Preparation of nuclear matrices from cultured cells: subfractionation of nuclei in situ

Analyses of the different structural systems of the nucleus and the proteins associated with them pose many problems. Because these systems are largely overlapping, in situ localization studies that preserve the in vivo location of proteins and cellular structures often are not satisfactory. In contrast, biochemical cell fractionation may provide artifactual results due to cross-contamination of extracts and structures. To overcome these problems, we have developed a method that combines biochemical cell fractionation and in situ localization and leads to the preparation of a residual cellular skeleton (nuclear matrix and cytoskeletal elements) from cultured cells. This method's main feature is that cell fractionation is performed in situ. Therefore, structures not solubilized in a particular extraction step remain attached to the substrate and retain their morphology. Before and after each extraction step they can be analyzed for the presence and location of the protein under study by using immunological or cytochemical techniques. Thereby the in vivo origin of a protein solubilized in a particular extraction step is determined. The solubilized protein then may be further characterized biochemically. In addition, to allow analyses of proteins associated with the residual cellular skeleton, we have developed conditions for its solubilization that do not interfere with enzymatic and immunological studies

Biases in the Experimental Annotations of Protein Function and their Effect on Our Understanding of Protein Function Space

The ongoing functional annotation of proteins relies upon the work of curators to capture experimental findings from scientific literature and apply them to protein sequence and structure data. However, with the increasing use of high-throughput experimental assays, a small number of experimental studies dominate the functional protein annotations collected in databases. Here we investigate just how prevalent is the "few articles -- many proteins" phenomenon. We examine the experimentally validated annotation of proteins provided by several groups in the GO Consortium, and show that the distribution of proteins per published study is exponential, with 0.14% of articles providing the source of annotations for 25% of the proteins in the UniProt-GOA compilation. Since each of the dominant articles describes the use of an assay that can find only one function or a small group of functions, this leads to substantial biases in what we know about the function of many proteins. Mass-spectrometry, microscopy and RNAi experiments dominate high throughput experiments. Consequently, the functional information derived from these experiments is mostly of the subcellular location of proteins, and of the participation of proteins in embryonic developmental pathways. For some organisms, the information provided by different studies overlap by a large amount. We also show that the information provided by high throughput experiments is less specific than those provided by low throughput experiments. Given the experimental techniques available, certain biases in protein function annotation due to high-throughput experiments are unavoidable. Knowing that these biases exist and understanding their characteristics and extent is important for database curators, developers of function annotation programs, and anyone who uses protein function annotation data to plan experiments.Comment: Accepted to PLoS Computational Biology. Press embargo applies. v4: text corrected for style and supplementary material inserte

Positionally dependent ^(15)N fraction factors in the UV photolysis of N_2O determined by high resolution FTIR spectroscopy

Positionally dependent fractionation factors for the photolysis of isotopomers of N_2O in natural abundance have been determined by high resolution FTIR spectroscopy at three photolysis wavelengths. Fractionation factors show clear 15N position and photolysis wavelength dependence and are in qualitative agreement with theoretical models but are twice as large. The fractionation factors increase with photolysis wavelength from 193 to 211 nm, with the fractionation factors at 207.6 nm for ^(14)N^(15)N^916)O, ^(15)N^(14)N^(16)O and ^(14)N^(14)N^(18)O equal to −66.5±5‰,−27.1±6‰ and −49±10‰, respectively

Kinetic study of pilot-scale supercritical CO2 extraction of rosemary (Rosmarinus officinalis) leaves

NOTICE: This is the author’s version of a work that was accepted for publication in Journal of Supercritical Fluids. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Supercritical Fluids, 55 (2011). http://dx.doi.org/10.1016/j.supflu.2010.09.030Rosmarinus officinalis (rosemary) extracts were obtained in a supercritical pilot-scale plant. Based on experimental information available in the literature for analytical or low-scale processes, extraction temperature and pressure were selected to be 313 K and 30 MPa. At these extraction conditions, the kinetic behavior of the pilot-scale overall extraction curve were determined with respect to yield, antioxidant activity and carnosic acid content. The overall extraction curve was represented using Sovova’s model; the average deviation between measured and calculated yields was lower than 2%. Mass transfer coefficients in the fluid and solid phases were determined and were compared with previous data reported in the literature for low-scale rosemary supercritical extraction. A two-stage depressurization procedure was accomplished and the effect of both on-line fractionation and extraction time on the antioxidant activity of the samples collected was studied. The antioxidant activity of the different fractions could be straight correlated with the carnosic acid content with a regression coefficient of 0.92This work has been financed by Universidad Autónoma de Madrid and Comunidad Autónoma de Madrid (ALIBIRD-S2009/AGR-1469) and project FUN-C-FOOD, CSD2007-00063 (CONSOLIDER-INGENIO) from Ministerio de Ciencia e Innovación, Spain

Can Mg isotopes be used to trace cyanobacteria-mediated magnesium carbonate precipitation in alkaline lakes?

The fractionation of Mg isotopes was determined during the cyanobacterial mediated precipitation of hydrous magnesium carbonate precipitation in both natural environments and in the laboratory. Natural samples were obtained from Lake Salda (SE Turkey), one of the few modern environments on the Earth's surface where hydrous Mg-carbonates are the dominant precipitating minerals. This precipitation was associated with cyanobacterial stromatolites which were abundant in this aquatic ecosystem. Mg isotope analyses were performed on samples of incoming streams, groundwaters, lake waters, stromatolites, and hydromagnesite-rich sediments. Laboratory Mg carbonate precipitation experiments were conducted in the presence of purified Synechococcus sp cyanobacteria that were isolated from the lake water and stromatolites. The hydrous magnesium carbonates nesquehonite (MgCO3·3H2O) and dypingite (Mg5(CO3)4(OH)25(H2O)) were precipitated in these batch reactor experiments from aqueous solutions containing either synthetic NaHCO3/MgCl2 mixtures or natural Lake Salda water, in the presence and absence of live photosynthesizing Synechococcus sp. Bulk precipitation rates were not to affected by the presence of bacteria when air was bubbled through the system. In the stirred non-bubbled reactors, conditions similar to natural settings, bacterial photosynthesis provoked nesquehonite precipitation, whilst no precipitation occurred in bacteria-free systems in the absence of air bubbling, despite the fluids achieving a similar or higher degree of supersaturation. The extent of Mg isotope fractionation (?26Mgsolid-solution) between the mineral and solution in the abiotic experiments was found to be identical, within uncertainty, to that measured in cyanobacteria-bearing experiments, and ranges from ?1.4 to ?0.7 ‰. This similarity refutes the use of Mg isotopes to validate microbial mediated precipitation of hydrous Mg carbonate

Circular DNA's from HeLa cell nuclei and mitochondria

An electron microscopic observation was made on the DNA's extracted from purified HeLa cell nuclei, mitochondria, and the whole cell, and fractionated by ethidium bromide-cesium chloride density gradient method or sucrose density gradient method. Nuclear DNA presents mainly long linear DNA derived from fragmented chromosomal DNA. In addition to this, the existence of small circular DNA molecules measuring 0.32 -1.78 &#956;, was confirmed. Mitochondrial DNA was mainly circular DNA, which measured 4.87 &#956; in the mean value of the contour lengths in the highest frequency group, and small circular DNA molecules, measuring 0.3-1.01 &#956; in contour length, were also found in an extremely low frequency.</p