganisms capable of producing cephalosporin C (CPC) acylase and/or 7-(4-carboxybutanamido)cephalosporanic acid (GL-7-ACA) acylase has been developed. The method is based on the degradation of 2-nitro-5-(6-bromohexanoylamino)benzoic acid (NBHAB), a chromogenic substrate, into yellow 2-nitro-5-aminobenzoic acid by the action of the CPC acylase or the GL-7-ACA acylase. This method is very sensitive and quite specific, and has been successfully applied to screen the acylases from a variety of bacteria. A large number of colonies isolated on a plate surface from more than 67 samples and several known bacteria were tested by the NBHAB paper. Five NBHAB-positive strains and isolates were obtained. They were further examined by the reaction of their bacterial cells upon CPC and GL-7-ACA, respectively, and by thin-layer chromatography in order to distinguish the CPC acylase from the GL-7-ACA acylase.

The photobleaching herbicide, acifluorfen-methyl (AFM), has been reported to be an inhibitor of the heme and chlorophyll biosynthetic enzyme protoporphyrinogen oxidase (Protox) in several plant species. However, AFM had no effect on the levels of Protox activity measured in a mitochondrial fraction from soybean roots. In contrast, AFM inhibited Protox activity in etioplasts from barley leaves and in mitochondria from barley roots, but the extent of inhibition varied depending upon the assay conditions and was maximal only in the presence of 5 mM dithiothreitol (DTT). AFM inhibition was enhanced by preincubation of barley organelle extract in the presence of DTT. Preincubation of barley extract with DTT and AFM together (but not with AFM alone) caused extensive enzyme inhibition which was not reversible by dialysis. These findings have implications for the mechanism of AFM action and for the differential effect of these herbicides on crop and weed species. AFM had no effect on the Protox activity of membranes from free-living bacterial cell of Bradyrhizobium japonicum or Escherichia coli, or on the high levels of Protox activity associated with the plant-derived membrane surrounding the symbiotic bacteria within the soybean root nodule.

Replacement of intracellular Cl- by impermeant anions, as well as treatment of insulinoma cells by the Cl- channel blocker, NPPB, leads to activation of ATP-dependent K+ (KATP) channels. Activation of KATP channels by C1- substitution is eliminated (i) when intracellular ATP is replaced by non-hydrolyzable ATP analogs, (ii) when the perfusion medium contains an ATP regenerating system, (iii) when the mitochondrial ATPase is blocked by oligomycin. Dinitrophenol and GDP have the same activating effects on KATP channels as NPPB or intracellular Cl- substitution. Our interpretation of the results is that NPPB and intracellular Cl- replacement produce an uncoupling of oxidative phosphorylation by acting on mitochondrial anion channels, which leads to rapid degradation of ATP and to activation of KATP channels. KATP channels are useful sensors of cytoplasmic ATP variations.

In order to examine effects of oral contraceptives (OC) on the microsomal drug matabolizing enzymes in a state of protein malnutrition, weanling Sprague-Dwayley female rats were fed diets containing 3.5% (low protein, LP), 26% (normal protein, NP) or 42% (high protein, HP) casein for 224 days and norethynodrel plus mestranol for the last 182 days. LP rats were smaller in body weight than NP and HP rats and the latter two groups showed depressed weight gain and hypertrophy of liver and kidney due to OC. Concentrations of microsomal proteins and cytochrome P-450 were lowered by LP diet and OC did not induce cytochrome P-450. Activity of biphenyl-4-hydroxylase was lowered in LP and HP rats compared to NP group in which activity of this enzyme was significantly decreased due to OC. The diets alone had no effect on the activity of p-nitrobenzoate reductase, however, its activity was enhanced by OC only in NP rats. LP diet caused reduction of 4-methylumbelliferone glucuronly transferase activity which was lowered by OC in NP as well as in LP rats. It is concluded that NP diet more than either LP or HP diet exposed rats to the modification of microsomal drug metabolizing enzymes by OC.

Homogeneous lignin peroxidase (diarylpropane oxygenase) oxidized veratryl alcohol to veratryl aldehyde under anaerobic conditions in the presence of either H2O2, m-chloroperoxybenzoic acid (mCPBA), or p-nitroperoxybenzoic acid (pNPBA). Lignin peroxidase also oxidized the 1-(3',4'-diethoxyphenyl)-1,2-dihydroxy-(4"-methoxyphenyl)-propane I under anaerobic conditions in the presence of mCPBA to yield 3,4-diethoxybenzaldehyde III and 1-(4'-methoxyphenyl)-1,2-dihydroxyethane IV. In contrast to what occurs under aerobic conditions, under anaerobic conditions no 2-hydroxy-1-(4'-methoxyphenyl)-1-oxoethane V was obtained. During the diarylpropane I cleavage under anaerobic conditions, 18O from H2(18)O was incorporated into the alpha-position of the phenylglycol IV. Lignin peroxidase also hydroxylated 1-(4'-ethoxy-3'-methoxyphenyl)propane II at the alpha-position to yield 1-(4'-ethoxy-3'-methoxyphenyl)-1-hydroxypropane VI under anaerobic conditions in the presence of mCPBA. During the phenylpropane II hydroxylation under anaerobic conditions, 18O from H2(18)O was incorporated into the alpha-position of VI. These results are rationalized according to a mechanism involving an initial one-electron oxidation of the diarylpropane I by the lignin peroxidase compound I to form a benzene pi cation radical which undergoes alpha, beta cleavage to produce a benzaldehyde and a C6C2 benzylic radical. The latter is then attacked by O2 to form a hydroperoxy radical which may decompose through a tetroxide to form the phenylglycol IV and phenylketol V. Under anaerobic conditions the C6C2 benzylic radical is probably oxidized to a carbonium ion which would be subsequently attacked by H2O to yield the phenylglycol V.

A sensitive quantitative method has been developed to determine the number of disulfide bonds in peptides and proteins. The disulfide bonds of several peptides and proteins were cleaved quantitatively by excess sodium sulfite at pH 9.5 and room temperature. Guanidine thiocyanate (2 M) was added to the protein solutions in order to denature them and thereby make the disulfide bonds accessible. The reaction with sulfite leads to a thiosulfonate and a free sulfhydryl group; the concentration of the latter was determined by reaction with disodium 2-nitro-5-thiosulfobenzoate (NTSB) in the presence of excess sodium sulfite. The synthesis, purification, and characterization of NTSB are described. The assay is rapid, requiring 3-5 min for oligopeptides and 20 min for proteins, and is as sensitive and quantitative as the sulfhydryl group assay employing 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). It can be used for the analysis of as little as 10(8) mol of disulfide bonds, with an error of +/- 3%.

A new method for the determination of serum creatine, based on its reaction with 3,5-dinitrobenzoyl chloride in an organic medium is described. Various analytical parameters are studied and comparisons are made with the routine picrate procedures, as well as the dinitrobenzoic acid methods reported in the literature. On the basis of the data submitted, the proposed method is recommended for routine clinical use.

We developed a method to measure disulfides which is applicable to biological fluids. It consisted of two parts. First, certain thiols and disulfides were separated by HPLC. Second, the eluted materials were submitted to postcolumn reaction with 2-nitro-5-thiosulfobenzoate in the presence of sulfite. The resultant yellow product, 2-nitro-5-thiobenzoate, was measured by its absorbance at 412 nm. We determined the elution characteristics of the thiols and disulfides derived from cysteine, glutathione, alpha-mercaptopropionylglycine (Thiola), and cysteamine. Penicillamine and its disulfide did not react. Cystine in the urine of 22 cystinuric patients, measured by this method, was compared with results obtained by automatic amino acid analysis.

A simple, rapid assay for screening penicillin G acylase-producing bacteria is presented. The method is based on the formation of yellow 2-nitro-5-aminobenzoic acid by penicillin G acylase acting on 2-nitro-5-phenylacetaminobenzoic acid (NIPAB). NIPAB test paper is briefly applied to bacterial colonies on the agar surface, which are subsequently scored individually on the paper by color; bright yellow indicates the presence of penicillin G acylase, natural color its absence. The present method is suitable not only for screening penicillin G acylase-production by a variety of bacteria but also for detection from a large number of transformant colonies of clones containing a gene encoding for the enzyme.

The use of 96-well microtiter plates and a programmable microplate reader to measure glutathione reductase in an assay based on reduction of 5,5'-dithiobis(2-nitrobenzoic acid) by GSH generated from an excess of GSSG is described. Samples are prepared in 96-well plates and absorbance at 415 nm with a reference wavelength of 595 is determined every 30 s for 3 min. The rate of increase in absorbance is directly proportional to the amount of glutathione reductase in the sample. Activity in an unknown sample is determined from a standard curve. The assay is rapid and allows many small samples to be analyzed in replicates of two or more at the same time.

A method for specific labeling of cysteine-containing peptides has been developed using Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Prior to cleavage with proteases or chemical reagents, proteins are reacted with DTNB, resulting in the formation of a mixed disulfide between the protein sulfhydryl group and 2-nitro-5-thiobenzoic acid (TNB). The formation of the mixed disulfide introduces a chromophore, with an absorbance peak at 328 nm. By monitoring peptide maps generated by HPLC at 210 and 328 nm, peptides containing cysteine residues are readily identified. The stability of the derivative was tested using glutathione-TNB as a model compound. Glutathione-TNB is stable to conditions used for CNBr cleavage, as well as those for tryptic cleavage. The TNB label may also increase the hydrophobicity of small peptides, which otherwise might not bind to reverse-phase matrices. This was the case for an oxidatively modified tetrapeptide isolated from Escherichia coli glutamine synthetase.