Detection of halogenated organic compounds using immobilized thermophilic dehalogenase.

Environmental pollutants containing halogenated organic compounds can cause a plethora of health problems. Detection, quantification, and eventual remediation of halogenated pollutants in the environment are important to human well-being. Toward this end, we previously identified a haloacid dehalogenase, L-HAD(ST), from the thermophile Sulfolobus tokodaii. This thermophilic enzyme is extremely stable and catalyzes, stereospecifically, the dehalogenation of L-2-haloacids. In the current study, we covalently linked L-HAD(ST) to an N-hydroxysuccinimidyl Sepharose resin to construct a highly specific sensor with long shelf life for the detection of L-2-haloacids. The enzyme-modified resin was packed into disposable columns. Samples containing L-2-haloacids were first incubated in the column, and were then collected to quantify the chloride produced through the breakdown of the substrate. The optimum pH of the immobilized enzyme is around 9.5, similar to that of the soluble protein. Its catalytic activity increased with temperature up to the highest temperature measured (50 degrees C). The resin could be fully regenerated after multiple reaction cycles and retained 70% of the initial activity after being stored at 4 degrees C for 6 months. The L-HAD(ST)-modified resin could be used to breakdown and quantify L-2-haloacids spiked in the simulated environmental samples, indicating dehalogenases from extremophiles can potentially be employed in the detection and decontamination of L-2-haloacids.

Do Cancer Cell Lines Have Fixed or Fluctuating Stem Cell Phenotypes? - Studies with the NTera2 Cell Line.

One of the important questions when studying established cancer cell lines is whether such cells contain a subpopulation of primitive cancer stem cells that maintains the expansion of the cell line. To address this issue, we performed studies on the established human embryonal carcinoma cell line NTera2 by evaluating the potential stemness of cells sorted according to their expression of the cell surface stem cell markers CD133 and SSEA4. By performing in vitro and in vivo assays, we observed different properties of cells expressing both, one, or neither of these antigens. While sorted SSEA4+ subpopulations exhibited the greatest propensity for migration toward normal serum and the highest seeding efficiency in the lungs of immunodeficient mice, CD133-SSEA4- cells displayed high seeding efficiency to the bone marrow after injection in vivo. It is worth noting that these properties did not depend on the size of the evaluated cells. To address the question of whether cancer stem cell phenotypes in cell lines are fixed or fluctuating, we sorted single cells according to their expression of CD133 and SSEA4 antigens and observed that cells which did not express these cancer stem cell markers gave rise to cells that express these markers after expansion in vitro. Therefore, our results support the idea that within established cancer cell lines, the phenotype of the cell subpopulation expressing cancer stem cell markers is not fixed but fluctuates during cell line expansion, and cells negative for these markers may acquire their expression.

Evidence that vitronectin is a potent migration-enhancing factor for cancer cells chaperoned by fibrinogen: a novel view of the metastasis of cancer cells to low-fibrinogen lymphatics and body cavities.

Diluted (1%) plasma induces migration of malignant cell lines much more strongly than potent pro-metastatic factors. To characterize the factor(s) present in diluted plasma responsible for this phenomenon we performed i) heat inactivation, ii) dialysis, iii) proteinase K treatment, and iv) molecular size filtration studies. We found that this remarkable pro-migratory activity of diluted normal plasma is associated with a ~50-100-kD protein that interacts with GαI protein-coupled receptors and activates p42/44 MAPK and AKT signaling in target cells. Since this pro-migratory activity of 1% plasma decreases at higher plasma concentrations (> 20%), but is retained in serum, we hypothesized that fibrinogen may be involved as a chaperone of the protein(s). To identify the pro-migratory protein(s) present in diluted plasma and fibrinogen-depleted serum, we performed gel filtration and hydrophobic interaction chromatography followed by mass spectrometry analysis. We identified several putative protein candidates that were further tested in in vitro experiments. We found that this pro-migratory factor chaperoned by fibrinogen is vitronectin, which activates uPAR, and that this effect can be inhibited by fibrinogen. These results provide a novel mechanism for the metastasis of cancer cells to lymphatics and body cavities, in which the concentration of fibrinogen is low, and thus suggests that free vitronectin stimulates migration of tumor cells.