Enhanced degradation of polychlorinated biphenyls by directed evolution of biphenyl dioxygenase

Data characterizing is considered the first and main stage of the statistical analysis. Rather than characterizing each biomechanical signal through one or few global indicators, such as the mean or the root mean square, this paper suggests first to cut the scale into several fuzzy windows and to summarize the data within each window through an occurrence indicator. These indicators become the analysis variables. They can be analyzed through the multiple correspondence analysis, which shows the most discriminant variables, connections between them, empirical situation classes and correspondences between these classes and the most discriminant variables. An example is considered for arguing our point of view; it concerns characterizing and analysis of forces situated at the hand, foot and back level in a load lifting task.     

Purification and characterization of an esterase involved in poly(vinyl alcohol) degradation by Pseudomonas vesicularis PD

An esterase catalyzing the hydrolysis of acetyl ester moieties in poly(vinyl alcohol) was purified 400-fold to electrophoretic homogeneity from the cytoplasmic fraction of Pseudomonas vesicularis PD, which was capable of assimilating poly(vinyl alcohol) as the sole carbon and energy source. The purified enzyme was a homodimeric protein with a molecular mass of 80 kDa and the isoelectric point was 6.8. The pH and temperature optima of the enzyme were 8.0 and 45 degrees C. The enzyme catalyzed the hydrolysis of side chains of poly(vinyl alcohol), short-chain p-nitrophenyl esters, 2-naphthyl acetate, and phenyl acetate, and was slightly active toward aliphatic esters. The enzyme was also active toward the enzymatic degradation products, acetoxy hydroxy fatty acids, of poly(vinyl alcohol). The K(m) and Vmax of poly(vinyl alcohol) (degree of polymerization, 500; saponification degree, 86.5-89.0 mol%) and p-nitrophenyl acetate were 0.381% (10.6 mM as acetyl content in the polymer) and 2.56 microM, and 6.52 and 12.6 mumol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate at a concentration of 5 mM, which indicated that the enzyme was a serine esterase. The pathway for the metabolism of poly(vinyl alcohol) is also discussed.     

Purification and characterization of the esterases involved in aflatoxin biosynthesis in Aspergillus parasiticus

The esterases from the cell-free extracts (CFEs) of Aspergillus parasiticus ATCC15517, an aflatoxin-producing strain, catalyzing the hydrolytic conversion of versiconal hemiacetal acetate (VHA) to versiconal was biochemically studied. The specific activity of the enzymes increased 2.5-fold during incubation of mycelia through 40-55 h. No metal ions were required for enzyme stability, but EDTA at 1 mM and dithiothreitol at 0.5-5 mM increased its stability. Three peaks of VHA esterase activity were resolved when the proteins in the CFEs prepared from the mycelia of different ages were separated by anion-exchange column chromatography, suggesting that at least three VHA esterases were present in the eluate of this purification step. One of these esterases extracted from the mycelia of a 55-h culture was partially purified in five steps by means of preparative chromatography and fast protein liquid chromatography. The partially purified enzyme when reacted with [14C]diisopropylfluorophosphate followed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis gave a single radiolabelled band, which corresponded to a protein of 32 kDa. The molecular mass of the partially purified VHA esterase determined with gel filtration was around 60 kDa. The results suggested that the enzyme consists of two isomeric subunits.     

Purification and characterization of a 39,000-Da serine proteinase from the hemolymph of a solitary ascidian, Halocynthia roretzi

The melanocortin-4 receptor (MC4-R) is a G protein-coupled, seven-transmembrane receptor expressed in the brain. Inactivation of this receptor by gene targeting results in mice that develop a maturity onset obesity syndrome associated with hyperphagia, hyperinsulinemia, and hyperglycemia. This syndrome recapitulates several of the characteristic features of the agouti obesity syndrome, which results from ectopic expression of agouti protein, a pigmentation factor normally expressed in the skin. Our data identify a novel signaling pathway in the mouse for body weight regulation and support a model in which the primary mechanism by which agouti induces obesity is chronic antagonism of the MC4-R.     

Purification and characterization of a membrane-bound hydrogenase from Sporomusa sphaeroides involved in energy-transducing electron transport

Three experiments were conducted to assess the influence of prolactin on lipolysis in rabbits. In vivo, a single injection of 1 mg of ovine prolactin induces increased plasma glycerol and nonesterified fatty acids concentrations within 30 min (P < 0.01). On the contrary, in vitro, oPRL did not stimulate glycerol release in isolated adipocytes at physiological concentrations (under 10(-8) M). In a third experiment, the effect of chronic hyperprolactinemia on the adrenergic control of lipolysis was studied (daily subcutaneous injections of 1 mg ovine prolactin for 12 days). The weight of perirenal adipose tissue at the end of the period of injections was 27% lower in the prolactin-injected (PRL) rabbits than in the control (CTL) rabbits (88 +/- 15 g vs. 120 +/- 25 g; P < 0.05). Food intake during the period of injections was 28% lower in the PRL group than in the CTL group (177 +/- 21 g/d vs. 246 +/- 13 g/d; P < 0.05). Basal glycerol release was 157% higher in adipocytes from PRL rabbits than in those from CTL rabbits (P < 0.05). Stimulation of lipolysis with different adrenergic agonists was similar in both groups. These results suggested an indirect influence of prolactin on adipose tissue lipolysis in rabbits, but mechanisms implicated in this effect remain to be elucidated.     

Purification and characterization of protein D/E, a putative sperm-binding protein involved in fertilization

We describe a method for the efficient purification of a 32 Kd glycoprotein from rat epididymal tissue. The glycoprotein was purified by gel filtration, ion-exchange, affinity, and reverse phase high pressure liquid chromatography. The highly purified glycoprotein was radiolabeled with an iodinatable, cleavable, photoreactive cross-linking agent, 1-[N-(2-hydrox-5-azidobenzoyl)-2-aminoethyl]-4-(-hydroxysuccini mid yl)-succinate (HAHS). The soluble radiolabeled glycoprotein was bound to washed epididymal spermatozoa in a time-dependent, saturable, and reversible manner. Scatchard analysis demonstrates that there are approximately 3,403 binding sites/spermatozoon. The binding efficiency (Kd) for spermatozoa was approximately 2.0 x 10(-10) M. The function of this glycoprotein was verified by using an in vivo artificial insemination fertilization assay. The fertility rate for control spermatozoa was approximately 53%, but the rate for spermatozoa exposed to polyclonal anti-glycoprotein antibodies was only 5%. These data suggest that the binding of the glycoprotein to the surface of rat spermatozoa is mediated by a receptor-type mechanism and is involved in the fertilization process.     

Hydrocarbons and polychlorinated biphenyls from the unsaponifiable fraction of anhydrous milk fat

Using a combination of gas-liquid chromatography and mass spectrometry, the presence of 39 aliphatic hydrocarbons was firmly established in the unsaponifiable fraction of anhydrous milk fat. The hydrocarbons were the C(14) to C(27) and the C(29) to C(31) straight-chain paraffins, their monoolefin analogs, and the C(25) to C(29) branched alkanes. Phytene (3,7,11,15-tetramethyl-n-hexadec-2-ene), identified for the first time in milk fat, was isolated and identified by high-resolution mass spectrometry and infrared analysis. The total hydrocarbon content amounted to 30 ppm of the milk fat. Polychlorinated biphenyls also were detected in trace amounts in the area of the chromatogram between the C(18) and the C(23) hydrocarbons.     

Purification and characterization of endothelin-1 degradation activity from porcine kidney

In order to identify the membrane-bound peptidase that is responsible for the degradation of endothelin (ET), an endothelin-1 (ET-1) degradation enzyme was solubilized from membrane fractions of porcine kidney with 1% Triton X-100, and subsequently purified by column chromatographies, i.e., diethylamino-Sepharose ion exchange, gel permeation, Con A Sepharose and hydroxyapatite chromatography. On DEAE-Toyopearl ion exchange column chromatography, the ET degradation enzyme and aminopeptidase were separated, but ET degradation enkephalinase activities were not separable. In order to separate ET degradation enzyme and enkephalinase, the active fractions were loaded on each of the column chromatographies: sephacryl S-200, Con A Sepharose or hydroxyapatite. The ET degradation activities were co-migrated with enkephalinase activities on all of the three chromatographies. In addition, the ET degradation activities were inhibited by thiorphan, phosphoramidon and EDTA, which are known to inhibit enkephalinase. These results suggest that ET degradation activity in the membrane fractions of the kidney is related to enkephalinase and may be involved in the degradation of ET-1 in vivo.     

贵阳市花溪河水体及沉积物中多氯联苯的污染状况研究

[目的]调查贵阳市花溪河水体及沉积物中的多氯联苯(PCBs)的污染状况.[方法]样品分别于丰水期及桔水期在6个采样点采集,经过处理后,利用GC-ECD检测PCBs的含量.[结果]花溪河水体及沉积物中PCBs含量分别为0.01～0.09、0.82～2.76 ng/g.[结论]花溪河水体及沉积物中均受到一定程度的PCBs污染,与国内外污染区相比,PCBs的含量较低.     

Gas chromatography determination of polychlorinated biphenyls in powdered and liquid soybean milks

OBJECTIVE: To compare the inhibitory effects of propiverine HCl(BUP-4) with those of atropine and oxybutynin on the detrusor instability induced by partial obstruction of the bladder neck of female rats. MATERIALS AND METHODS: Partial obstruction was created using partial ligation of the proximal urethra in 20 female Sprague-Dawley rats. Both the obstructed rats and a control group of 15 rats were evaluated cystometrically about 6 weeks later and the values compared both baseline and after injection with BUP-4, atropine or oxybutynin. During cystometry, the bladder capacity (BC), residual volume (RV), compliance and frequency of spontaneous activity (SA) were determined. RESULTS: The BC, RV and frequency of SA were significantly increased, and compliance markedly decreased, in obstructed compared with normal rats. The micturition pressure was significantly decreased only after injection with BUP-4 in both normal and obstructed rats. For both, the BC was increased significantly after injection with atropine or BUP-4 (P < 0.05), with the increase greater after BUP-4 than after atropine in both groups (P < 0.01). After injecting BUP-4, the RV was significantly increased in both groups (P < 0.05); atropine increased the RV only in normal rats (P < 0.01) and oxybutynin had no significant effect on RV. Increases in compliance after the administration of each drug were significant only in obstructed rats (P < 0.01) and were markedly higher after oxybutynin (780%) than after the other drugs (180-250%). The frequencies and amplitude of SA after injection with each drug were significantly lower only in obstructed rats, but in these rats, there were no significant differences in this reduction after injecting oxybutynin or BUP-4, whereas they were significantly greater after injecting oxybutynin than after atropine. CONCLUSION: Partial bladder outlet obstruction successfully created a hyperactive (unstable) bladder, typified by increased BC, RV, frequency of SA and a marked decrease in compliance. The greater BC, lower MP and frequency and amplitude of SA were prominent after the administration of BUP-4. Thus it is suggested that BUP-4 effectively inhibited bladder instability in rats induced by infravesical outlet obstruction and was more effective than oxybutynin in increasing BC.     

Purification and characterization of a glucuronyltransferase involved in the biosynthesis of the HNK-1 epitope on glycoproteins from rat brain

The glucuronyltransferase involved in the biosynthesis of the HNK-1 epitope on glycoproteins was purified to an apparent homogeneity from the Nonidet P-40 extract of 2-week postnatal rat forebrain by sequential chromatographies on CM-Sepharose CL-6B, UDP-GlcA-Sepharose 4B, asialo-orosomucoid-Sepharose 4B, Matrex gel Blue A, Mono Q, HiTrap chelating, and HiTrap heparin columns. The purified enzyme migrated as a 45-kDa protein upon SDS-polyacrylamide gel electrophoresis under reducing conditions, but eluted as a 90-kDa protein upon Superose gel filtration in the presence of Nonidet P-40, suggesting that the enzyme forms homodimers under non-denatured conditions. The enzyme transferred glucuronic acid to various glycoprotein acceptors bearing terminal N-acetyllactosamine structure such as asialo-orosomucoid, asialo-fetuin, and asialo-neural cell adhesion molecule, whereas little activity was detected to paragloboside, a precursor glycolipid of the HNK-1 epitope on glycolipids. These results suggested that the enzyme is specifically associated with the biosynthesis of the HNK-1 epitope on glycoproteins. Sphingomyelin was specifically required for expression of the enzyme activity. Stearoyl-sphingomyelin (18:0) was the most effective, followed by palmitoyl-sphingomyelin (16:0) and lignoceroyl-sphingomyelin (24:0). Interestingly, activity was demonstrated only for sphingomyelin with a saturated fatty acid, i.e. not for that with an unsaturated fatty acid, regardless of the length of the acyl group.     

Purification and some properties of maleylpyruvate hydrolase and fumarylpyruvate hydrolase from Pseudomonas alcaligenes

Hydrolysis of the gentisate ring-cleavage product, maleylpyruvate (cis-2,4-diketohept-5-enedioic acid), was shown to be catalyzed by an enzyme, maleylpyruvate hydrolase 11, in Pseudomonas alcaligenes (P25X1) after growth with 3-hydroxybenzoate. This activity was separated from fumarylpyruvate hydrolase activity during the course of its purification which accomplished an approximately 50-fold increase in specific activity. An apparent molecular weight of 77,000 was assigned on the basis of Sephadex G-200 chromatography. Despite the presence of up to three similarly migrating bands of protein on polyacrylamide-gel electrophoresis of the purified enzyme, at least two of these bands possessed maleylpyruvate hydrolase activity. Electrophoresis on sodium dodecyl sulfate-polyacrylamide before and after reduction with mercaptoethanol gave a principal band of molecular weight of 33,000 (and a minor band of molecular weight 50,000). A number of substituted maleylpyruvates also served as substrates for maleylpyruvate hydrolase 11, but maleylacetoacetate and fumarylpyruvate were not attacked. Fumarylpyruvate hydrolase was purified approximately 40-fold to give a single band on polyacrylamide gels and with an apparent molecular weight of 73,000 by Sephadex G-200 chromatography. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis before or after reduction with mercaptoethanol, a subunit molecular weight of 25,000 was obtained. Neither maleylpyruvate nor fumarylacetoacetate served as substrates for fumarylpyruvate hydrolase. The activities of both maleyl- and fumarylpyruvate hydrolases were stimulated by Mn(2+) ions. Reasons are discussed for the presence of both enzyme activities, one of which appears to be redundant.     

Purification and characterization of beta 2-tomatinase, an enzyme involved in the degradation of alpha-tomatine and isolation of the gene encoding beta 2-tomatinase from Septoria lycopersici

Lycopersicon species often contain the toxic glycoalkaloid alpha-tomatine, which is proposed to protect these plants from general microbial infection. however, fungal pathogens of tomato often are tolerant to alpha-tomatine and detoxification of alpha-tomatine may be how these pathogens avoid this potential barrier. As an initial step to evaluate this possibility, we have purfied to homogeneity a beta-1,2-D glucosidase from the tomato pathogen Septoria lycopersici that hydrolyzes the beta-1,2-D glucosyl bond on the tetrasaccharide moiety of alpha-tomatine to produce beta2-tomatine. The enzyme is a 110-kDa protein with a pI of 4.5 and a Km for alpha-tomatine of 62 microM. Little or no activity was detected on a variety of other glycosides. The gene encoding this protein was isolated and contains an open reading frame of 803 amino acids that shares sequence homology with several other beta-D-glucosidases. When S. lycopersici was incubated with alpha-tomatine, beta2-tomatinase mRNA accumulated, suggesting that the enzyme is substrate inducible. Aspergillus nidulans expressed ?beta2-tomatinase? activity when transformed with this gene but transformants were still sensitive to alpha-tomatine.     

Purification and characterization of an NADH-rubredoxin oxidoreductase involved in the utilization of oxygen by Desulfovibrio gigas

An NADH--rubredoxin oxidoreductase previously isolated from Desulfovibrio gigas [LeGall, J. (1968) Ann. Inst. Pasteur 114, 109-115] has now been fully purified and further characterized. It contains two subunits of 27 kDa and 32 kDa. With two mid-point redox potentials of -295 mV and -325 mV, this FMN- and FAD-containing protein can induce the specific reduction of D. gigas rubredoxin. In contrast, rubredoxins from the other Desulfovibrio species or desulforedoxin from D. gigas show very low reaction rates with the same enzyme. The phylogenetic significance of the narrow specificity of the enzyme toward the rubredoxin from the same organism is discussed. The purified enzyme has NADH oxidase activity with H2O2 as a final product of O2 reduction. The reaction is half-inhibited by 4.2 microM p-chloromercuribenzoate, whereas cyanide and azide are not significant inhibitors in this reaction. The role of this protein as a part of the enzymic equipment that allows the formation of ATP in the presence of oxygen from the degradation of carbon reserves is discussed.     

The effect of chlorine substitution on the dermal absorption of polychlorinated biphenyls

This study investigated the characteristics of computer-based case simulations (CCS) that may be associated with case difficulty. Difficulty was defined as the average rating by physicians of examinee performance on a nine-point scale or the passing rate on the cases. Two data sets were used, one from an administration of 18 cases, the other from an administration of 22 cases with 13 cases used on both occasions. Stepwise regression procedures were used separately for case properties and for analytic scoring of key variables to identify the best sets of predictors of case difficulty. Because of the small number of cases, regression results were evaluated for consistency across both data and both difficulty measures. For key variables, the best set of predictors included the number of different serious errors of commission, risk items, and benefit items. In general, cases were more difficult for higher values of these variables. For case variables, the only consistent variable was the length of the paragraph that provided patient history, with longer paragraphs associated with more difficult cases. Other variables were less consistent, but were often related to the structure of the simulation or the severity of the patient condition. Although the findings for case variables were limited, the analyses were very helpful in illuminating the interconnections among the variables within cases.     

Liquid-gel partitioning using Lipidex in the determination of polychlorinated biphenyls in cod liver oil

A technique was developed for transfer of fat and polychlorinated biphenyls from cod liver oil into the lipophilic gel Lipidex 5000. Subsequent elution of the gel separated about 60% of the fat from the sample. Following further purification on aluminium oxide and silica gel, toxic non-ortho- and mono-ortho-PCB congeners were isolated in two separate fractions on charcoal. Recoveries were studied by addition of twelve different PCB congeners to 0.2 g of fat. The non-ortho-PCBs were labelled with 13C. The recoveries of 5-50 ng of the unlabelled compounds were 80-100% and those of 50-100 pg of the labelled compounds were 76-106%.     

Levels of polychlorinated biphenyls in some waste motor and transformer oils from Poland

Waste motor and transformer oils are considered as a main source of polychlorinated biphenyls (PCBs) emission into the environment. The levels of total PCB in twenty six randomly selected samples of waste motor and transformer oils from different regions of Poland were studied. The clean-up of the extracts on two solid phase extraction columns system and determination of PCBs by capillary gas chromatography with electron capture detection were performed. The determined content of PCBs in motor oils was by one order of magnitude higher than in transformer oils, but in the both types of analysed samples, except one sample, did not exceed 50 micrograms/g.     

Interactive effects of three structurally different polychlorinated biphenyls in a rat liver tumor promotion bioassay

PURPOSE: Our goal was to validate linear and nonlinear intersubject image registration using an automated method (AIR 3.0) based on voxel intensity. METHOD: PET and MRI data from 22 normal subjects were registered to corresponding averaged PET or MRI brain atlases using several specific linear and nonlinear spatial transformation models with an automated algorithm. Validation was based on anatomically defined landmarks. RESULTS: Automated registration produced results that were superior to a manual nine parameter variant of the Talairach registration method. Increasing the degrees of freedom in the spatial transformation model improved the accuracy of automated intersubject registration. CONCLUSION: Linear or nonlinear automated intersubject registration based on voxel intensities is computationally practical and produces more accurate alignment of homologous landmarks than manual nine parameter Talairach registration. Nonlinear models provide better registration than linear models but are slower.