Degradation of fluorene by Brevibacterium sp. strain DPO 1361: a novel C-C bond cleavage mechanism via 1,10-dihydro-1,10-dihydroxyfluoren-9-one

Angular dioxygenation has been established as the crucial step in dibenzofuran degradation by Brevibacterium sp. strain DPO 1361 (V. Strubel, K. H. Engesser, P. Fischer, and H.-J. Knackmuss, J. Bacteriol. 173:1932-1937, 1991). The same strain utilizes biphenyl and fluorene as sole sources of carbon and energy. The fluorene degradation sequence is proposed to be initiated by oxidation of the fluorene methylene group to 9-fluorenol. Cells grown on fluorene exhibit pronounced 9-fluorenol dehydrogenase activity. Angular dioxygenation of the 9-fluorenone thus formed yields 1,10-dihydro-1,10-dihydroxyfluoren-9-one (DDF). A mechanistic model is presented for the subsequent C-C bond cleavage by an NAD(+)-dependent DDF dehydrogenase, acting on the angular dihydrodiol. This enzyme was purified and characterized as a tetramer of four identical 40-kDa subunits. The following Km values were determined: 13 microM for DDF and 65 microM for 2,3-dihydro-2,3-dihydroxybiphenyl. The enzyme also catalyzes the production of 3-(2'-carboxyphenyl)catechol, which was isolated, and structurally characterized, in the form of the corresponding lactone, 4-hydroxydibenzo-(b,d)-pyran-6-one. Stoichiometry analysis unequivocally demonstrates that angular dioxygenation constitutes the principal pathway in Brevibacterium sp. strain DPO 1361

Knock-down of the 37kDa/67kDa laminin receptor LRP/LR impedes telomerase activity.

Cancer has become a major problem worldwide due to its increasing incidence and mortality rates. Both the 37kDa/67kDa laminin receptor (LRP/LR) and telomerase are overexpressed in cancer cells. LRP/LR enhances the invasiveness of cancer cells thereby promoting metastasis, supporting angiogenesis and hampering apoptosis. An essential component of telomerase, hTERT is overexpressed in 85-90% of most cancers. hTERT expression and increased telomerase activity are associated with tumor progression. As LRP/LR and hTERT both play a role in cancer progression, we investigated a possible correlation between LRP/LR and telomerase. LRP/LR and hTERT co-localized in the perinuclear compartment of tumorigenic breast cancer (MDA-MB231) cells and non-tumorigenic human embryonic kidney (HEK293) cells. FLAG® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT. In addition, flow cytometry revealed that both cell lines displayed high cell surface and intracellular LRP/LR and hTERT levels. Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity. These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity.This work was supported by the National Research Foundation, the Republic of South Africa.NCS201

Microbial degradation of biaryl structures: relationships between fluorene, dibenzofuran and biphenyl pathways

Initial dioxygenation of fluorene by dibenzofuran degrading strains occurs in the unusual angular position. The resulting dihydrodiendiol is converted to 3-(2-carboxyphenyl)catechol by action of a dehydrogenase. This is a novel activity for a dehydrogenase causing a C-C-bond cleavage. After growth with dibenzofuran and biphenyl respectively two different initial dioxygenases are expressed. The first enzyme shows a broad substrate range, the second enzyme only converts biphenyl. Strains degrading fluorene, dibenzofuran and biphenyl may constitute a unique physiological group

Anti-LRP/LR specific antibody IgG1-iS18 impedes adhesion and invasion of liver cancer cells.

Two key events, namely adhesion and invasion, are pivotal to the occurrence of metastasis. Importantly, the 37 kDa/67 kDa laminin receptor (LRP/LR) has been implicated in enhancing these two events thus facilitating cancer progression. In the current study, the role of LRP/LR in the adhesion and invasion of liver cancer (HUH-7) and leukaemia (K562) cells was investigated. Flow cytometry revealed that the HUH-7 cells displayed significantly higher cell surface LRP/LR levels compared to the poorly-invasive breast cancer (MCF-7) control cells, whilst the K562 cells displayed significantly lower cell surface LRP/LR levels in comparison to the MCF-7 control cells. However, Western blotting and densitometric analysis revealed that all three tumorigenic cell lines did not differ significantly with regards to total LRP/LR levels. Furthermore, treatment of liver cancer cells with anti-LRP/LR specific antibody IgG1-iS18 (0.2 mg/ml) significantly reduced the adhesive potential of cells to laminin-1 and the invasive potential of cells through the ECM-like Matrigel, whilst leukaemia cells showed no significant differences in both instances. Additionally, Pearson's correlation coefficients suggested direct proportionality between cell surface LRP/LR levels and the adhesive and invasive potential of liver cancer and leukaemia cells. These findings suggest the potential use of anti-LRP/LR specific antibody IgG1-iS18 as an alternative therapeutic tool for metastatic liver cancer through impediment of the LRP/LR- laminin-1 interaction

Invasive potential of oesophageal and breast cancer cells.

<p>Invasive potentials were determined using the absorbance measured at 620 nm. A percentage was then determined by calculating the difference between the respective cell line and the invasive potential of MCF-7 cell line. p-values were calculated using the two-tailed Students t-test with a 95%confidence interval.</p

Effect of anti-LRP/LR specific antibody IgG1-iS18 on the invasive potential of breast and oesophageal cancer cells.

<p>Cells were incubated with IgG1-iS18 (0.2 mg/ml) or CAT (negative control) (0.2 mg/ml) on Matrigel™ coated inserts for an 18 h period. Toulidine blue was used to stain the invaded cells and following extraction of the dye using SDS absorbance was measured at 620 nm. A significant decrease in invasion potential was noted for both WHCO1 and MDA-MB 231 cells upon application of IgG1-iS18 antibody (light gray) compared to the no antibody control (black). Data are representative of three experiments carried out in triplicate from different cell cultures.</p

IgG1-iS18 reduces invasive potential of breast and oesophageal cancer cells.

<p>The % reduction in invasion in the difference in invasive potential between cells without antibody compared to cells treated with IgG1-iS18 antibody. p-values were calculated using the two-tailed Students <i>t-test</i> with a 95% confidence interval.</p

Oesophageal and breast cancer cells display significantly increased invasive potential compared to non-invasive MCF-7 cells.

<p>Cells were incubated in Matrigel™ coated inserts for an18 h period and stained with toulidine. The dye was extracted using SDS and measured the absorbance of the resulting solution at 620 nm. Data are representative of three experiments carried out in triplicate.</p