A high-throughput screening assay for distinguishing nitrile hydratases from nitrilases

A modified colorimetric high-throughput screen based on pH changes combined with an amidase inhibitor capable of distinguishing between nitrilases and nitrile hydratases. This enzymatic screening is based on a binary response and is suitable for the first step of hierarchical screening projects.

Dyskerin, tRNA genes, and condensin tether pericentric chromatin to the spindle axis in mitosis.

Condensin is enriched in the pericentromere of budding yeast chromosomes where it is constrained to the spindle axis in metaphase. Pericentric condensin contributes to chromatin compaction, resistance to microtubule-based spindle forces, and spindle length and variance regulation. Condensin is clustered along the spindle axis in a heterogeneous fashion. We demonstrate that pericentric enrichment of condensin is mediated by interactions with transfer ribonucleic acid (tRNA) genes and their regulatory factors. This recruitment is important for generating axial tension on the pericentromere and coordinating movement between pericentromeres from different chromosomes. The interaction between condensin and tRNA genes in the pericentromere reveals a feature of yeast centromeres that has profound implications for the function and evolution of mitotic segregation mechanisms.

[Dependence of protective effect of alpha-tocopherol on its concentration and time of action on PC12 cell under conditions of oxidative stress].

At the short-term incubation (0.5 and 1.5 h) of cells of the PC12 neuronal line with alpha-tocopherol, its protective effect against the cytotoxic hydrogen peroxide action was increased with rise of its concentration in samples; the protection was practically absent at action of nanomolar antioxidant concentrations, but was well expressed at its micromolar concentrations. These data agree with the concept that alpha-tocopherol increases the cell living activity by reacting directly with free radicals, which leads to formation of the less reactive compounds deprived of non-paired electron. The evidence is obtained that at the long-term action on PC12 cells, alpha-tocopherol not only in micro-, but also in nanomolar concentrations increases statistically significantly the cell living activity under conditions of oxidative stress. As follows from the obtained data, an important role in realization of the alpha-tocopherol protective effect at the long-term incubation with it seems to be played by modulation by this antioxidant of activity of protein kinase activated by extracellular signaling, phosphatidylinosite 3-kinase, and protein kinase C.

Ferrous and ferric ions-based high-throughput screening strategy for nitrile hydratase and amidase.

Rapid and direct screening of nitrile-converting enzymes is of great importance in the development of industrial biocatalytic process for pharmaceuticals and fine chemicals. In this paper, a combination of ferrous and ferric ions was used to establish a novel colorimetric screening method for nitrile hydratase and amidase with α-amino nitriles and α-amino amides as substrates, respectively. Ferrous and ferric ions reacted sequentially with the cyanide dissociated spontaneously from α-amino nitrile solution, forming a characteristic deep blue precipitate. They were also sensitive to weak basicity due to the presence of amino amide, resulting in a yellow precipitate. When amino amide was further hydrolyzed to amino acid, it gave a light yellow solution. Mechanisms of color changes were further proposed. Using this method, two isolates with nitrile hydratase activity towards 2-amino-2,3-dimethyl butyronitrile, one strain capable of hydrating 2-amino-4-(hydroxymethyl phosphiny) butyronitrile and another microbe exhibiting amidase activity against 2-amino-4-methylsulfanyl butyrlamide were obtained from soil samples and culture collections of our laboratory. Versatility of this method enabled it the first direct and inexpensive high-throughput screening system for both nitrile hydratase and amidase.

Protective effect of maternal prenatal melatonin administration on rat pups born to mothers submitted to constant light during gestation

We studied the effects of adverse conditions such as constant light (LL) on the circadian rhythm of malate (MDH, EC 1.1.1.37) and lactate (LDH, EC 1.1.1.27) dehydrogenase activities of the testes of male Wistar rats on postnatal day 28 (PN28), anxiety-like behavior (elevated plus-maze test) at PN60 and sexual behavior at PN120. The rats were assigned to mother groups on day 10 of pregnancy: control (12-h light/dark), LL (light from day 10 to 21 of pregnancy), and LL+Mel (LL and sc injection to the mothers of a daily dose of melatonin, 1 mg/kg body weight at circadian time 12, from day 17 to 21 of pregnancy). LL offspring did not show circadian rhythms of MDH (N = 62) and LDH (N = 63) activities (cosinor and ANOVA-LSD Fisher). They presented a 44.7 percent decrease in open-arm entries and a 67.9 percent decrease in time (plus-maze test, N = 15, P < 0.001, Mann-Whitney U-test and Kruskal-Wallis test), an increase in mounting (94.4 percent), intromission (94.5 percent) and ejaculation (56.6 percent) latencies (N = 12, P < 0.01, Mann-Whitney U-test and Kruskal-Wallis test) and lower numbers of these events (61, 59 and 73 percent, respectively; P < 0.01, N = 12) compared to controls. The offspring of the LL+Mel group presented MDH and LDH circadian rhythms (P < 0.05, N = 50, cosinor and ANOVA-LSD Fisher), anxiety-like and sexual behaviors similar to control. These findings supported the importance of the melatonin signal and provide evidence for the protective effects of hormones on maternal programming during gestation. This protective action of melatonin is probably related to its entrainment capacity, favoring internal coupling of the fetal multioscillatory system.

Comprehensive analysis of elastase-induced pulmonary emphysema in mice: effects of ambient existing particulate matters.

Pulmonary exposure of rodents to porcine pancreatic elastase (PPE) induces lesions that morphologically resemble human panacinar emphysema. However, there has been little work on the comprehensive analysis of this model. The present study was designed to extensively examine the biological effects of PPE on inflammation, cell damage, emphysematous change, and cholinergic reactivity in the lungs of mice. Furthermore, we evaluated the effects of pulmonary exposure to diesel exhaust particles (DEP) on the disease model. Intratracheal administration of PPE induced (1) proinflammatory response in the lungs that was characterized by significant infiltration of leukocytes such as macrophages, eosinophils, and lymphocytes and an increased level of interleukin-1ß in lung homogenates, (2) lung cell damage, indicated by higher levels of total protein, lactate dehydrogenase, and alkaline phosphatase in lung homogenates, (3) emphysema-related morphological changes including airspace enlargement and progressive destruction of alveolar wall structures, and (4) airway responsiveness to methacholine in the context of the compliance value of the respiratory system in a dose-dependent manner showing an overall trend. A single intratracheal administration of DEP did not significantly facilitate the hallmark of the disease. This is the first study to extensively analyze PPE-induced lung emphysema in mice with evaluation of the effects of DEP. Furthermore, this bioassay may be applied to future investigations that evaluate new therapeutic agents or risk factors for pulmonary emphysema.

Comparative study about the effects of pollution on glass and yellow eels (Anguilla anguilla) from the estuaries of Minho, Lima and Douro Rivers (NW Portugal).

The health status of eels (Anguilla anguilla) developing in three estuaries of the NW Portuguese coast with different types and levels of pollution was compared in relation to morphometric parameters, Fulton condition index (F index) and several biomarkers. Relatively to the reference population, glass eels from the Lima estuary had lower weight and length, cholinesterase (ChE) and lactate dehydrogenase (LDH) inhibition, and lower levels of some anti-oxidant parameters, while yellow eels also showed a decreased F index, and increased Na(+)/K(+)-ATPase and lipid peroxidation (LPO) levels. Relatively to the reference population, glass eels from the Douro estuary had increased Na(+)/K(+)-ATPase and glutathione-S-transferase activities and LDH inhibition, while yellow eels also had ChE inhibition and increased LPO. Overall, these results indicate that eels from polluted estuaries showed a poor health status than those from a reference estuary, and that adverse effects become more pronounced after spending several years in polluted estuaries.

RNAi and iTRAQ reagents united: targeted quantitation of siRNA-mediated protein silencing in human cells.

Bridging the gap between functional genomics and traditional molecular cell biology is a challenge of the next decade. Here, we are aiming to find routines for targeted quantitation of protein silencing in response to RNAi based on complex cellular lysates. A workflow was established adapting siRNA treatment, processing the sample, generating isobaric iTRAQ-reagent-labeled peptides, and analyzing the sample applying MRM-based peptide quantitation to verify protein silencing on a 4000 QTRAP LC/MS/MS mass spectrometer. Subsequently, eight targets were analyzed, mostly with two siRNA designs. Although transcript and protein silencing correlated, the downregulation on the protein level was less pronounced. A time-course analysis of the chaperon HSPA9/mortalin indicated a delayed kinetic of protein versus transcript silencing. Further, the analysis of the functional response on the example of HSD17B4, a multifunctional enzyme essential to generate precursors for cholesterol biosynthesis, confirmed that strong silencing on the transcript level accompanied by moderate reduction of protein is sufficient to generate a physiological significant response. Fifty percent protein silencing resulted in a 3.5-fold induction of low-density lipoprotein and therefore cholesterol uptake in human liver cells. The established routines pave the way for the development of targeted protein quantitation assays suitable for target and biomarker validation.

Pores formation on cell membranes by hederacolchiside A1 leads to a rapid release of proteins for cytosolic subproteome analysis.

Hederacolchiside A1 was used to progressively permeabilize the membrane of human melanoma MEL-5 cells. Holes formation was followed by Scanning Electron Microscopy and interaction of the saponin with cholesterol and phospholipids by TOF-SIMS. 2D-LC-MS/MS and 2D-SDS-PAGE show that the release of soluble proteins into serum-free culture media increases with time. This can lead to a new rapid and efficient strategy to analyze the cytosolic subproteome and it opens the door to get information from the cytosolic compartment for clinical proteomic studies.

6-phosphogluconate dehydratase from Zymomonas mobilis: an iron-sulfur-manganese enzyme.

The enzyme 6-phosphogluconate dehydratase has been isolated in a stable form by a simple one-step procedure using dye ligand chromatography. The role of metal ions in the activity and stability of the enzyme was investigated. As with aconitase and several other dehydratase enzymes, the active site includes an Fe4S4 cluster. In addition, the purified enzyme has been shown to contain one manganese ion per subunit, which is also essential for activity. Rapid inactivation by superoxide radical was observed, which could only partly be protected by manganous ions The purified enzyme could be stabilised by alpha-glycerophosphate in place of manganese; glycerophosphate mimics the carbon atoms 4 to 6 of the natural substrate. This suggests that the manganous ion may involved in binding this part of the substrate.

[Screening methods for the diagnosis of lysosomal storage disease].

Lysosomal storage disease is one of the inborn errors of metabolism caused by a deficiency of lysosomal acid hydrolase activity. We describe here the details of screening methods for the diagnosis of this disorder. It is definitely important to perform both enzyme assay of acid hydrolases and the detection of accumulated materials in patient's tissues. Leukocytes (lymphocytes), serum or plasma, and cultured skin fibroblasts are commonly used as the enzyme source for the assay. Although most lysosomal storage diseases can be diagnosed using leukocytes as the enzyme source, enzymatic activities of beta-glucosidase and sialidase in leukocytes are sometimes normal even in patients. At present, the most reliable enzyme source is considered to be cultured skin fibroblasts. Nevertheless, we should remind that we cannot detect a deficiency of galactocerebroside beta-galactosidase activity even using fibroblasts, if we use synthetic substrate. Natural substrates should be employed for the correct diagnosis and for the study of the nature of patient's enzyme. Deficiency of the enzymatic activity in patients should be confirmed by the demonstration of accumulated materials due to the enzyme defect in patient's tissues and urine. The accumulation of mucopolysaccharides and oligosaccharides in urine is obvious in patients with mucopolysaccharidoses and mucolipidoses, respectively. In case of sphingolipidoses, rectal biopsy specimen and blood could be a target of the investigation. In final, the choice of these screening methods should be made solely based on the detailed clinical manifestation of patients.

Nitrilase of Rhodococcus rhodochrous J1. Purification and characterization.

Nitrilase was purified from an extract of isovaleronitrile-induced cells of Rhodococcus rhodochrous J1 in seven steps. In the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous by polyacrylamide electrophoresis, ampholyte electrofocusing and double immunodiffusion in agarose. The enzyme has a molecular mass of about 78 kDa and consists of two subunits identical in molecular mass. The purified enzyme exhibits a pH optimum of 7.6 and a temperature optimum of 45 degrees C. The enzyme catalyzed stoichiometrically the hydrolysis of benzonitrile to benzoic acid and ammonia, and no formation of amide was detected. The enzyme required thiol compounds such as dithiothreitol, L-cysteine or reduced glutathione to exhibit maximum activity. The enzyme was specific for nitrile groups attached to an aromatic or heteroaromatic ring, e.g. benzonitrile, 3-chlorobenzonitrile, 4-tolunitrile, 2-furonitrile and 2-thiophenecarbonitrile. The comparison of the properties of the enzyme with other nitrilases and nitrile hydratases has been also discussed.

beta-Hydroxydecanoylthioester dehydrase: a rapid, convenient, and accurate product distribution assay.

High-performance liquid chromatography on silica gel has been used to separate the products from incubation of substrates with beta-hydroxydecanoylthioester dehydrase (Escherichia coli). Peaks are detected by their absorbances at 230 nm. Following correction for differences in extinction coefficients, comparison of the peak areas reveals the relative amounts of beta-hydroxydecanoate, E-2-decenoate, and Z-3-decenoate thioesters of N-acetylcysteamine.

Low-molecular-weight O-acetylserine sulfhydrylase and serine sulfhydrylase of Saccharomyces cerevisiae are the same protein.

Low-molecular-weight O-acetyl-l-serine sulfhydrylase was purified from a cysteine auxotroph of Saccharomyces cerevisiae and was demonstrated to be identical with l-serine sulfhydrylase.

Immunochemical identity of peroxisomal enoyl-CoA hydratase with the peroxisome-proliferation-associated 80,000 mol wt polypeptide in rat liver.

Peroxisome proliferators, which induce proliferation of hepatic peroxisomes, have been shown previously to cause a marked increase in an 80,000 mol wt polypeptide predominantly in the light mitochondrial and microsomal fractions of liver of rodents. We now present evidence to show that this hepatic peroxisome-proliferation-associated polypeptide, referred to as polypeptide PPA-80, is immunochemically identical with the multifunctional peroxisome protein displaying heat-labile enoyl-CoA hydratase activity. This conclusion is based on the following observations: (a) the purified polypeptide PPA-80 and the heat- labile enoyl-CoA hydratase from livers of rats treated with the peroxisome proliferators Wy-14,643 {[4-chloro-6(2,3-xylidino)-2-pyrimidinylthio]acetic acid} exhibit identical minimum molecular weights of approximately 80,000 on SDS polyacrylamide gel electrophoresis; (b) these two proteins are immunochemically identical on the basis of ouchterlony double diffusion, immunotitration, rocket immunoelectrophoresis, and crossed immunoelectrophoresis analysis; and (c) the immunoprecipitates formed by antibodies to polypeptide PPA-80 when dissociated on a sephadex G-200 column yield enoyl-CoA hydratase activity. Whether the polypeptide PPA-80 exhibits the activity of other enzyme(s) of the peroxisomal beta-oxidation system such as fatty acyl-CoA oxidase activity or displays immunochemical identity with such enzymes remains to be determined. The availability of antibodies to polypeptide PPA-80 and enoyl-CoA hydratase facilitated immunofluorescent and immunocytochemical localization of the polypeptide PPA- 80 and enoyl-CoA hydratase in the rat liver. The indirect immunofluorescent studies with these antibodies provided direct visual evidence for the marked induction of polypeptide PPA-80 and enoyl-CoA hydratase in the livers of rats treated with Wy-14,643. The present studies also provide immunocytochemical evidence for the localization of polypeptide PPA- 80 and the heat-labile enoyl-CoA hydratase in the peroxisome, but not in the mitochondria, of hepatic parenchymal cells. These studies, therefore, provide morphological evidence for the existence of fatty acyl-CoA oxidizing system in peroxisomes. An increase of polypeptide PPA-80 on SDS polyacrylamide gel electrophoretic analysis of the subcellular fractions of liver of rodents treated with lipid-lowering drugs should serve as a reliable and sensitive indicator of enhanced peroxisomal beta- oxidation system.

Gas chromatographic and mass fragmentographic assays of carcinogenic polycyclic hydrocarbon epoxide hydratase activity.

A specific and very sensitive procedure for the determination of epoxide hydratase activity in hepatic microsomes is described. Any polycyclic hydrocarbon epoxide can be used as a substrate; in this study, benzo(a)anthracene-5,6-oxide, benzo(a)pyrene-4,5-oxide and 3-methylcholanthrene-11,12-oxide were utilized. The corresponding trans-diols formed during incubation are separated and evaluated using either an electron-capture gas chromatographic method for the determination of their chloromethyldimethylsilylated derivatives or gas chromatographic-mass fragmentographic measurement of their trimethylsilylated derivatives. Concentrations as low as 1 ng per millilitre of incubation mixture can be estimated.

Chorismate mutase/prephenate dehydratase from Escherichia coli K12. 2. Evidence for identical subunits catalysing the two activities.

On the basis of amino acid composition, tryptic fingerprints and the determination of amino acid sequences around the four cysteine residues, it can be concluded that chorismate mutase/prephenate dehydratase from Escherichia coli K12 consists of identical, or closely similar subunits. It follows from this that the mutase and dehydratase activities of the enzyme are probably catalysed on the one subunit.

Methionine adenosyltransferase, cystathionine beta-synthase and cystathionine gamma-lyase activity of rat liver subcellular particles, human blood cells and mixed white cells from rat bone marrow.

1. Methionine adenosyltransferase (ATP:L-methionine-S-adenosyl transferase, EC 2.5.1.6), cystathionine beta-synthase F1L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] and cystathionine gamma-lyase [L-cystathionine cysteine-lyase (deaminating), EC 4.4.1.1] activities were found only in the cytosol fraction of rat liver cells. None was found in the mitochondrial or endoplasmic reticulum fractions as judged by the distribution of marker enzymes on a density gradient after centrifugation of the cytoplasmic fraction of a liver homogenate, or in a preparation of liver cell nuclei. 2. Polymorphs, lymphocytes (with admixed monocytes) and mixed bone marrow white cells contained no methionine adenosyl transferase, cystathionine beta-synthase or cystathionine gamma-lyase activities. 3. The possible bearing of these results on the problem of abnormal cystine storage in cystinosis is briefly discussed.