Optimal selection of learning data for highly accurate QSAR prediction of chemical biodegradability: a machine learning-based approach.

Prior to the manufacture of new chemicals, regulations mandate a thorough review of the chemicals under risk management. This review involves evaluating their effects on the environment and human health. To assess these effects, a review report that conforms to the OECD Test Guidelines must be submitted to the regulatory body. One of the essential components of the report is an assessment of the biodegradability of chemicals in the environment. In addition to conventional methods, quantitative structure-activity relationship (QSAR) models have been developed to predict the properties of chemicals based on their structural features. Although a greater number of chemicals in the learning set may enhance the prediction accuracy, it may also lead to a decrease in accuracy due to the mixing of different structural features and properties of the chemicals. To improve the prediction performance, it is recommended to use only the appropriate data for biodegradability prediction as a training set. In this study, we propose a novel approach for the optimal selection of training set that enables a highly accurate prediction of the biodegradability of chemicals by QSAR. Our findings indicate that the proposed method effectively reduces the root mean squared error and improves the prediction accuracy.

Three-dimensional structures of free form and two substrate complexes of an extradiol ring-cleavage type dioxygenase, the BphC enzyme from Pseudomonas sp. strain KKS102.

The crystal structure of an enzyme having polychlorinated-biphenyl degrading activity, the BphC enzyme from Pseudomonas sp. strain KKS102, has been solved as a free form at 1.8 A resolution. This is the first three-dimensional structure among the extradiol-type dioxygenases. Based on 34,387 reflections (10.0 to 1.8 A, completeness 87.8%), a current R-factor of 20.4% (with a free R-factor of 24.3%) was obtained with a model obeying standard geometry within 0.011 A in bond lengths and 1.91 degrees in bond angles. The BphC enzyme is a homo-octamer and each subunit is composed of two domains: Domain 1 (N-terminal part) and Domain 2 (C-terminal part). Each domain contains two repetitions of a novel folding motif (the "beta alpha beta beta beta" motif) each consisting of ca 55 amino acid residues. A single Fe ion in the active site coordinates the side-chains of three amino acid residues (His145, His209 and Glu260) and two solvent molecules. The coordination geometry is that of a square pyramid. In addition to the free form of the BphC enzyme, we have solved two three-dimensional structures of the BphC enzyme complexed with its substrates, 2,3-dihydroxybiphenyl (2,3-DHBP) or 3-methylcatechol (3-MCT). These substrates were found intact in the active site probably because of the oxidation of the Fe ion into ferric form (as judged by EPR spectra) in the present crystals. In both of the two substrate complexes, the two hydroxyl groups of the substrate, together with the three enzymatic side-chain ligands, were found to form a penta-coordinated system around the Fe ion roughly arranged in a trigonal bipyramidal configuration. The active site structures appear to be essentially consistent with the reaction mechanism proposed so far.

Expression of the bph genes involved in biphenyl/PCB degradation in Pseudomonas sp. KKS102 induced by the biphenyl degradation intermediate, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid.

The bph genes involved in PCB/biphenyl degradation in Pseudomonas sp. KKS102 are clustered as bphEGFA1A2A3BCDA4R. The bph genes are inducibly expressed in the presence of biphenyl. In order to understand the induction more fully, the inducer of bph gene expression was investigated. To identify the inducer molecule, we constructed four deletion mutants of the structural genes and analyzed the inducibility of the bphE gene in each mutant strain. In the wild-type cell and the bphD deletion mutant, the levels of the bphE transcript were enhanced in the presence of biphenyl. On the other hand, in the bphA, bphB, and bphC deletion mutants, levels of the bphE transcript were not enhanced in the presence of biphenyl. These results demonstrated that the series of reactions catalyzed by biphenyl dioxygenase (BphA), dihydrodiol dehydrogenase (BphB), and 2, 3-dihydroxybiphenyl dioxygenase (BphC) are necessary to convert biphenyl to the inducer. It is known that these reactions convert biphenyl to 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), and it was found that the expression of the bph genes was induced by purified HOPDA. These results clearly indicate that HOPDA is the inducer of the bph genes in KKS102.

Nucleotide sequence of a regulatory region controlling alginate synthesis in Pseudomonas aeruginosa: characterization of the algR2 gene.

Alginate (Alg), an exopolysaccharide with strong gelling properties, is produced by Pseudomonas aeruginosa primarily during its infection of the cystic fibrosis (CF) lung. The alg genes are normally not expressed in other environments. The promoter for a critical Alg biosynthetic gene, algD, encoding GDP-mannose dehydrogenase, is activated only under conditions reminiscent of the CF lung (i.e., under high osmolarity), and at least two regulatory genes, algR1 and algR2, have been implicated in this activation process. The physical mapping of a 4.4-kb region harboring algR2 has been accomplished and the complete nucleotide sequence of this fragment, including that of algR2, is presented. The cloning and complementation experiments also demonstrate the presence, on this fragment, of regulatory gene(s) different from algR1 and algR2. The expression of the algR2 gene allows a high level of activation of the algD promoter in Escherichia coli, in the presence of algR1 in a high osmotic environment, suggesting that the AlgR2 and AlgR1 proteins act cooperatively to activate the algD promoter. Hyperexpression of the algR2 gene from the tac promoter also allows the conversion of nonmucoid cells of strain 8822, a spontaneous revertant of the mucoid CF isolate strain 8821, back to mucoidy, but not that of the clinical isolate, strain PAO1.

The bphDEF meta-cleavage pathway genes involved in biphenyl/polychlorinated biphenyl degradation are located on a linear plasmid and separated from the initial bphACB genes in Rhodococcus sp. strain RHA1.

The bphACB genes responsible for the initial oxidation of the aromatic ring of biphenyl/polychlorinated biphenyls (PCB) to meta-cleavage product in Rhodococcus sp. RHA1 have been characterized. We cloned the 6.1 kb EcoRI fragment containing another extradiol dioxygenase gene (etbC) which was induced during the growth on ethylbenzene. The bphD, bphE and bphF encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase, 2-hydroxypenta-2,4-dienoate hydratase and 4-hydroxy-2-oxovalerate aldolase, respectively, were found downstream of etbC. The deduced amino acid (aa) sequence of RHA1 bphD and bphE had 27-33% and 32-38% identity, respectively, with those of the corresponding genes in Pseudomonas. BphE and BphF are closely related to the corresponding homoprotocatechuate meta-cleavage pathway enzymes of Escherichia coli C. The bphD and bphF were expressed in E. coli and the BphD activity was detected. The etbCphDEF genes were transcribed in biphenyl and ethylbenzene growing cells. Pulsed field gel electrophoresis (PFGE) analysis indicated that RHA1 contains three large linear plasmids. Southern blot analysis indicated that the meta-cleavage pathway for biphenyl/PCB catabolism in RHA1 is directed by the 390 kb plasmid borne bphDEF genes located separately from bphACB gene cluster on the 1100 kb plasmid.

Concurrent daily carboplatin and accelerated hyperfractionated thoracic radiotherapy in locally advanced nonsmall cell lung cancer.

PURPOSE: This study was designed to evaluate the feasibility and efficacy of accelerated hyperfractionated thoracic radiotherapy concurrently combined with daily carboplatin in patients with locally advanced, unresectable nonsmall cell lung cancer. METHODS AND MATERIALS: Thirty-one patients with locally advanced nonsmall cell lung cancer were treated with continuous course, twice daily thoracic radiotherapy (1.5 Gy each) to a total of 60 Gy over 4 weeks. Carboplatin (25 mg/m2) i.v. was given immediately before each morning thoracic radiotherapy. Blood samples were taken to measure the blood free platinum pharmacokinetics on day 1. RESULTS: All 31 patients completed the protocol treatment without delay. The median age was 73 years, and the majority had Stage IIIA (32%) or IIIB (48%) disease. Major acute toxicity (> or = Grade 3) included 17 patients (55%) with leukopenia, 5 patients (16%) with thrombocytopenia, and 7 patients (23%) with esophagitis. One possible treatment-related death due to diffuse pneumonitis was observed. There were three complete responses (CRs) and 23 partial responses (PRs) in the radiation field, for a response rate of 84%. The relapse pattern was predominantly loco-regional, and the median survival time was 9.8 months. The area under the plasma level-time curve (AUC) of free platinum correlated significantly (r = -0.41, p = 0.04) with the surviving fraction of leukocytes, but not with the severity of the esophagitis. Responders had significantly (p = 0.04 by Welch's t-test) higher AUCs than nonresponders. CONCLUSIONS: This combination therapy was feasible and efficacious against locally advanced nonsmall cell lung cancer. Although long-term local control still remains unsatisfactory, pharmacokinetic data are suggestive of a role for platinum in enhancing the radiation effect.

Isolation and characterization of a thermophilic Bacillus sp. JF8 capable of degrading polychlorinated biphenyls and naphthalene.

Bacillus sp. strain JF8, which was isolated from compost, utilizes naphthalene and biphenyl as carbon sources at 60 degrees C. Biphenyl grown cells of strain JF8 barely degraded naphthalene while naphthalene grown cells did not degrade p-chlorobiphenyl, suggesting the existince of two independent degradation pathways. Isolation of JF8N, a mutant strain which can not utilize biphenyl as a carbon source while retaining the ability to utilize naphthalene, supports this hypothesis. Biphenyl grown cells of strain JF8 can degrade several polychlorinated biphenyl congeners including tetra- and pentachlorobiphenyl. bph and nah probes from mesophilic organisms failed to hybridize to strain JF8 DNA.

[Effect of ultraviolet-filtering contact lens on rabbit eyes after exposure to ultraviolet light].

We evaluated the protective effect of a newly designed soft contact lens (SCL) on rabbit eyes. This SCL absorbs the ultraviolet B light of wavelengths between 280 nm and 320 nm. Three weeks after resecting the nictitating membrane, the rabbit corneas were covered either by the new SCL or a conventional SCL. The eyes were exposed to ultraviolet light from two 15 W lamps placed 20 cm anteriorly for 3 hours. Another three hours after the exposure, the cornea was examined first with a slitlamp biomicroscope after staining with fluorescein and then by histopathological means after enucleation. Six eyes wearing the new SCL showed moderate superficial punctate keratopathy. Six eyes wearing conventional SCL showed severe superficial keratopathy or corneal erosion. These findings were in good agreement with the histopathological findings. The new ultraviolet-filtering SCL was thus effective in protecting the rabbit cornea under our experimental conditions.

Oxidative stress by biphenyl metabolites induces inhibition of bacterial cell separation.

Cell-cell separation of a polychlorinated biphenyl (PCB)-degrading bacterium Comamonas testosteroni TK102 was monitored by flow cytometry. When monohydroxy metabolites of biphenyl (BP) (2-hydroxybiphenyl and 3-hydroxybiphenyl) were added to the culture, cell-cell separation of strain TK102 was inhibited at stationary phase. This inhibition was reproduced on non-PCB degrading bacteria such as Pseudomonas putida PpY101 and Escherichia coli MV1184, but was not observed on Pseudomonas aeruginosa PAO1. An opportunistic pathogen, P. aeruginosa PAO1, produces exopolysaccharide, which is known to scavenge damaging chemicals such as reactive oxygen species (ROS). The higher level of ROS and lipid peroxidants were detected in the cells treated by monohydroxybiphenyls. Fat-soluble vitamin E, which is a lipid radical scavenger, maintained bacterial cell separation during monohydroxybiphenyls treatment. Our results demonstrated that intracellular oxidative stress played an important role in the inhibition of bacterial cell separation during BP metabolism. This study shows that metabolites of environmental pollutants, such as monohydroxylated BP, inhibit bacterial cell separation by oxidative stress.

A novel nicotinoprotein aldehyde dehydrogenase involved in polyethylene glycol degradation.

A gene (pegC) encoding aldehyde dehydrogenase (ALDH) was located 3.4 kb upstream of a gene encoding polyethylene glycol (PEG) dehydrogenase (pegA) in Sphingomonas macrogoltabidus strain 103. ALDH was expressed in Escherichia coli and purified on a Ni-nitrilotriacetic acid agarose column. The recombinant enzyme was a homotetramer consisting of four 46.1-kDa subunits. The alignment of the putative amino acid sequence of the cloned enzyme showed high similarity with a group of NAD(P)-dependent ALDHs (identity 36-52%); NAD-binding domains (Rossmann fold and four glycine residues) and catalytic residues (Glu225 and Cys259) were well conserved. The cofactor, which was extracted from the purified enzyme, was tightly bound to the enzyme and identified as NADP. The enzyme contained 0.94 mol NADP per subunit. The enzyme was activated by Ca(2+), but by no other metals; no metal (Zn, Fe, Mg, or Mn) was detected in the purified recombinant enzyme. Activity was inhibited by p-chloromercuric benzoate, and heavy metals such as Hg, Cu, Pb and Cd, indicating that a cysteine residue is involved in the activity. Enzyme activity was independent of N,N-dimethyl-p-nitrosoaniline as an electron acceptor. Trans-4-(N,N-dimethylamino)-cinnamaldehyde was not oxidized as a substrate, but the compound worked as an inhibitor for the enzyme, as did pyrazole. The enzyme acted on n-aldehydes C(2)-C(14)) and PEG-aldehydes. Thus the enzyme was concluded to be a novel Ca(2+)-activating nicotinoprotein (NADP-containing) PEG-aldehyde dehydrogenase involved in the degradation of PEG in S. macrogoltabidus strain 103.

The delivery of health care and eye care in Japan.

Japan has developed a socialized system of health care delivery operated under a governmental social insurance plan. It is all inclusive in that no matter what the working status, each individual is covered. Outcomes of the system are impressive, reporting the highest life expectancy and the lowest infant mortality rates in the world. Optometry as a legalized profession is not presently recognized in Japan, even though it is common practice for the public to visit optical shops to receive routine eye examinations and lens prescriptions. There is a movement in Japan to provide optometric education and qualifying examinations comparable to international standards.

Control of alginate synthesis in Pseudomonas aeruginosa: regulation of the algR1 gene.

The triggering of mucoidy (formation of the exopolysaccharide alginate) in Pseudomonas aeruginosa is accomplished primarily in Cystic Fibrosis lung environment through activation of the promoter of a gene algD, which encodes GDP-mannose dehydrogenase, by the product of a regulatory gene algR1. Both algD and algR1 promoter regions have significant homology, including the presence of sequences recognized by the RNA polymerase sigma 54. We demonstrate that the algR1 promoter is partly constitutive and its activation, similar to that of algD, is dependent on high osmolarity as well as the presence of its own gene product and is repressed by high concentrations of AlgR1. The RpoN sigma factor also plays a critical role in the transcription of both algD and algR1 genes.

Pulmonary dehydration and infection in cystic fibrosis: evidence that ethanol activates alginate gene expression and induction of mucoidy in Pseudomonas aeruginosa.

Mortality among cystic fibrosis (CF) patients is most commonly attributed to pulmonary infection by mucoid, alginate-producing Pseudomonas aeruginosa. The initial infecting P. aeruginosa are typically non-mucoid; however, upon continued exposure to the CF lung environment, they become highly mucoid. The CF lung is an osmotically high environment because of the presence of substantial concentrations of electrolytes and dehydrated mucus. In this report we demonstrate that ethanol (a commonly used dehydrating agent) activates transcription from a critical alginate promoter, algD, and show that prolonged exposure to ethanol allows switching to the mucoid form. This activation appears to be dependent on DNA gyrase. Analysis of alginate gene activation, and the subsequent reversal of the activation process by bacterial DNA gyrase inhibitors, should aid the development of treatment strategies for CF patients infected with this organism.

Analysis of changes in congener selectivity during PCB degradation by Burkholderia sp. strain TSN101 with increasing concentrations of PCB and characterization of the bphBCD genes and gene products.

We isolated and characterized a gram-negative bacterium, Burkholderia sp. strain TSN101, that can degrade polychlorinated biphenyls (PCBs) at concentrations as high as 150 microg Kaneclor 300/ml, a PCB mixture equivalent to Aroclor 1242. Growing cells of strain TSN101 degraded most of the tri- and tetrachlorobiphenyls in medium containing 25 microg Kaneclor 300/ml. Using PCB concentrations of 50-150 microg of Kaneclor 300/ml, the congener selectivity pattern was different and the pattern of chlorine substitution strongly affected degradation of some congeners. At 25 microg Kaneclor 300/ml, strain TSN101 degraded di- and trichlorinated congeners with chlorine substitutions at both the ortho and the para positions. At higher concentrations of Kaneclor 300, di- and trichlorobiphenyls with ortho substituents in both phenyl rings were not degraded well. Trichlorobiphenyls with para and meta substitutents were degraded equally well at all concentrations studied. The ability of strain TSN101 to degrade ortho and para-substituted congeners was confirmed using a defined PCB mixture with chlorine substituents at 2'- and 4'-positions. A 5-kb DNA fragment containing the bphBCD genes was cloned and sequenced. Comparison of the deduced amino acid sequences of these genes with related proteins indicated 99 and 98% sequence similarity to the BphB and BphD of Comamonas testosteroni strain B-356, respectively. The bphC gene product showed 74% sequence similarity to the BphC of Burkholderia cepacia strain LB400 and exhibited a narrow substrate specificity with strong affinity for 2, 3-dihydroxybiphenyl. A bphC-disrupted mutant of Burkholderia sp. strain TSN101, constructed by gene replacement, lost the ability to utilize biphenyl, thus supporting the role of the cloned bph gene in biphenyl metabolism.

BphS, a key transcriptional regulator of bph genes involved in polychlorinated biphenyl/biphenyl degradation in Pseudomonas sp. KKS102.

The bph genes in Pseudomonas sp. KKS102, which are involved in the degradation of polychlorinated biphenyl/biphenyl, are induced in the presence of biphenyl. In this study our goal was to understand the regulatory mechanisms involved in the inducible expression. The bph genes (bphEGF(orf4)A1A2A3BCD(orf1)A4R) constitute an operon, and its expression is strongly dependent on the pE promoter located upstream of the bphE gene. A bphS gene, whose deduced amino acid sequence showed homology with the GntR family transcriptional repressors, was identified at the upstream region of the bphE gene. Disruption of the bphS gene resulted in constitutive expression of bph genes, suggesting that the bphS gene product negatively regulated the pE promoter. The gel retardation and DNase footprinting analyses demonstrated specific binding of BphS to the pE promoter region and identified four BphS binding sites that were located within and immediately downstream of the -10 box of the pE promoter. The four binding sites were functional in repression because their respective elimination resulted in derepression of the pE promoter. The binding of BphS was abolished in the presence of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, an intermediate compound in the biphenyl degradation pathway. We concluded that the negative regulator BphS plays a central role in the regulation of bph gene expression through its action at the pE promoter.

Cloning and sequencing of two tandem genes involved in degradation of 2,3-dihydroxybiphenyl to benzoic acid in the polychlorinated biphenyl-degrading soil bacterium Pseudomonas sp. strain KKS102.

Two genes involved in the degradation of biphenyl were isolated from a gene library of a polychlorinated biphenyl-degrading soil bacterium, Pseudomonas sp. strain KKS102, by using a broad-host-range cosmid vector, pKS13. When a 3.2-kilobase (kb) PstI fragment of a 29-kb cosmid DNA insert was subcloned into pUC18 at the PstI site downstream of the lacZ promoter, Escherichia coli cells carrying this recombinant plasmid expressed 2,3-dihydroxybiphenyl dioxygenase activity. Nucleotide sequencing of the 3.2-kb PstI fragment revealed that there were two open reading frames (ORFI [882 base pairs] and ORFII [834 base pairs], in this gene order). Results of analysis of Tn5 insertion mutants and unidirectional deletion mutants suggested that the ORFI coded for 2,3-dihydroxybiphenyl dioxygenase. When the sequence of ORFI was compared with that of bphC of Pseudomonas pseudoalcaligenes KF707 (K. Furukawa, N. Arima, and T. Miyazaki, J. Bacteriol. 169:427-429, 1987), the homology was 68%, with both strains having the same Shine-Dalgarno sequence. The result of gas chromatography-mass spectrometry analysis of the metabolic product suggested that the ORFII had meta cleavage compound hydrolase activity to produce benzoic acid. DNA sequencing suggested that these two genes were contained in one operon.

Identification of the bphA4 gene encoding ferredoxin reductase involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102.

The nucleotide sequence of the downstream region of the bph operon from Pseudomonas sp. strain KKS102 was determined. Two open reading frames (ORF1 and ORF2) were found in this region, and the deduced amino acid sequence of ORF2 showed homology with the sequences of four ferredoxin reductases of dioxygenase systems. When this region was inserted just upstream of the bph operon, which does not contain a gene encoding ferredoxin reductase, biphenyl dioxygenase activity was detected. The 24- and 44-kDa polypeptides predicted from the two open reading frames were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Crude extract which contained the products of ORF2 and bphA1A2A3 showed cytochrome c reduction activity. These data clearly suggest that ORF2 encodes ferredoxin reductase. The deduced amino acid sequence of ORF1 does not show significant homology with the sequences of any other proteins in the SWISS-PROT data bank, and the function of ORF1 is unknown.

A Novel Transformation of Polychlorinated Biphenyls by Rhodococcus sp. Strain RHA1.

We have characterized a biphenyl degrader, Rhodococcus sp. strain RHA1. Biphenyl-grown cells of strain RHA1 efficiently transformed 45 components in the 62 major peaks of a polychlorinated biphenyl (PCB) mixture of Kanechlors 200, 300, 400, and 500 within 3 days, which includes mono- to octachlorobiphenyls. Among the intermediate metabolites of PCB transformation, di- and trichlorobenzoic acids were identified. The gradual decrease of these chlorobenzoic acids during incubation indicated that these chlorobenzoic acids would also be degraded by this strain. The effect of the position of chlorine substitution was determined by using PCB mixtures that have chlorine substitutions mainly at either the ortho or the meta position. This strain transformed both types of congeners, and strong PCB transformation activity of RHA1 was indicated. RHA1 accumulated 4-chlorobenzoic acid temporally during the transformation of 4-chlorobiphenyl. The release of most chloride in the course of 2,2(prm1)-dichlorobiphenyl degradation was observed. These results suggested that RHA1 would break down at least some PCB congeners into smaller molecules to a considerable extent.

Multiple Polychlorinated Biphenyl Transformation Systems in the Gram-Positive Bacterium Rhodococcus sp. Strain RHA1.

The cloned bphA gene of the polychlorinated biphenyl (PCB) degrader Rhodococcus sp. strain RHA1 was expressed in Rhodococcus erythropolis IAM1399 cells, resulting in the transformation of di-, tri-, and tetrachlorobiphenyls. Disruption of the bphA1 gene in RHA1 resulted in a lack of growth on biphenyl and a loss of PCB transformation activity. However, the bphA1 insertion mutant of RHA1, designated RDA1, retained the ability to transform PCB congeners when grown on ethylbenzene as its carbon source. It also transformed 4-chlorobiphenyl to 4-chlorobenzoate, although it was suspected to be deficient in bphB and bphC gene activities as well as bphA. This suggested that an alternative PCB degradation system distinct from the one encoded by the cloned bph genes was present.

Identification of the bphA and bphB genes of Pseudomonas sp. strains KKS102 involved in degradation of biphenyl and polychlorinated biphenyls.

The nucleotide sequence of the upstream region of the bphC gene from Pseudomonas sp. strain KKS102 was determined. Four genes were found in this region. Deduced amino acid sequences of the first, second, third and fourth genes showed significant homology with a large subunit of iron-sulfur protein, a small subunit of iron-sulfur protein, ferredoxin and dihydrodiol dehydrogenase, respectively, from other bacteria which degrade biphenyl/polychlorinated biphenyls, toluene and benzene. E. coli, in which the four genes, bphC and the gene for ferredoxin reductase from benzene degrading bacterium were expressed, was able to produce meta-cleavage compounds from chlorinated biphenyls. These results show that these gene products are functional in both biphenyl and polychlorinated biphenyls degradation.