Biodesulfurization of naphthothiophene and benzothiophene through selective cleavage of carbon-sulfur bonds by Rhodococcus sp. strain WU-K2R

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following hydrodesulfurization treatment. Rhodococcus sp. strain WU-K2R was newly isolated from soil for its ability to grow in a medium with NTH as the sole source of sulfur, and growing cells of WU-K2R degraded 0.27 mM NTH within 7 days. WU-K2R could also grow in the medium with NTH sulfone, benzothiophene (BTH), 3-methyl-BTH, or 5-methyl-BTH as the sole source of sulfur but could not utilize DBT, DBT sulfone, or 4,6-dimethyl-DBT. On the other hand, WU-K2R did not utilize NTH or BTH as the sole source of carbon. By gas chromatography-mass spectrometry analysis, desulfurized NTH metabolites were identified as NTH sulfone, 2'-hydroxynaphthylethene, and naphtho[2,1-b]furan. Moreover, since desulfurized BTH metabolites were identified as BTH sulfone, benzo[c][1,2]oxathiin S-oxide, benzo[c][1,2]oxathiin S,S-dioxide, o-hydroxystyrene, 2-(2'-hydroxyphenyl)ethan-1-al, and benzofuran, it was concluded that WU-K2R desulfurized NTH and BTH through the sulfur-specific degradation pathways with the selective cleavage of carbon-sulfur bonds. Therefore, Rhodococcus sp. strain WU-K2R, which could preferentially desulfurize asymmetric heterocyclic sulfur compounds such as NTH and BTH through the sulfur-specific degradation pathways, is a unique desulfurizing biocatalyst showing properties different from those of DBT-desulfurizing bacteria.     

Thermophilic biodesulfurization of naphthothiophene and 2-ethylnaphthothiophene by a dibenzothiophene-desulfurizing bacterium, Mycobacterium phlei WU-F1

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and NTH derivatives can be detected in diesel oil following hydrodesulfurization treatment, in addition to DBT derivatives. Mycobacterium phlei WU-F1, which possesses high desulfurizing ability toward DBT and its derivatives over a wide temperature range (20-50 degrees C), could also grow at 50 degrees C in a medium with NTH or 2-ethylNTH, an alkylated derivative, as the sole source of sulfur. At 50 degrees C, the resting cells of WU-Fl degraded 67% and 83% of 0.81 mM NTH and 2-ethylNTH, respectively, within 8 h. By GC-MS analysis, 2-ethylNTH-desulfurized metabolites were identified as 2-ethylNTH sulfoxide, 1-(2'-hydroxynaphthyl)-1-butene and 1-naphthyl-2-hydroxy-1-butene, and it was concluded that WU-F1 desulfurized 2-ethylNTH through a sulfur-specific degradation pathway with the selective cleavage of carbon-sulfur bonds. Therefore, M. phlei WU-Fl can effectively desulfurize asymmetric organosulfur compounds, NTH and 2-ethylNTH, as well as symmetric DBT derivatives under high-temperature conditions, and it may be a useful desulfurizing biocatalyst possessing a broad substrate specificity toward organosulfur compounds.     

Infiltration anesthetic lidocaine inhibits cancer cell invasion by modulating ectodomain shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF)

Although the mechanism is unknown, infiltration anesthetics are believed to have membrane-stabilizing action. We report here that such a most commonly used anesthetic, lidocaine, effectively inhibited the invasive ability of human cancer (HT1080, HOS, and RPMI-7951) cells at concentrations used in surgical operations (5-20 mM). Ectodomain shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF) from the cell surface plays an important role in invasion by HT1080 cells. Lidocaine reduced the invasion ability of these cells by partly inhibiting the shedding of HB-EGF from the cell surface and modulation of intracellular Ca2+ concentration contributed to this action. The anesthetic action of lidocaine (sodium channel blocking ability) did not contribute to this anti-invasive action. In addition, lidocaine (5-30 mM), infiltrated around the inoculation site, inhibited pulmonary metastases of murine osteosarcoma (LM 8) cells in vivo. These data point to previously unrecognized beneficial actions of lidocaine and suggest that lidocaine might be an ideal infiltration anesthetic for surgical cancer operations.     

Monocyte chemoattractant protein-1 selectively inhibits the acquisition of CD40 ligand-dependent IL-12-producing capacity of monocyte-derived dendritic cells and modulates Th1 immune response

Accumulating evidence indicates that monocyte chemoattractant protein-1 (MCP-1), a CC chemokine, also displays immunoregulatory functions and may be involved in Th subset differentiation. In this study, we examined the effects of MCP-1 on the cytokine-driven differentiation of monocytes into dendritic cells (DCs), the most potent APCs for naive T cells. We found that DCs generated in the presence of MCP-1 displayed a markedly reduced production of IL-12 in response to CD40 ligand but not in response to Staphylococcus aureus stimulation in the presence or absence of IFN-gamma. The production of IL-10, a potent endogenous IL-12 inhibitor, was not affected by MCP-1. Whereas the inhibitory activity of MCP-1 on IL-12 production by monocytes was sensitive to pertussis toxin, its effects on DC differentiation were pertussis toxin resistant. MCP-1 did not affect the surface phenotype and T cell-stimulating activity of DCs, but most interestingly, naive T cells stimulated with MCP-1-primed DCs produced much less IFN-gamma but the same levels of IL-13. Taken together, our results indicated that MCP-1 modulates the differentiation of monocytes into DCs and may thereby inhibit Th1 cell development.     

Time-dependent aromatase inactivation by 4 beta,5 beta-epoxides of the natural substrate androstenedione and its 19-oxygenated analogs

Aromatase catalyzes the conversion of androgens to estrogens through three sequential oxygenations. To gain insight into the catalytic function of aromatase and its aromatization mechanism, we studied the inhibition of human placental aromatase by 4 beta,5 beta-epoxyandrostenedione (5) as well as its 19-hydroxy and 19-oxo derivatives (6 and 7, respectively), and we also examined the biochemical aromatization of these steroids. All of the epoxides were weak competitive inhibitors of aromatase with apparent K(i) values ranging from 5.0 microM to 30 microM. The 19-methyl and 19-oxo compounds 5 and 7 inactivated aromatase in a time-dependent manner with k(inact) of 0.048 and 0.110 min(-1), respectively, in the presence of NADPH. In the absence of NADPH, only the former inhibited aromatase with a k(inact) of 0.091 min(-1). However, 19-hydroxy steroid 6 did not cause irreversible inactivation either in the presence or absence of NADPH. Gas chromatography-mass spectrometric analysis of the metabolite produced by a 5-min incubation of the three epoxides with human placental microsomes in the presence of NADPH under air revealed that all three compounds were aromatized to produce estradiol with rates of 8.82, 0.51, and 1.62 pmol/min/mg protein for 5, 6, and 7, respectively. In each case, the aromatization was efficiently prevented by 19-hydroxyandrost-4-en-17-one, a potent aromatase inhibitor. On the basis of the aromatization and inactivation results, it seems likely that the two pathways, aromatization and inactivation, may proceed, in part, through a common intermediate, 19-oxo compound 7, although they may be principally different.     

Subtle mutations in the cysteine region of HIV-1 Vif drastically alter the viral replication phenotype

Mutations were introduced into the region encoding the two cysteine and nearby amino acid residues of human immunodeficiency virus type 1 (HIV-1) Vif protein and, 12 single-amino-acid viral mutants were constructed. Determination of their growth characteristics in two lymphocytic cell lines revealed that only a single amino acid change in the cysteine region greatly altered the replication phenotype. In particular, the four mutants of amino acid 132 of Vif were grouped into three categories on the basis of their growth potentials. These results indicate that the cysteine region of Vif is critical for the cell-dependent replication efficiency of HIV-1.     

Pyrobaculum calidifontis sp. nov., a novel hyperthermophilic archaeon that grows in atmospheric air

A novel, facultatively aerobic, heterotrophic hyperthermophilic archaeon was isolated from a terrestrial hot spring in the Philippines. Cells of the new isolate, strain VA1, were rod-shaped with a length of 1.5 to 10 microm and a width of 0.5 to 1.0 microm. Isolate VA1 grew optimally at 90 to 95 degrees C and pH 7.0 in atmospheric air. Oxygen served as a final electron acceptor under aerobic growth conditions, and vigorous shaking of the medium significantly enhanced growth. Elemental sulfur inhibited cell growth under aerobic growth conditions, whereas thiosulfate stimulated cell growth. Under anaerobic growth conditions, nitrate served as a final electron acceptor, but nitrite or sulfur-containing compounds such as elemental sulfur, thiosulfate, sulfate and sulfite could not act as final electron acceptors. The G+C content of the genomic DNA was 51 mol%. Phylogenetic analysis based on 16S rRNA sequences indicated that strain VA1 exhibited close relationships to species of the genus Pyrobaculum. A DNA-DNA hybridization study revealed a low level of similarity (< or = 18%) between strain VA1 and previously described members of the genus Pyrobaculum. Physiological characteristics also indicated that strain VA1 was distinct from these Pyrobaculum species. Our results indicate that isolate VA1 represents a novel species, named Pyrobaculum calidifontis.     

High-performance liquid chromatographic assay for amiodarone N-deethylation activity in human liver microsomes using solid-phase extraction

A selective and sensitive assay for amiodarone N-deethylation activity in human liver microsomes by high-performance liquid chromatography (HPLC) with UV detection is reported. The extraction of desethylamiodarone from incubation samples was performed by means of an original solid-phase extraction (SPE) procedure using a polymeric reversed-phase sorbent (Oasis HLB). The method was validated for the determination of desethylamiodarone with respect to specificity, linearity, precision, accuracy, recovery, limit of quantitation and stability. Amiodarone N-deethylation activity from low to high substrate concentrations using human liver microsomes was precisely determined without a concentration step. This method is applicable to the study in vitro of the metabolism of amiodarone.