Morphological characterization by microscopy remains the gold standard for accurately identifying apoptotic cells using characteristics such as nuclear condensation, nuclear fragmentation, and membrane blebbing. However, quantitative measurement of apoptotic morphology using microscopy can be time consuming and can lack objectivity and reproducibility, making it difficult to identify subtle changes in large populations. Thus the apoptotic index of a sample is commonly measured by flow cytometry using a variety of fluorescence intensity based (photometric) assays which target hallmarks of apoptosis with secondary markers such as the TUNEL (Terminal Deoxynucleotide Transferase dUTP Nick End Labeling) assay for detection of DNA fragmentation, the Annexin V assay for surface phosphatidylserine (PS) exposure, and fluorogenic caspase substrates to detect caspase activation. Here a novel method is presented for accurate quantitation of apoptosis based on nuclear condensation, nuclear fragmentation, and membrane blebbing using automated image analysis on large numbers of images collected in flow by the ImageStream multispectral imaging cytometer. Additionally the measurement of nuclear fragmentation correlates with the secondary methods of detection of apoptosis over time, indicating that it is also an early marker for apoptosis. False-positive and false-negative events associated with each photometric flow cytometry based method are quantitated and can be automatically removed/included where appropriate. Acquisition of multi-spectral imagery on large numbers of cells couples the quantitative advantage of flow cytometry with the accuracy of morphology-based algorithms allowing more complete and robust analysis of apoptosis.
Methane monooxygenase (MMO) enzymes catalyze the oxidation of methane to methanol in methanotrophic bacteria. Several strains of methanotrophs, including Methylococcus capsulatus (Bath), express a membrane-bound or particulate MMO (pMMO) at high copper-to-biomass ratios and a soluble MMO (sMMO) form when copper is limited. The mechanism of this "copper switch" is not understood. The mmoS gene, located downstream of the sMMO operon, encodes a sensor protein that is part of a two-component signaling system and has been proposed to play a role in the copper switch. MmoS from M. capsulatus (Bath) has been cloned, expressed, and purified. The purified protein is a tetramer of molecular mass 480 kDa. Optical spectra indicate that MmoS contains a flavin cofactor, identified as flavin adenine dinucleotide (FAD) by fluorescence spectroscopy and chromatographic analysis. The redox potential of the MmoS-bound FAD, which binds within the N-terminal PAS-PAC domains, is -290 +/- 2 mV at pH 8.0 and 25 degrees C. Despite extensive efforts, MmoS could not be loaded with Cu(I) or Cu(II), indicating that MmoS does not sense copper directly. These data suggest that MmoS functions as a redox sensor and provide new insight into the copper-mediated regulation of sMMO expression.
Titanium dioxide and a 100 W mercury spotlamp were used to photoreduce 100 ppm Hg aqueous mercuric chloride solutions. The solution's basicity and temperature were varied. Two optimum photoreduction conditions were determined: pH 9, 0 °C and pH 11, 40 °C. TiO 2 -assisted photoreduction at these two conditions lowered the concentration of mercury left in the solution to below 200 ppb. Methodology was developed to perform an overall mercury mass balance on the process. The overall mercury balance revealed that more than 97% (average 103% ( 6%) of the mercury removed from solution was deposited as mercury metal on the surface of the TiO 2 for the pH 9, 0 °C treatment conditions. This mercury could be driven off the TiO 2 surface by heating to 100 °C for half of an hour under nitrogen flow. The pH and temperature information under light and dark conditions is consistent with a pH-dependent adsorption of a dissociated mercuric species by hydroxide ions on the TiO 2 surface followed by nucleation of the reduced species. The TiO 2 assisted photoreduction process shows promise for remediation of mercuric waste below the EPA 200 ppb mercury disposal limit as well as the potential for recycling the mercury and TiO 2 catalyst.
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