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Nitroaromatic compounds are essential materials for chemical industry, but they are also potentially toxic environmental pollutants. Therefore, their sensitive detection and degradation are important concerns. The microbial degradation pathways of nitroaromatic compounds have been studied in detail, but their usefulness needs to be evaluated to understand their potential applications in bioremediation. Here, we developed a rapid and relatively sensitive assay system to evaluate the activities and substrate specificities of nitroaromatic dioxygenases involved in the oxidative biodegradation of nitroaromatic compounds. In this system, nitrous acid, which was released from the nitroaromatic compounds by the dioxygenases, was detected and quantified using the Saltzman reagent. Escherichia coli producing the 3-nitrobenzoic acid dioxygenase complex MnbAB from Comamonas sp. JS46 clearly showed the apparent substrate specificity of MnbAB as follows. MnbAB accepted not only 3-nitrobenzoic acid but also several other p- and m-nitrobenzoic acid derivatives as substrates, although it much preferred 3-nitrobenzoic acid to others. Furthermore, the presence of a hydroxy or an amino group at the ortho position of the nitro group decreased the activity of MnbAB. In addition, MnbAB accepted 2-(4-nitrophenyl)acetic acid as a substrate, which has one additional methylene group between the aromatic ring and the carboxy group of 3-nitrobenzoic acid. This is the first report about the detailed substrate specificity of MnbAB. Our system can be used for other nitroaromatic dioxygenases and contribute to their characterization., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

In photosynthetic organisms chlorophyll and heme biosynthesis is tightly regulated at various levels in response to environmental adaptation and plant development. The formation of 5-aminolevulinic acid (ALA) is the key regulatory step and provides adequate amounts of the common precursor molecule for the Mg and Fe branches of tetrapyrrole biosynthesis. Pathway control prevents accumulation of metabolic intermediates and avoids photo-oxidative damage. In angiosperms reduction of protochlorophyllide (Pchlide) to chlorophyllide is catalyzed by the light-dependent NADPH:Pchlide oxidoreductase (POR). Although a correlation between down-regulated ALA synthesis and accumulation of Pchlide in the dark was proposed a long time ago, the time-resolved mutual dependency has never been analyzed. Taking advantage of the high metabolic activity of young barley (Hordeum vulgare L.) seedlings, in planta ALA synthesis could be determined with high time-resolution. ALA formation declined immediately after transition from light to dark and correlated with an immediate accumulation of POR-bound Pchlide within the first 60 min in darkness. The flu homologous barley mutant tigrina d(12) uncouples ALA synthesis from dark-suppression and continued to form ALA in darkness without a significant change in synthesis rate in this time interval. Similarly, inhibition of protoporphyrinogen IX oxidase by acifluorfen resulted in a delayed accumulation of Pchlide during the entire dark period and a weak repression of ALA synthesis in darkness. Moreover, it is demonstrated that dark repression of ALA formation relies rather on rapid post-translational regulation in response to accumulating Pchlide than on changes in nuclear gene expression.

Background: Human ejaculated sperm undergo volume regulation, and swollen cells fail to penetrate mucus. Study of an infertile mouse model indicates maturation of volume regulation mechanism in the epididymis., Methods: Sperm from the ejaculate and three regions of the epididymis of the cynomolgus monkey (Macaca fascicularis) were dispersed in BWW medium and changes in the cell volume and kinematics, and their responses to ion channel blockers, were monitored by flow cytometry and motion analysis., Results: Initially swollen cauda epididymidal spermatozoa regained their original volume within 20 min, but not in the presence of 0.25 mM quinine. Corpus epididymidal spermatozoa underwent such regulatory volume decrease (RVD) to a lesser extent, with a similar response to quinine. Caput sperm showed no swelling throughout incubation. The chloride channel inhibitor NPPB also caused swelling of cauda spermatozoa and both quinine and NPPB decreased the efficiency of forward progression. RVD of ejaculated spermatozoa was inhibited by the K+ channel blockers quinine and 4-aminopyridine (4-AP) but not by tetraethylammonium, Ba2+ or Gd3+, or the specific potassium channel blockers charybdotoxin, margatoxin, dendrotoxin, apamin, glybenclamide or clofilium. Quinine and 4-AP also altered ejaculated sperm kinematics as reported in human ejaculated spermatozoa., Conclusions: Quinine- and 4-AP-sensitive (implying K+) and NPPB-sensitive (implying Cl-) channels are involved in RVD of primate sperm, which develop this volume regulatory ability in the epididymis.

Selective separation of tryptophan-containing peptides has been attempted using 2-nitro-4-carboxyphenylsulfenyl chloride (NCps)-C1 as a reagent for hydrophobic modification of tryptophan. S-Carboxymethylated proteins were modified with NCps-C1 in 70% acetic acid and digested with TPCK-trypsin, and the digests were fractionated, directly or after partial fractionation on a Sephadex G-25 column, by high performance liquid chromatography using a reverse phase column. The tryptophan-containing peptides from the tryptic digests of S-carboxymethylated hen-egg white lysozyme and Trimeresurus flavoviridis phospholipase A2 were thus selectively separated and the amino acid sequences were determined, showing the validity of the method. The phenylthiohydantoin derivative of 2-(2'-nitro-4'-carboxyphenylthio)-L-tryptophan was synthesized and its spectroscopic and chromatographic properties determined, enabling us to identify the derivative on Edman sequencing.

The proteins precipitated with ammonium sulfate from the urine of a patient (Mat) with multiple myeloma were separated into three components by ion-exchange and gel chromatographies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, amino acid analyses, immunochemical tests, and measurement of circular dichroism showed that these components were a dimer with a disulfide bond, a stable monomer, and a variable fragment, respectively. All three protein components reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) in Tris-HCl buffer at pH 8.0, indicating that they contained free sulhydryl groups. Partial reduction with dithiothreitol in the absence of denaturants yielded two SH groups per molecule from both the monomer and the dimer, and one SH group per molecule from the fragment. This indicates that the monomer of Mat protein contains a cysteinyl residue in the variable region in addition to a cysteinyl residue at the COOH terminus. The reactivities of the two SH groups of the partially reduced monomer toward iodoacetamide and iodoacetic acid were studied by polyacrylamide gel electrophoresis. The two SH groups had similar reactivities with iodacetamide, but the SH group at the COOH terminus was more reactive with iodoacetic acid than that in the variable region. The extrinsic Cotton effects of an azobenzene-2-sulfenyl group introduced into the SH group in the variable region were different from those of dye attached to the COOH terminal SH group, indicating that the two SH groups had different environments. The states of the SH groups of the intact monomer are discussed on the basis of these findings.

The reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent) with thiols is sensitive to daylight, in particular to ultraviolet radiation at wavelengths around 325 nm. Exposure to light at the absorbance maximum of the yellow product (the thionitrobenzoate ion) at 410 nm had no effect on the reaction. The light-sensitive species is apparently the DTNB, because a spectral-irradiation experiment showed that the wavelength of light that produced the maximum rate of absorbance change coincided with the peak absorbance of DTNB, and it was well separated from the thionitrobenzoate absorbance peak. Ascorbate is ineffective as a stabilizer and can produce an apparent increase in the rate of DTNB destruction. In a practical example we found the light interference to be severe when hydrolysis of propionylthiocholine by plasma cholinesterase (EC 3.1.1.8) was measured after a 20-min incubation. The apparent cholinesterase activity in clear glass or plastic tubes exposed to diffuse daylight could be decreased to 25% of the value obtained for samples in light-excluded tubes. We recommend the reaction be carried out in artificial room light, with total elimination of daylight, because window glass does not sufficiently attenuate 325-nm wavelength irradiation.

Measurement of creatinine has many applications. We review the determination of urinary creatinine as a valid index of completeness of 24-h urine collection, the clinical utility of the determination of creatinine clearance ratios, and measurement of the ratio of the clearance of specific analytes, such as amylase, to the ratio of clearance of creatinine. The chemistry and variables that affect the Jaffé reaction are reviewed, and attempts at improvement of specificity are discussed. We also review and assess techniques other than the Jaffé reaction for measurement of creatinine.

Very low levels of lipase can easily be measured by a new serum lipase assay method (the BALB-DTNB method), using BAL-tributyrate (BALB) as a substrate, 5,5'-dithiobis(2-nitrobenzoic acid) as a chromogenic SH reagent, phenylmethylsulfonylfluoride as an inhibitor of esterases and sodium dodecyl sulfate as a surfactant. The BALB-DTNB method has a higher sensitivity than the conventional serum lipase assay methods, and proved useful for analyzing the properties of serum lipases in combination with gel-filtration on a Sephacryl S 200 column and isoelectrofocusing in an Ampholine column. Serum samples containing high levels of lipases from patients with pancreatic diseases or patients in whom the pancreatic exocrine gland had been stimulated by injecting caerulein and secretin were analyzed by these methods. The lipolytic profiles obtained indicated the presence of a lipase with an estimated molecular weight of 46,000 and isoelectric points of 7.4, 6.8, or/and 6.4. A lipase with properties similar to those of the serum lipase was found to be present in human pancreatic juice.

We describe the process of certification for a gamma-glutamyltransferase reference material (CRM no. 319). Fifteen laboratories participated to this interlaboratory evaluation. All steps of the measurements were controlled in an effort to locate potential sources of variations. In particular, the exclusion of some data was strictly documented or justified by the non-observance of the IFCC method and (or) discrepancies in instrumentation, reconstitution of the lyophilized samples, or measurement technique. Inaccuracy in the reconstitution of the lyophilized material was +/- 0.68%, and the molar absorptivity of the 5-amino-2-nitrobenzoate reported by each laboratory was within +/- 2% limits of the value reported by the IFCC. Calculated from the sets of accepted results, the total CV among samples was 2.6% and the overall CV was 3.2%. Within-day and between-day CVs were 1.1% and 1.4%, respectively. The greatest variation for a single component was the between-laboratory variability (CV 3.1%); the within-laboratory CV, including the day effect, was 1.8%. Finally, the certified value for the catalytic concentration of this enzyme in the reconstituted lyophilized reference material was 86.8 U/L with an uncertainty of +/- 2.1 U/L (0.95 confidence interval). The uncertainty appeared to be compatible with the end-use of this reference material.

The procedure of Dietz et al. (Clin. Chem. 19: 1309-1313, 1973) for plasma cholinesterase (EC 3.1.1.7) gives a background absorbance of 1.4 A when extended to erythrocyte cholinesterase (EC 3.1.1.8) measurement, because the peak absorbance of the reaction product, 5-thionitrobenzoate, coincides with the hemoglobin Soret band at 410 nm. Consequently, the precision of erythrocyte cholinesterase measurements is poor, and the test is restricted to laboratories with a spectrophotometer having a high signal-to-noise ratio. Use of the detergent benzethonium chloride (Hyamine 1622) instead of quinidine sulfate to stop enzyme action allows readings to be made at 440 nm because the hemoglobin band is shifted to 405 nm and its peak intensity is decreased. Moreover, detergent micelle interactions shift the peak absorbance of the 5-thionitrobenzoate from 410 to 435 nm. Overall, the blank absorbance is decreased to about 0.4 A. This results in an assay that is twice as precise as the previous version and is suited for use in a routine laboratory with a moderate-quality spectrophotometer. Thus erythrocyte cholinesterase measurements can readily be made, to complement plasma cholinesterase in the investigation of exposure to organophosphates.

The inducing effect of certain barbiturates (secobarbitone, thiopentone, pentobarbitone, allobarbitone, phenobarbitone and barbitone) on the levels of the hepatic microsomal drug-metabolizing enzymes has been studied in the rat both in vivo and in vitro. The extent of induction was related to the plasma half-lives of the barbiturates; compounds with low rates of metabolism and long half-lives were the most potent inducing agents. The latter (phenobarbitone, pentobarbitone and allobarbitone) were shown by spectral technique to interact with cytochrome P-450 suggesting that their mechanism of enzyme induction was 'substrate induction' in type. Barbiturates containing an allyl group (secobarbitone and allobarbitone) had a weaker inducing effect than expected, possibly due to their destruction of cytochrome P-450. Despite its short plasma half-life of 0-5 h thiopentone was a relatively potent inducer probably due to its metabolism to pentobarbitone, which has a much longer plasma half-life (1-3 h). Barbitone is an effective inducer of the drug-metabolizing enzymes, yet does not interact spectrally with cytochrome P-450; this is in accord with the observations that although there are increases in NADPH-cytochrome c reductase and cytochrome b5, following administration of barbitone there is no increase in cytochrome P-450. Barbiturate pretreatment does not affect the activities of glucose-6-phosphatase, glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase.

We studied the effect of Ca2+ on the reactivities of two SH groups of DTNB light chain (Cys 128 and Cys 157) using a fluorogenic thiol reagent. It was found that a Ca2+-induced change in reactivity occurred only with Cys 128 when the light chain was in an isolated state, whereas it occurred with both Cys 128 and Cys 157 when the light chain was incorporated in myosin. These results indicate that the Ca2+-induced change in the conformation of DTNB light chain in the isolated state was different from that of the light chain in myosin. It may therefore be difficult to relate the Ca2+-induced conformational change observed in the isolated DTNB light chain to the molecular mechanism of myosin-linked Ca2+ regulation.

Methyl-3,5-dinitrobenzoate and 3,5-dinitrobenzoyl chloride are demonstrated to react with creatinine through their conversion to 3,5-dinitrobenzoate. Analytical recovery of creatinine added to serum is improved by an "acid-supernate" modification, which is also much less susceptible to a combination of known interfering agents than is the picrate procedure. The "neutral-supernate" procedure shows negligible interference by cephalothin as compared to commonly used picrate procedures. Replacement of tetramethylammonium hydroxide by sodium hydroxide was also tested. The interrelationships between dimethyl sulfoxide concentration, recoveries of creatinine, and slopes of correlations (r greater than .992) of various dinitrobenzoate and picrate methods are critically assessed. Because of its precision, greater specificity, and other criteria, we recommend the methyl-3,5-dinitrobenzoate (or 3,5-dinitrobenzoic acid) neutral-supernate method for routine applications.