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Publication
Journal: Analytical Biochemistry
July/23/1987
Abstract
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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Publication
Journal: Analytical Biochemistry
August/30/1987
Abstract
A fast and efficient method for the isolation of RNA from plant tissues is described. Tuber tissue is homogenized in a guanidine hydrochloride-containing buffer followed by direct extraction with phenol/chloroform. The RNA is precipitated from the aqueous phase, washed with 3 M sodium acetate and 70% ethanol, and finally dissolved in water. The yield of RNA is up to 500 micrograms/g of tissue and several tests indicate intact and nondegraded RNA. This method can be adapted to a small-scale version by the use of 1.5-ml tubes, allowing rapid isolation of RNA from a larger number of samples. Finally, this method is of particular use for isolating RNA from tissues with a high polysaccharide and nuclease content such as wounded potato tubers.
Publication
Journal: BioTechniques
November/25/1993
Abstract
This report describes a new method for simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. The method is based on the use of a reagent containing phenol and guanidine thiocyanate. A biological sample is homogenized in the reagent and the simultaneous isolation of RNA, DNA and proteins is accomplished in a single step by a liquid-phase separation. The isolation of RNA can be completed in about 1 h, and DNA and proteins in about 3 h. The simultaneously isolated RNA, DNA and proteins are ready for Northern, Southern and Western blotting. The complete recovery of DNA from samples used for the RNA and protein isolation makes it possible to normalize the results of gene expression studies based on DNA content instead of on the more variable total RNA, protein content or tissue weight.
Publication
Journal: Biochimica et Biophysica Acta - General Subjects
November/30/1996
Publication
Journal: Nature Protocols
September/20/2007
Abstract
Since its introduction, the 'single-step' method has become widely used for isolating total RNA from biological samples of different sources. The principle at the basis of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol and can be used for several applications. The original protocol, enabling the isolation of RNA from cells and tissues in less than 4 hours, greatly advanced the analysis of gene expression in plant and animal models as well as in pathological samples, as demonstrated by the overwhelming number of citations the paper gained over 20 years.
Publication
Journal: Journal of Immunology
August/2/2000
Abstract
Toll-like receptor (TLR) 2 has recently been associated with cellular responses to numerous microbial products, including LPS and bacterial lipoproteins. However, many preparations of LPS contain low concentrations of highly bioactive contaminants described previously as "endotoxin protein," suggesting that these contaminants could be responsible for the TLR2-mediated signaling observed upon LPS stimulation. To test this hypothesis, commercial preparations of LPS were subjected to a modified phenol re-extraction protocol to eliminate endotoxin protein. While it did not influence the ability to stimulate cells from wild-type mice, repurification eliminated the ability of LPS to activate cells from C3H/HeJ (Lpsd) mice. Additionally, only cell lines transfected with human TLR4, but not human or murine TLR2, acquired responsiveness to both re-extracted LPS and to a protein-free, synthetic preparation of lipid A. These results suggest that neither human nor murine TLR2 plays a role in LPS signaling in the absence of contaminating endotoxin protein.
Publication
Journal: Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)
April/30/2003
Publication
Journal: Journal of Clinical Pathology
April/30/2003
Publication
Journal: Environmental Health Perspectives
September/9/2008
Abstract
BACKGROUND
Many phthalates and phenols are hormonally active and are suspected to alter the course of development.
OBJECTIVE
We investigated prenatal exposures to phthalate and phenol metabolites and their associations with body size measures of the infants at birth.
METHODS
We measured 5 phenol and 10 phthalate urinary metabolites in a multiethnic cohort of 404 women in New York City during their third trimester of pregnancy and recorded size of infants at birth.
RESULTS
Median urinary concentrations were>> 10 microg/L for 2 of 5 phenols and 6 of 10 phthalate monoester metabolites. Concentrations of low-molecular-weight phthalate monoesters (low-MWP) were approximately 5-fold greater than those of high-molecular-weight metabolites. Low-MWP metabolites had a positive association with gestational age [0.97 day gestational age per ln-biomarker; 95% confidence interval (CI), 0.07-1.9 days, multivariate adjusted] and with head circumference. Higher prenatal exposures to 2,5-dichlorophenol (2,5-DCP) predicted lower birth weight in boys (-210 g average birth weight difference between the third tertile and first tertile of 2,5-DCP; 95% CI, 71-348 g). Higher maternal benzophenone-3 (BP3) concentrations were associated with a similar decrease in birth weight among girls but with greater birth weight in boys.
CONCLUSIONS
We observed a range of phthalate and phenol exposures during pregnancy in our population, but few were associated with birth size. The association of 2,5-DCP and BP3 with reduced or increased birth weight could be important in very early or small-size births. In addition, positive associations of urinary metabolites with some outcomes may be attributable partly to unresolved confounding with maternal anthropometric factors.
Publication
Journal: Applied and Environmental Microbiology
December/16/2002
Abstract
Identifying microorganisms responsible for recognized environmental processes remains a great challenge in contemporary microbial ecology. Only in the last few years have methodological innovations provided access to the relationship between the function of a microbial community and the phylogeny of the organisms accountable for it. In this study stable-isotope-labeled [13C]phenol was fed into a phenol-degrading community from an aerobic industrial bioreactor, and the 13C-labeled RNA produced was used to identify the bacteria responsible for the process. Stable-isotope-labeled RNA was analyzed by equilibrium density centrifugation in concert with reverse transcription-PCR and denaturing gradient gel electrophoresis. In contradiction with findings from conventional methodologies, this unique approach revealed that phenol degradation in the microbial community under investigation is dominated by a member of the Thauera genus. Our results suggest that this organism is important for the function of this bioreactor.
Publication
Journal: Biochimica et Biophysica Acta - General Subjects
April/5/1995
Publication
Journal: Journal of Proteome Research
September/22/2009
Abstract
Protein tyrosine nitration (PTN) is a post-translational modification occurring under the action of a nitrating agent. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group (NO2). In the present article, we review the main nitration reactions and elucidate why nitration is not a random chemical process. The particular physical and chemical properties of 3-nitrotyrosine (e.g., pKa, spectrophotometric properties, reduction to aminotyrosine) will be discussed, and the biological consequences of PTN (e.g., modification of enzymatic activity, sensitivity to proteolytic degradation, impact on protein phosphorylation, immunogenicity and implication in disease) will be reviewed. Recent data indicate the possibility of an in vivo denitration process, which will be discussed with respect to the different reaction mechanisms that have been proposed. The second part of this review article focuses on analytical methods to determine this post-translational modification in complex proteomes, which remains a major challenge.
Publication
Journal: Proceedings of the National Academy of Sciences of the United States of America
April/19/2006
Abstract
Ca(2+)-ATPase of sarcoplasmic reticulum is an ATP-powered Ca(2+) pump but also a H(+) pump in the opposite direction with no demonstrated functional role. Here, we report a 2.4-A-resolution crystal structure of the Ca(2+)-ATPase in the absence of Ca(2+) stabilized by two inhibitors, dibutyldihydroxybenzene, which bridges two transmembrane helices, and thapsigargin, also bound in the membrane region. Now visualized are water and several phospholipid molecules, one of which occupies a cleft between two transmembrane helices. Atomic models of the Ca(2+) binding sites with explicit hydrogens derived by continuum electrostatic calculations show how water and protons fill the space and compensate charge imbalance created by Ca(2+)-release. They suggest that H(+) countertransport is a consequence of a requirement for maintaining structural integrity of the empty Ca(2+)-binding sites. For this reason, cation countertransport is probably mandatory for all P-type ATPases and possibly accompanies transport of water as well.
Publication
Journal: Journal of Bone and Joint Surgery - Series A
June/2/2008
Abstract
BACKGROUND
The use of adjuvants after curettage has been well established for the treatment of giant cell tumor of bone. The purpose of this study was to analyze the rates of recurrence following different types of treatment as well as the influence of various factors of tumor presentation on those rates.
METHODS
The data regarding benign giant cell tumors of the appendicular skeleton from ten bone tumor centers were evaluated. Axial and malignant tumors were excluded. The recurrence rates associated with the different treatment modalities were analyzed, and hazard ratios for a recurrence were calculated for multiple factors of tumor presentation.
RESULTS
The study included 384 surgical procedures, involving 256 primary and 128 recurrent tumors. The mean duration of follow-up was 64.2 months. Wide excision was performed in seventy-eight cases (20.3%), and an intralesional procedure was done in 306 (79.7%). Of the intralesional procedures, 103 (33.7%) were performed without the use of adjuvants, 102 (33.3%) included filling with polymethylmethacrylate, seventy-four (24.2%) included polymethylmethacrylate filling after phenolization, and twenty-seven (8.8%) included use of local toxins. The overall recurrence rate after the intralesional procedures was 49% when no adjuvants had been used, 22% when polymethylmethacrylate only had been used as an adjuvant, 27% when polymethylmethacrylate had been used after phenolization, and 15% when phenol or other local toxins had been used (without polymethylmethacrylate). The highest rate of recurrence (36%) after curettage with adjuvants was associated with extracompartmental tumors. Recurrent tumors were not at increased risk for another recurrence, even when they were extracompartmental. The recurrence rate following curettage of a primary tumor without the use of adjuvants (55%) was higher than that following the same treatment of a recurrent tumor (39%) (p = 0.033).
CONCLUSIONS
Use of polymethylmethacrylate as an adjuvant significantly reduces the recurrence rate following intralesional treatment of benign giant cell tumors, and it appears to be the therapy of choice for primary as well as recurrent giant cell tumors of bone. The significantly better results following treatment of recurrent tumors without adjuvants compared with the results of the same treatment of primary tumors were probably related to increased surgical thoroughness brought about by the surgeon's awareness of dealing with a riskier tumor.
Publication
Journal: Applied and Environmental Microbiology
August/2/1987
Abstract
Biodegradation of trichloroethylene (TCE) by bacterial strain G4 resulted in complete dechlorination of the compound, as indicated by the production of inorganic chloride. A component of the water from which strain G4 was isolated that was required for TCE degradation was identified as phenol. Strain G4 degraded TCE in the presence of chloramphenicol only when preinduced with phenol. Toluene, o-cresol. and m-cresol could replace the phenol requirement. Two of the inducers of TCE metabolism, phenol and toluene, apparently induced the same aromatic degradative pathway that cleaved the aromatic ring by meta fission. Cells induced with either phenol or toluene had similar oxidation rates for several aromatic compounds and had similar levels of catechol-2,3-dioxygenase. The results indicate that one or more enzymes of an inducible pathway for aromatic degradation in strain G4 are responsible for the degradation of TCE.
Publication
Journal: Applied and Environmental Microbiology
August/5/1992
Abstract
A trans unsaturated fatty acid was found as a major constituent in the lipids of Pseudomonas putida P8. The fatty acid was identified as 9-trans-hexadecenoic acid by gas chromatography, argentation thin-layer chromatography, and infrared absorption spectrometry. Growing cells of P. putida P8 reacted to the presence of sublethal concentrations of phenol in the medium with changes in the fatty acid composition of the lipids, thereby increasing the degree of saturation. At phenol concentrations which completely inhibited the growth of P. putida, the cells were still able to increase the content of the trans unsaturated fatty acid and simultaneously to decrease the proportion of the corresponding 9-cis-hexadecenoic acid. This conversion of fatty acids was also induced by 4-chlorophenol in nongrowing cells in which the de novo synthesis of lipids had stopped, as shown by incorporation experiments with labeled acetate. The isomerization of the double bond in the presence of chloramphenicol indicates a constitutively operating enzyme system. The cis-to-trans modification of the fatty acids studied here apparently is a new way of adapting the membrane fluidity to the presence of phenols, thereby compensating for the elevation of membrane permeability induced by these toxic substances.
Publication
Journal: Applied and Environmental Microbiology
June/20/1990
Abstract
Intact cells of Pseudomonas cepacia G4 completely degraded trichloroethylene (TCE) following growth with phenol. Degradation kinetics were determined for both phenol, used to induce requisite enzymes, and TCE, the target substrate. Apparent Ks and Vmax values for degradation of phenol by cells were 8.5 microM and 466 nmol/min per mg of protein, respectively. At phenol concentrations greater than 50 microM, phenol degradation was inhibited, yielding an apparent second-order inhibitory value, KSI, of 0.45 mM as modeled by the Haldane expression. A partition coefficient for TCE was determined to be 0.40 +/- 0.02, [TCEair]/[TCEwater], consistent with Henry's law. To eliminate experimental problems associated with TCE volatility and partitioning, a no-headspace bottle assay was developed, allowing for direct and accurate determinations of aqueous TCE concentration. By this assay procedure, apparent Ks and Vmax values determined for TCE degradation by intact cells were 3 microM and 8 nmol/min per mg of protein, respectively. Following a transient lag period, P. cepacia G4 degraded TCE at concentrations of at least 300 microM with no apparent retardation in rate. Consistent with Ks values determined for degradation, TCE significantly inhibited phenol degradation.
Publication
Journal: Cell
April/28/1997
Abstract
The Lls1 (lethal leaf spot1) locus of maize is defined by a recessive mutation characterized by the initiation, in a developmentally programmed manner, of necrotic lesions that expand to kill leaves cell autonomously. The loss-of-function nature of all Lls1 mutants implies that the Lls1 gene is required to limit the spread of cell death in mature leaves. We have cloned the Lls1 gene by tagging with Mutator, a transposable element system in maize, and we show that it encodes a novel protein highly conserved in plants. Two consensus binding motifs of aromatic ring-hydroxylating dioxygenases are present in the predicted LLS1 protein, suggesting that it may function to degrade a phenolic mediator of cell death.
Publication
Journal: Journal of Chemical Physics
July/26/2007
Abstract
Alchemical free energy calculations are becoming a useful tool for calculating absolute binding free energies of small molecule ligands to proteins. Here, we find that the presence of multiple metastable ligand orientations can cause convergence problems when distance restraints alone are used. We demonstrate that the use of orientational restraints can greatly accelerate the convergence of these calculations. However, even with this acceleration, we find that sufficient sampling requires substantially longer simulations than are used in many published protocols. To further accelerate convergence, we introduce a new method of configuration space decomposition by orientation which reduces required simulation lengths by at least a factor of 5 in the cases examined. Our method is easily parallelizable, well suited for cases where a ligand cocrystal structure is not available, and can utilize initial orientations generated by docking packages.
Publication
Journal: Journal of Biological Chemistry
April/28/2013
Abstract
The roots of plants have the ability to influence its surrounding microbiology, the so-called rhizosphere microbiome, through the creation of specific chemical niches in the soil mediated by the release of phytochemicals. Here we report how these phytochemicals could modulate the microbial composition of a soil in the absence of the plant. For this purpose, root exudates of Arabidopsis were collected and fractionated to obtain natural blends of phytochemicals at various relative concentrations that were characterized by GC-MS and applied repeatedly to a soil. Soil bacterial changes were monitored by amplifying and pyrosequencing the 16 S ribosomal small subunit region. Our analyses reveal that one phytochemical can culture different operational taxonomic units (OTUs), mixtures of phytochemicals synergistically culture groups of OTUs, and the same phytochemical can act as a stimulator or deterrent to different groups of OTUs. Furthermore, phenolic-related compounds showed positive correlation with a higher number of unique OTUs compared with other groups of compounds (i.e. sugars, sugar alcohols, and amino acids). For instance, salicylic acid showed positive correlations with species of Corynebacterineae, Pseudonocardineae and Streptomycineae, and GABA correlated with species of Sphingomonas, Methylobacterium, Frankineae, Variovorax, Micromonosporineae, and Skermanella. These results imply that phenolic compounds act as specific substrates or signaling molecules for a large group of microbial species in the soil.
Publication
Journal: Current Protocols in Molecular Biology
March/9/2008
Abstract
This unit provides two protocols for extraction of RNA from yeast that differ primarily in the method for lysing the yeast cells. The first protocol isolates RNA directly from intact yeast cells by extraction with hot acidic phenol. This yields RNA that is relatively free of contaminating DNA, is convenient to perform with multiple samples, and gives little or no sample-to-sample variation. In contrast, an alternate protocol relies upon disruption of cells by vigorous mixing with glass beads and denaturing agents. Although this procedure results in efficient breaking of the cells, the product is associated with residual DNA, and the procedure itself is troublesome when one is working with multiple samples. A second alternate protocol describes the scaling up of the first two procedures to isolate enough total RNA for poly (A)+ RNA preparation.
Publication
Journal: European journal of biochemistry
April/2/1984
Abstract
The phenol-phase soluble cellular lipopolysaccharide isolated by the phenol/water extraction method from Yersinia enterocolitica serotype O:9 cells was shown by hydrolytic, periodate oxidation, methylation and nuclear magnetic resonance studies to be an S-type lipopolysaccharide with a linear O-antigenic polysaccharide of 1,2-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units. The serological cross-reactivity between Y. enterocolitica serotype O:9 and the lipopolysaccharides of Vibrio cholerae and Brucella species can now be related to the presence of N-acylated 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl residues in their respective O-antigenic chains.
Publication
Journal: FEBS Letters
April/24/2006
Abstract
Plants produce a large number of secondary metabolites, such as alkaloids, terpenoids, polyphenols, quinones and many further compounds having combined structures of those groups. Physiological roles of those metabolites for plants are still under investigation, but they play, at least in part, important functions as protectants for plant bodies against herbivores and pathogens, as well as from physical stresses like ultraviolet light and heat. In order to accomplish these functions, biosyntheses and accumulation of secondary metabolites are highly regulated in a temporal and spatial manner in plant organs, where they can appropriately accumulate. In this mini-review, I introduce the mechanism of accumulation and membrane transport of these metabolites, in particular, focusing on ATP-binding cassette transporters involved.
Publication
Journal: Nature
May/10/1989
Abstract
SINCE insulin was first shown by Scott to crystallize in the presence of zinc ions in 1934, a variety of Zn-containing insulin crystals have been grown. The structures of insulin in the related rhombohedral crystals of 2Zn-insulin and 4Zn-insulin have been solved and reveal that the molecule is a hexamer, organized as three dimers, each containing a 2-fold symmetry axis and held together by Zn ions. In 2Zn-insulin the hexamer is nearly symmetrical with the two axial Zn ions and the two molecules of the dimer related closely by a local 2-fold axis. But in 4Zn-insulin the two molecules in the dimer differ remarkably, creating an asymmetric 4Zn-hexamer in which one trimer is essentially equivalent to that in 2Zn-insulin and the other is different by virtue of an additional stretch of N-terminal helix between residues B1 and B8 (refs 6, 7). We report here the structure of a new symmetrical hexamer, in which all six molecules have the B1-B8 helix seen in 4Zn-insulin. Phenol molecules, found bonding specifically to each molecule, evidently stabilize this new helical conformation.
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