Hydrolysis of benzonitrile herbicides by soil actinobacteria and metabolite toxicity

The soil actinobacteria Rhodococcus rhodochrous PA-34, Rhodococcus sp. NDB 1165 and Nocardia globerula NHB-2 grown in the presence of isobutyronitrile exhibited nitrilase activities towards benzonitrile (approx. 1.1–1.9 U mg^?1 dry cell weight). The resting cell suspensions eliminated benzonitrile and the benzonitrile analogues chloroxynil (3,5-dichloro-4-hydroxybenzonitrile), bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodo-4-hydroxybenzonitrile) (0.5 mM each) from reaction mixtures at 30°C and pH 8.0. The products were isolated and identified as the corresponding substituted benzoic acids. The reaction rates decreased in the order benzonitrile ? chloroxynil > bromoxynil > ioxynil in all strains. Depending on the strain, 92–100, 70–90 and 30–51% of chloroxynil, bromoxynil and ioxynil, respectively, was hydrolyzed after 5 h. After a 20-h incubation, almost full conversion of chloroxynil and bromoxynil was observed in all strains, while only about 60% of the added ioxynil was converted into carboxylic acid. The product of ioxynil was not metabolized any further, and those of the other two herbicides very slowly. None of the nitrilase-producing strains hydrolyzed dichlobenil (2,6-dichlorobenzonitrile). 3,5-Dibromo-4-hydroxybenzoic acid exhibited less inhibitory effect than bromoxynil both on luminescent bacteria and germinating seeds of Lactuca sativa. 3,5-Diiodo-4-hydroxybenzoic acid only exhibited lower toxicity than ioxynil in the latter test.     

Electronic waste—a modern form of risk? On the consequences of the delay between the increasing generation of electronic waste and regulations to manage this increase

This article addresses the relation between the current proliferation of Information and Communication Technology (ICT) goods and services and the development of different kinds of risk assessments and policy documents. The ambition is to shed light on a, hithertho, less-discussed aspect of the development of risk assessments and policy documents, namely the time span between the development of new technologies and the development of regulatory frameworks. The concept of risk and danger can be seen as a potential means through which we can start to think about the consequences of the delay between the increased generation of electronic waste (e-waste) and the regulations to manage this increase. By using e-waste as a case study, this article provides the basis for a more general understanding of the relation between the development of new technologies and the development of regulatory frameworks. While it might be difficult to pin down the effects that this delay had/has for the subsequent development of ICTs, this article highlights the importance of taking into account not only how and by whom risk assessments and policy documents are developed, but also when they are developed in relation to the technologies that they serve to regulate.     

Interaction of TonB with the outer membrane receptor FpvA of Pseudomonas aeruginosa

Pyoverdine-mediated iron uptake by the FpvA receptor in the outer membrane of Pseudomonas aeruginosa is dependent on the inner membrane protein TonB1. This energy transducer couples the proton-electrochemical potential of the inner membrane to the transport event. To shed more light upon this process, a recombinant TonB1 protein lacking the N-terminal inner membrane anchor (TonB(pp)) was constructed. This protein was, after expression in Escherichia coli, purified from the soluble fraction of lysed cells by means of an N-terminal hexahistidine or glutathione S-transferase (GST) tag. Purified GST-TonB(pp) was able to capture detergent-solubilized FpvA, regardless of the presence of pyoverdine or pyoverdine-Fe. Targeting of the TonB1 fragment to the periplasm of P. aeruginosa inhibited the transport of ferric pyoverdine by FpvA in vivo, indicating an interference with endogenous TonB1, presumably caused by competition for binding sites at the transporter or by formation of nonfunctional TonB heterodimers. Surface plasmon resonance experiments demonstrated that the FpvA-TonB(pp) interactions have apparent affinities in the micromolar range. The binding of pyoverdine or ferric pyoverdine to FpvA did not modulate this affinity. Apparently, the presence of either iron or pyoverdine is not essential for the formation of the FpvA-TonB complex in vitro.     

Role of heme in intracellular trafficking of thyroperoxidase and involvement of H2O2 generated at the apical surface of thyroid cells in autocatalytic covalent heme binding

Thyroperoxidase (TPO) is a glycosylated hemoprotein that plays a key role in thyroid hormone synthesis. We previously showed that in CHO cells expressing human TPO (hTPO) only 2% of synthesized hTPO reaches the cell surface. Herein, we investigated the role of heme moiety insertion in the exit of hTPO from the endoplasmic reticulum. Peroxidase activity at the cell surface and cell surface expression of hTPO were decreased by approximately 30 and approximately 80%, respectively, with succinyl acetone, an inhibitor of heme biosynthesis, and were increased by 20% with holotransferrin and aminolevulinic acid, precursors of heme biosynthesis. Results were similar with holotransferrin plus aminolevulinic acid or hemin, but hemin increased cell surface activity more efficiently (+120%) relative to the control. It had been suggested (DePillis, G., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 8857-8960) that covalent attachment of heme to mammalian peroxidases could be an H2O2-dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H2O2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 microM H2O2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H2O2 was synthesized at the apical cell surface and allowed covalent attachment of heme. Two-day incubation of primocultures of thyroid cells with catalase led to a 30% decrease in TPO activity at the cell surface. In conclusion, we provide compelling evidence for an essential role of 1) heme incorporation in the intracellular trafficking of hTPO and of 2) H2O2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule.     

Submerged cultivation of Ganoderma lucidum biomass and immunostimulatory effects of fungal polysaccharides

Original Ganoderma lucidum strain MZKI G97 isolated from Slovenian forests was cultivated in a liquid substrate based on potato dextrose and olive oil. The influences of inoculum and oxygen partial pressure in batch and fed batch cultivation in a 10-l laboratory stirred tank reactor were studied. Fungal biomass was found to be oxygen and shear sensible. Using a 17% (wet weight) 6 days old vegetative inoculum, 9.6 g l(-1) of dry biomass in batch cultivation and 15.2 g l(-1) in fed batch process were obtained. Extracellular (9.6 g l(-1)) and intracellular (6.3 g l(-1)) polysaccharide fractions were isolated. Extracellular polysaccharide fraction and four intracellular polysaccharide fractions were obtained. Polysaccharides were further separated by ion-exchange, gel and affinity chromatography. The isolated polysaccharides were mainly beta-D-glucanes. Immunostimulatory effects of isolates were tested on induction of cytokine (tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma)) synthesis in primary cultures of human peripheral blood mononuclear cells (PBMC) isolated from a buffy coat. The TNF-alpha inducing activity is comparable with romurtide, which has been used as a supporting therapy in cancer patients treated with radiotherapy and/or chemotherapy.     

PP1/PP2A phosphatases inhibitors okadaic acid and calyculin A block ERK5 activation by growth factors and oxidative stress

Okadaic acid is an inhibitor of the protein Ser/Thr phosphatases PP1 and PP2A, which blocks the activation of extracellular signal-regulated protein kinase 5 (ERK5), a member of the MAP kinase family activated by growth factors and several types of stressors. The blocking of ERK5 activation by okadaic acid was observed in HeLa cells exposed to epidermal growth factor and H(2)O(2) as well as in PC12 cells stimulated by nerve growth factor and H(2)O(2). Calyculin A, another PP1 and PP2A inhibitor, behaved similarly although these compounds are not structurally related. This suggests that either PP1 or PP2A or both are necessary for ERK5 activation. Protein kinase C (PKC) acts as a negative regulator of the ERK5 activation pathway, however our data suggest that the effects of PKC and the phosphatase are unrelated.     

Heme Binding Proteins of Bartonella henselae Are Required when Undergoing Oxidative Stress During Cell and Flea Invasion

Bartonella are hemotropic bacteria responsible for emerging zoonoses. These heme auxotroph alphaproteobacteria must import heme for their growth, since they cannot synthesize it. To import exogenous heme, Bartonella genomes encode for a complete heme uptake system enabling transportation of this compound into the cytoplasm and degrading it to release iron. In addition, these bacteria encode for four or five outer membrane heme binding proteins (Hbps). The structural genes of these highly homologous proteins are expressed differently depending on oxygen, temperature and heme concentrations. These proteins were hypothesized as being involved in various cellular processes according to their ability to bind heme and their regulation profile. In this report, we investigated the roles of the four Hbps of Bartonella henselae, responsible for cat scratch disease. We show that Hbps can bind heme in vitro. They are able to enhance the efficiency of heme uptake when co-expressed with a heme transporter in Escherichia coli. Using B. henselae Hbp knockdown mutants, we show that these proteins are involved in defense against the oxidative stress, colonization of human endothelial cell and survival in the flea.