Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases

Five extracellular PHB depolymerases of bacteria isolated from various sources were purified to electrophoretic homogeneity and compared with known extracellular PHB depolymerase fromAlcaligenes faecalis T1. The molecular mass of these enzymes were all around 40–50 kDa. Nonionic detergent, diisopropylfluorophosphate and dithiothreitol inhibited the PHB depolymerase activity of all these enzymes. Trypsin abolished PHB depolymerase activity, but not theD-3-hydroxybutyric acid dimer hydrolase activity of all the enzymes. These results showed that the basic properties of these PHB depolymerases resemble those of theA. faecalis T1 enzyme. Analysis ofN-terminal amino acid sequence of the purified enzymes revealed that these enzymes includingA. faecalis T1 enzyme fall into three groups.     

Relative importance and interactive effects of photosynthesis and food in two solar-powered sea slugs

Sacoglossans use chloroplasts taken from algal food for photosynthesis (kleptoplasty), but the adaptive significance of this phenomenon remains unclear. Two con-generic sacoglossans (Elysia trisinuata and E. atroviridis) were collected in 2009–2011 from Shirahama (33.69°N, 135.34°E) and Mukaishima (34.37°N, 133.22°E), Japan, respectively. They were individually maintained for 16 days under four experimental conditions (combination of light/dark and with/without food), and their survival rate and relative (=final/initial) weights were measured. Both light and food had positive effects on the survival in E. trisinuata, whereas no positive effects of light or food on survival were detected in E. atroviridis. Both light and food had positive effects on relative weights in both species, but light had smaller effects than food. A significant interaction term between light and food was detected in E. trisinuata (but not in E. atroviridis) in that only the presence of both resulted in weight gains. This result suggests that E. trisinuata can obtain sufficient additional energy from photosynthesis for sustaining growth when fresh chloroplasts are continuously supplied from algal food. In addition, fluorescence yield measurements showed that unfed individuals of both E. trisinuata and E. atroviridis lost photosynthetic activity soon (<4 and 4–8 days, respectively). In conclusion, photosynthesis may function to obtain supplementary nutrition for sustaining growth when food is available in sacoglossans with short-term functional kleptoplasty.     

沈阳市降雪中PFOS和PFOA污染现状调查

通过调查降雪中PFOS和PFOA的浓度,阐明了沈阳市大气中PFOS和PFOA的污染状况和污染规律.2006-02-06采集沈阳市区和郊区共计36个采样点的降雪样品,2006-02-25在其中5个采样点再次采集降雪样品.固相萃取融雪水中的PFOS和PFOA,利用LC-MS-SIM法测定样品中PFOS和PFOA浓度.全部样品中均检出PFOS和PFOA.2006-02-06降雪中PFOS和PFOA的浓度几何平均值分别为2.0 ng·L^-1（范围：0.4～46.2 ng·L^-1）和3.6 ng·L^-1（范围：1.6～22.4 ng·L^-1）,95%置信区间分别为1.5～2.8 ng·L^-1和3.1～4.2 ng·L^-1.PFOS和PFOA的最高浓度同时出现在郊区采样点朱尔屯,市中心区2种物质的浓度呈显著正相关.2006-02-25的5个采样点降雪中PFOS和PFOA的浓度几何平均值均为2.2 ng·L^-1.2次降雪中PFOS浓度差异不显著,2006-02-25降雪中PFOA浓度高于2006-02-06.结果表明,沈阳市区和郊区降雪中广泛存在PFOS和PFOA污染,局部区域可能存在共同的PFOS和PFOA污染来源;沈阳地区有较稳定的PFOS来源持续向大气中输送该类物质;PFOS和PFOA的环境行为可能不同.     

Development of a accelerated solvent extraction and gel permeation chromatography analytical method for measuring persistent organohalogen compounds in adipose and organ tissue analysis

A new analytical method has been developed for the quantification of 59 different persistent organohalogen compounds, such as polybrominated diphenyl ethers (PBDEs), polychlorinated naphthalenes (PCNs), polychlorinated biphenyls (PCBs), PCB metabolites, organochlorine pesticides (OCPs) in biological organ tissues. The optimum extraction and cleanup procedures were examined using accelerated solvent extraction (ASE), automated gel permeation chromatography (GPC) on Biobeads S-X3 and automated solid phase extraction (SPE) on silica-gel. The target compounds were divided into two fractions, non-polar compounds and more polar compounds, which in the latter fraction was subsequently methylated using diazomethane. Detection can be achieved by GC/MS in negative chemical ionization (NCI) mode. The average recoveries of the compounds spiked in swine liver, heart, kidney, and cattle adipose tissues were considered satisfactory, and it was confirmed that the method could be used in routine analysis.     

Separation, synthesis and estrogenic activity of 4-nonylphenols: two sets of new diastereomeric isomers in a commercial mixture

Two sets of new diastereomeric 4-nonylphenol (NP) isomers [4-(3,4-dimethylheptan-4-yl)phenol (344NP, NP-J, L) and 4-(3,4-dimethylheptan-3-yl)phenol (343NP, NP-K, P)] were separated from a commercial NP mixture. The mixture of these diastereomers was synthesized at the same time by a single Friedel-Crafts reaction of 3,4-dimethyl-4-heptanol and phenol, and the mixture was separated into individual NPs by HPLC equipped with Hypercarb column. For the first time, in this study the stereostructure-estrogenic activity relationship of NP diastereomers was investigated. The NP isomers (NP-L and NP-P) having the beta-methyl group over the benzene ring were found to be 2-4 times more estrogenic than their diastereomers (NP-J and NP-K). In the case of the other set of diastereomer [4-(3,5-dimethylheptan-3-yl)phenol, (353NP, NP-E, G)] containing gamma-methyl group in the molecule, the gamma-methyl proton signal (delta 0.49) in the more estrogenic isomer (NP-G) also appeared in a higher field than the corresponding methyl signal (delta 0.76) of the less estrogenic isomer (NP-E).     

Improvement of an efficient separation method for chemicals in diesel exhaust particles: analysis for nitrophenols

GOAL, SCOPE, AND BACKGROUND: Diesel exhaust is believed to consist of thousands of organic constituents and is a major cause of urban pollution. We recently reported that a systematic separation procedure involving successive solvent extractions, followed by repeated column chromatography, resulted in the isolation of vasodilatory active nitrophenols. These findings indicated that the estimation of the amount of nitrophenols in the environment is important to evaluate their effect on human health. The isolation procedure, however, involved successive solvent extractions followed by tedious, repeated chromatography, resulting in poor fractionation and in a significant loss of accuracy and reliability. Therefore, it was crucial to develop an alternative, efficient, and reliable analytical method. Here, we describe a facile and efficient acid-base extraction procedure for the analysis of nitrophenols. MATERIALS AND METHODS: Diesel exhaust particles (DEP) were collected from the exhaust of a 4JB1-type engine (ISUZU Automobile Co., Tokyo, Japan). Gas chromatography-mass spectrometry (GC-MS) analysis was performed with a GCMS-QP2010 instrument (Shimadzu, Kyoto, Japan). RESULTS: A solution of DEP in 1-butanol was extracted with aqueous NaOH to afford a nitrophenol-rich oily extract. The resulting oil was methylated with trimethylsilyldiazomethane and subsequently subjected to GC-MS analysis, revealing that 4-nitrophenol, 3-methyl-4-nitrophenol, 2-methyl-4-nitrophenol, and 4-nitro-3-phenylphenol were present in significantly higher concentrations than those reported previously. DISCUSSION: Simple acid-base extraction followed by the direct analysis of the resulting extract by GC-MS gave only broad peaks of nitrophenols with a poor detection limit, while the GC-MS analysis of the sample pretreated with (trimethylsilyl)diazomethane gave satisfactorily clear chromatograms with sharp peaks and with a significantly lowered detection limit (0.5 ng/ml, approximately 100 times). CONCLUSION: The present method involving an acid-base extraction, in situ derivatization, and GC-MS analysis has shown to be a simple, efficient, and reliable method for the isolation and identification of the chemical substances in DEP.     

Research to develop a predicting system of mammalian subacute toxicity (3) construction of a predictive toxicokinetics model

A new predictive toxicokinetics model was developed to estimate subacute toxicity (target organs, severity, etc.) of non-congeneric industrial chemicals, where the chemical structures and physico-chemical properties are only available. Thus, a physiological pharmacokinetics model, which consists of blood, liver, kidney (these were experimentally found as major toxicological targets), muscle and fat compartments , was established to simulate the chemical concentrations in organs/tissues with pharmacokinetic parameters by means of Runge-Kutta-Gill algorithm. The pliarmacokinetic parameters, i.e. absorption rate, absorption ratio, hepatic extraction ratio of metabolism and renal clearance were calculated by using separately established Quantitative Structure-Pharmacokinetics Relationship equations. The developed predictive model was then applied to simulations of 43 non-congeneric industrial chemicals. The chemical concentrations in organs/tissues after single oral administration were simulated, and their maximum concentrations (Cmax's) and area tinder the concentration-time curves (AUC's) were calculated.Fast Inverse Laplace Transform was newly applied for the purpose of simulation of 28-day repeated dose toxicity.Simulated concentrations of 28 days repeated dose were, however, found to be the same as those of simple repetitions of a single administration per day because of the short half-lives of non-congeneric industrial chemicals.A comparison of subacute toxicity data with Cmax's and AUC's in a single dose scenario suggested that the organs/tissues with relatively high concentrations of tested chemical substances were the most sensitive targets within a chemical.Chemical concentrations in liver, for instance, were correlated with the severity of hepatotoxicity among the chemicals. It was also suggested that to improve and widen the present approach, data of metabolite and reactivity of non-congeneric industrial chemicals to organs/tissues, receptors, etc. should be incorporated into the model.     

Identification of 2,6-diisopropylnaphthalene metabolites in carp part 1. In vivo experiment

The metabolism of diisopropylnaphthalene (DIPN) in fish was studied from an ecotoxicological viewpoint to clarify the environmental fate of DIPN, a solvent of carbonless paper which is widely used as a substitute for PCB. This study identified the following metabolites in carp by in vivo experiment; 2-(1-hydroxy-1-methyl) ethyl-6-isopropylnaphthalene, 2-(1-methyl-2-hydroxyethyl)-6-isopropyl-naphthalene, 2,6-(1-hydroxy-1-methyl) ethyl-naphthalene, 2-(1-hydroxy-1-hydroxymethyl) ethyl-6-isopropylnaphthalene and α-[2-(6-isopropylnaphthyl)] propionic acid. Identification was based on UV, IR, MS, and NMR analyses. The metabolism of 2,6-DIPN in carp was concluded to mainly through oxidation of the isopropyl chain.     

Respiratory tract retention of inhaled air pollutants: Report 1 : Mercury absorption by inhaling through the nose and expiring through the mouth at various concentrations

To study atmospheric mercury absorption in human respiratory passage-ways, mercury in expired air was measured in three different states of breathing: steady breathing, deep breathing and breath held after inspiration.In this study, air containing mercury was inhaled through the nose and expired through the mouth. The concentration of mercury in the exhaled air was determined by the technique of gold-amalgam trapping, heat vaporization, and flameless atomic absorption measurement.The subjects were 13 male adults, aged 25–62 years, and 38 cases were observed. Four different concentrations of mercury, 1–3, 4–6, 10–11, and 20–30 μg/m³ were used, and absorption for each was determined. When the concentration was 1–3 μg/m₃, the absorption was 74–92%, the average being 82.5%. At concentrations of 4–6, 10–11, and 20–30 μg/m₃, the absorption was 76.6–100%, 75.5–99.2%, and 79.9–95.9% respectively, and the average was 88.8%, 85.2%, and 87.7% respectively.A slightly higher rate of mercury absorption was observed in deep breathing than in steady breathing, and when expiration was suppressed for some time after inspiration, the rate increased remarkably to 97.4–99.7%. Prolonged retention of inhaled air containing mercury in the respiratory tract is believed to have caused the increased absorption.     

Debromination of hexabromobenzene by its co-grinding with CaO

A hexabromobenzene (HBB, C6Br6) powder was subjected to co-grinding with excess CaO powder in air using a planetary ball mill to investigate mechanochemical debromination of the HBB sample. The reaction proceeds smoothly with an increase in both grinding time and molar ratio of CaO addition. The co-grinding enables us to debrominate HBB, forming CaBr2, C and CaCO3 besides excess CaO in the product. CaBr2 in the product tends to absorb moisture from the atmosphere to form CaBr2 x nH2O. The reaction yield is quantitatively evaluated by measuring the amount of water-soluble bromide through water washing and the amount of unreacted HBB through toluene washing. It is found that almost 100% of debromination is achieved by 6 h grinding with the molar ratio kept constant at 2:1. Radicals are detected in the ground products, possibly due to rupture in the covalent bonds of C-C and C-Br in HBB.