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Summary: Green fluorescent protein
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Green fluorescent protein Edit Wikipedia article
|Green fluorescent protein
Structure of the Aequorea victoria green fluorescent protein.[1]
|Pfam clan
The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues (26.9 kDa) that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range.[2][3] Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower green portion of the visible spectrum. The fluorescence quantum yield (QY) of GFP is 0.79. The GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm. GFP makes for an excellent tool in many forms of biology due to its ability to form internal chromophore without requiring any accessory cofactors, gene products, or enzymes / substrates other than molecular oxygen.[4]
In cell and molecular biology, the GFP gene is frequently used as a reporter of expression.[5] It has been used in modified forms to make biosensors, and many animals have been created that express GFP, which demonstrates a proof of concept that a gene can be expressed throughout a given organism, in selected organs, or in cells of interest. GFP can be introduced into animals or other species through transgenic techniques, and maintained in their genome and that of their offspring. To date, GFP has been expressed in many species, including bacteria, yeasts, fungi, fish and mammals, including in human cells. Scientists Roger Y. Tsien, Osamu Shimomura, and Martin Chalfie were awarded the 2008 Nobel Prize in Chemistry on 10 October 2008 for their discovery and development of the green fluorescent protein.
Wild-type GFP (wtGFP)
In the 1960s and 1970s, GFP, along with the separate luminescent protein aequorin (an enzyme that catalyzes the breakdown of luciferin, releasing light), was first purified from Aequorea victoria and its properties studied by Osamu Shimomura.[6] In A. victoria, GFP fluorescence occurs when aequorin interacts with Ca2+ ions, inducing a blue glow. Some of this luminescent energy is transferred to the GFP, shifting the overall color towards green.[7] However, its utility as a tool for molecular biologists did not begin to be realized until 1992 when Douglas Prasher reported the cloning and nucleotide sequence of wtGFP in Gene.[8] The funding for this project had run out, so Prasher sent cDNA samples to several labs. The lab of Martin Chalfie expressed the coding sequence of wtGFP, with the first few amino acids deleted, in heterologous cells of E. coli and C. elegans, publishing the results in Science in 1994.[9] Frederick Tsuji's lab independently reported the expression of the recombinant protein one month later.[10] Remarkably, the GFP molecule folded and was fluorescent at room temperature, without the need for exogenous cofactors specific to the jellyfish. Although this near-wtGFP was fluorescent, it had several drawbacks, including dual peaked excitation spectra, pH sensitivity, chloride sensitivity, poor fluorescence quantum yield, poor photostability and poor folding at 37 °C.
The first reported crystal structure of a GFP was that of the S65T mutant by the Remington group in Science in 1996.[11] One month later, the Phillips group independently reported the wild-type GFP structure in Nature Biotechnology.[12] These crystal structures provided vital background on chromophore formation and neighboring residue interactions. Researchers have modified these residues by directed and random mutagenesis to produce the wide variety of GFP derivatives in use today. Further research into GFP has shown that is resistant to detergents, proteases, guanidinium chloride (GdmCl) treatments, and drastic temperature changes.[13]
GFP derivatives
Due to the potential for widespread usage and the evolving needs of researchers, many different mutants of GFP have been engineered.[14][15] The first major improvement was a single point mutation (S65T) reported in 1995 in Nature by Roger Tsien.[16] This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostability, and a shift of the major excitation peak to 488 nm, with the peak emission kept at 509 nm. This matched the spectral characteristics of commonly available FITC filter sets, increasing the practicality of use by the general researcher. A 37 °C folding efficiency (F64L) point mutant to this scaffold, yielding enhanced GFP (EGFP), was discovered in 1995 by the laboratories of Thastrup[17] and Falkow.[18] EGFP allowed the practical use of GFPs in mammalian cells. EGFP has an extinction coefficient (denoted ε) of 55,000 M−1cm−1.[19] The fluorescence quantum yield (QY) of EGFP is 0.60. The relative brightness, expressed as ε•QY, is 33,000 M−1cm−1. Superfolder GFP, a series of mutations that allow GFP to rapidly fold and mature even when fused to poorly folding peptides, was reported in 2006.[20]
Many other mutations have been made, including color mutants; in particular, blue fluorescent protein (EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein (ECFP, Cerulean, CyPet, mTurquoise2), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet). BFP derivatives (except mKalama1) contain the Y66H substitution.They exhibit a broad absorption band in the ultraviolet centered close to 380 nanometers and an emission maximum at 448 nanometers. A green fluorescent protein mutant (BFPms1) that preferentially binds Zn(II) and Cu(II) has been developed. BFPms1 have several important mutations including and the BFP chromophore (Y66H),Y145F for higher quantum yield, H148G for creating a hole into the beta-barrel and several other mutations that increase solubility. Zn(II) binding increases fluorescence intensity, while Cu(II) binding quenches fluorescence and shifts the absorbance maximum from 379 to 444 nm. Therefore, they can be used as Zn biosensor.[21]
The critical mutation in cyan derivatives is the Y66W substitution, which causes the chromophore to form with an indole rather than phenol component. Several additional compensatory mutations in the surrounding barrel are required to restore brightness to this modified chromophore due to the increased bulk of the indole group. In ECFP and Cerulean, the N-terminal half of the seventh strand exhibits two conformations. These conformations both have a complex set of van der Waals interactions with the chromophore. The Y145A and H148D mutations in Cerulean stabilize these interactions and allow the chromophore to be more planar, better packed, and less prone to collisional quenching.[22] Additional site-directed random mutagenesis in combination with fluorescence lifetime based screening has further stabilized the seventh β-strand resulting in a bright variant, mTurquoise2, with a quantum yield (QY) of 0.93.[23] The red-shifted wavelength of the YFP derivatives is accomplished by the T203Y mutation and is due to π-electron stacking interactions between the substituted tyrosine residue and the chromophore.[3] These two classes of spectral variants are often employed for Förster resonance energy transfer (FRET) experiments. Genetically encoded FRET reporters sensitive to cell signaling molecules, such as calcium or glutamate, protein phosphorylation state, protein complementation, receptor dimerization, and other processes provide highly specific optical readouts of cell activity in real time.
Semirational mutagenesis of a number of residues led to pH-sensitive mutants known as pHluorins, and later super-ecliptic pHluorins. By exploiting the rapid change in pH upon synaptic vesicle fusion, pHluorins tagged to synaptobrevin have been used to visualize synaptic activity in neurons.[24]
Redox sensitive versions of GFP (roGFP) were engineered by introduction of cysteines into the beta barrel structure. The redox state of the cysteines determines the fluorescent properties of roGFP.[25]
The nomenclature of modified GFPs is often confusing due to overlapping mapping of several GFP versions onto a single name. For example, mGFP often refers to a GFP with an N-terminal palmitoylation that causes the GFP to bind to cell membranes. However, the same term is also used to refer to monomeric GFP, which is often achieved by the dimer interface breaking A206K mutation.[26] Wild-type GFP has a weak dimerization tendency at concentrations above 5 mg/mL. mGFP also stands for "modified GFP," which has been optimized through amino acid exchange for stable expression in plant cells.
In nature
The purpose of both the (primary) bioluminescence (from aequorin's action on luciferin) and the (secondary) fluorescence of GFP in jellyfish is unknown. GFP is co-expressed with aequorin in small granules around the rim of the jellyfish bell. The secondary excitation peak (480 nm) of GFP does absorb some of the blue emission of aequorin, giving the bioluminescence a more green hue. The serine 65 residue of the GFP chromophore is responsible for the dual-peaked excitation spectra of wild-type GFP. It is conserved in all three GFP isoforms originally cloned by Prasher. Nearly all mutations of this residue consolidate the excitation spectra to a single peak at either 395 nm or 480 nm. The precise mechanism of this sensitivity is complex, but, it seems, involves donation of a hydrogen from serine 65 to glutamate 222, which influences chromophore ionization.[3] Since a single mutation can dramatically enhance the 480 nm excitation peak, making GFP a much more efficient partner of aequorin, A. victoria appears to evolutionarily prefer the less-efficient, dual-peaked excitation spectrum. Roger Tsien has speculated that varying hydrostatic pressure with depth may affect serine 65's ability to donate a hydrogen to the chromophore and shift the ratio of the two excitation peaks. Thus, the jellyfish may change the color of its bioluminescence with depth. However, a collapse in the population of jellyfish in Friday Harbor, where GFP was originally discovered, has hampered further study of the role of GFP in the jellyfish's natural environment.
Other fluorescent proteins
Because of the great variety of engineered GFP derivatives, fluorescent proteins that belong to a different family, such as the bilirubin-inducible fluorescent protein UnaG, dsRed, eqFP611, Dronpa, TagRFPs, KFP, EosFP, Dendra, IrisFP and many others, are erroneously referred to as GFP derivatives. Several of these proteins display unique properties like red-shifted emission above 600 nm or photoconversion from a green-emitting state to a red-emitting state. These properties are so far unique to fluorescent proteins other than GFP derivatives.
FMN-binding fluorescent proteins (FbFPs) were developed in 2007[27] and are a class of small (11-16 kDa), oxygen-independent fluorescent proteins that are derived from blue-light receptors. They are intended especially for the use under anaerobic or hypoxic conditions, since the formation and binding of the Flavin chromophore does not require molecular oxygen, as it is the case with the synthesis of the GFP chromophore.
A new class of fluorescent protein was evolved from a cyanobacterial (Trichodesmium erythraeum) phycobiliprotein, α-allophycocyanin, and named small ultra red fluorescent protein (smURFP) in 2016. smURFP autocatalytically self-incorporates the chromophore biliverdin without the need of an external protein, known as a lyase.[28] Jellyfish- and coral-derived fluorescent proteins require oxygen and produce a stoichiometric amount of hydrogen peroxide upon chromophore formation.[29] smURFP does not require oxygen or produce hydrogen peroxide and uses the chromophore, bliverdin. smURFP has a large extinction coefficient (180,000 M−1 cm−1) and has a modest quantum yield (0.20), which makes it comparable biophysical brightness to eGFP and ~2-fold brighter than most red or far-red fluorescent proteins derived from coral. smURFP spectral properties are similar to the organic dye Cy5.[28]
A review of new classes of fluorescent proteins and applications can be found in Trends in Biochemical Sciences.[30]
GFP has a beta barrel structure consisting of eleven β-strands with a pleated sheet arrangement, with an alpha helix containing the covalently bonded chromophore 4-(p-hydroxybenzylidene)imidazolidin-5-one (HBI) running through the center.[3][11][12] Five shorter alpha helices form caps on the ends of the structure. The beta barrel structure is a nearly perfect cylinder, 42Å long and 24Å in diameter (some studies have reported a diameter of 30Å[13]),[11] creating what is referred to as a "β-can" formation, which is unique to the GFP-like family.[12] HBI, the spontaneously modified form of the tripeptide Ser65–Tyr66–Gly67, is nonfluorescent in the absence of the properly folded GFP scaffold and exists mainly in the un-ionized phenol form in wtGFP.[31] Inward-facing sidechains of the barrel induce specific cyclization reactions in Ser65–Tyr66–Gly67 that induce ionization of HBI to the phenolate form and chromophore formation. This process of post-translational modification is referred to as maturation.[32] The hydrogen-bonding network and electron-stacking interactions with these sidechains influence the color, intensity and photostability of GFP and its numerous derivatives.[33] The tightly packed nature of the barrel excludes solvent molecules, protecting the chromophore fluorescence from quenching by water. In addition to the auto-cyclization of the Ser65-Tyr66-Gly67, a 1,2-dehydrogenation reaction occurs at the Tyr66 residue.[13] Besides the three residues that form the chromophore, residues such as Gln94, Arg96, His148, Thr203, and Glu222 all act as stabilizers. The residues of Gln94, Arg96, and His148 are able to stabilize by delocalizing the chromophore charge. Arg96 is the most important stabilizing residue due to the fact that it prompts the necessary structural realignments that are necessary from the HBI ring to occur. Any mutation to the Arg96 residue would result in a decrease in the development rate of the chromophore because proper electrostatic and steric interactions would be lost. Tyr66 is the recipient of hydrogen bonds and does not ionize in order to produce favorable electrostatics.[34]
Reporter assays
Green fluorescent protein may be used as a reporter gene.[35][36]
For example, GFP can be used as a reporter for environmental toxicity levels. This protein has been shown to be an effective way to measure the toxicity levels of various chemicals including ethanol, p-formaldehyde, phenol, triclosan, and paraben. GFP is great as a reporter protein because of the fact that it has not effect on the host once it is introduced to its cellular environment of the host. Due to this ability, no external visualization stain, ATP, or cofactors are needed. With regards to pollutant levels, the fluorescence was measured in order to gauge the effect that the pollutants were have on the host cell. The cellular density of the host cell was also measured. Results from the study conducted by Song, Kim, & Seo (2016) showed that there was a decrease in both fluorescence and cellular density as pollutant levels increased. This was indicative of the fact that cellular activity had decreased. More research into this specific application in order to determine the mechanism by which GFP acts as a pollutant marker.[37] Similar results have been observed in zebrafish because zebrafish that were injected with GFP were approximately twenty times more susceptible to recognize cellular stresses than zebrafish that were not injected with GFP.[38]
The biggest advantage of GFP is that it can be heritable, depending on how it was introduced, allowing for continued study of cells and tissues it is expressed in. Visualizing GFP is noninvasive, requiring only illumination with blue light. GFP alone does not interfere with biological processes, but when fused to proteins of interest, careful design of linkers is required to maintain the function of the protein of interest. Moreover, if used with a monomer it is able to diffuse readily throughout cells.[39]
Fluorescence microscopy
The availability of GFP and its derivatives has thoroughly redefined fluorescence microscopy and the way it is used in cell biology and other biological disciplines.[40] While most small fluorescent molecules such as FITC (fluorescein isothiocyanate) are strongly phototoxic when used in live cells, fluorescent proteins such as GFP are usually much less harmful when illuminated in living cells. This has triggered the development of highly automated live-cell fluorescence microscopy systems, which can be used to observe cells over time expressing one or more proteins tagged with fluorescent proteins. For example, GFP had been widely used in labelling the spermatozoa of various organisms for identification purposes as in Drosophila melanogaster, where expression of GFP can be used as a marker for a particular characteristic. GFP can also be expressed in different structures enabling morphological distinction. In such cases, the gene for the production of GFP is incorporated into the genome of the organism in the region of the DNA that codes for the target proteins and that is controlled by the same regulatory sequence; that is, the gene's regulatory sequence now controls the production of GFP, in addition to the tagged protein(s). In cells where the gene is expressed, and the tagged proteins are produced, GFP is produced at the same time. Thus, only those cells in which the tagged gene is expressed, or the target proteins are produced, will fluoresce when observed under fluorescence microscopy. Analysis of such time lapse movies has redefined the understanding of many biological processes including protein folding, protein transport, and RNA dynamics, which in the past had been studied using fixed (i.e., dead) material. Obtained data are also used to calibrate mathematical models of intracellular systems and to estimate rates of gene expression.[41]
The Vertico SMI microscope using the SPDM Phymod technology uses the so-called "reversible photobleaching" effect of fluorescent dyes like GFP and its derivatives to localize them as single molecules in an optical resolution of 10 nm. This can also be performed as a co-localization of two GFP derivatives (2CLM).[42]
Another powerful use of GFP is to express the protein in small sets of specific cells. This allows researchers to optically detect specific types of cells in vitro (in a dish), or even in vivo (in the living organism).[43] Genetically combining several spectral variants of GFP is a useful trick for the analysis of brain circuitry (Brainbow).[44] Other interesting uses of fluorescent proteins in the literature include using FPs as sensors of neuron membrane potential,[45] tracking of AMPA receptors on cell membranes,[46] viral entry and the infection of individual influenza viruses and lentiviral viruses,[47][48] etc.
It has also been found that new lines of transgenic GFP rats can be relevant for gene therapy as well as regenerative medicine.[49] By using "high-expresser" GFP, transgenic rats display high expression in most tissues, and many cells that have not been characterized or have been only poorly characterized in previous GFP-transgenic rats.
GFP has been shown to be useful in cryobiology as a viability assay. Correlation of viability as measured by trypan blue assays were 0.97.[50] Another application is the use of GFP co-transfection as internal control for transfection efficiency in mammalian cells.[51]
A novel possible use of GFP includes using it as a sensitive monitor of intracellular processes via an eGFP laser system made out of a human embryonic kidney cell line. The first engineered living laser is made by an eGFP expressing cell inside a reflective optical cavity and hitting it with pulses of blue light. At a certain pulse threshold, the eGFP's optical output becomes brighter and completely uniform in color of pure green with a wavelength of 516 nm. Before being emitted as laser light, the light bounces back and forth within the resonator cavity and passes the cell numerous times. By studying the changes in optical activity, researchers may better understand cellular processes.[52][53]
GFP is used widely in cancer research to label and track cancer cells. GFP-labelled cancer cells have been used to model metastasis, the process by which cancer cells spread to distant organs.[54]
Macro-scale biological processes, such as the spread of virus infections, can be followed using GFP labeling.[55] In the past, mutagenic ultra violet light (UV) has been used to illuminate living organisms (e.g., see[56]) to detect and photograph the GFP expression. Recently, a technique using non-mutagenic LED lights[57] have been developed for macro-photography.[58] The technique uses an epifluorescence camera attachment[59] based on the same principle used in the construction of epifluorescence microscopes.
Transgenic pets
Alba, a green-fluorescent rabbit, was created by a French laboratory commissioned by Eduardo Kac using GFP for purposes of art and social commentary.[60] The US company Yorktown Technologies markets to aquarium shops green fluorescent zebrafish (GloFish) that were initially developed to detect pollution in waterways. NeonPets, a US-based company has marketed green fluorescent mice to the pet industry as NeonMice.[61] Green fluorescent pigs, known as Noels, were bred by a group of researchers led by Wu Shinn-Chih at the Department of Animal Science and Technology at National Taiwan University.[62] A Japanese-American Team created green-fluorescent cats as proof of concept to use them potentially as model organisms for diseases, particularly HIV.[63] In 2009 a South Korean team from Seoul National University bred the first transgenic beagles with fibroblast cells from sea anemones. The dogs give off a red fluorescent light, and they are meant to allow scientists to study the genes that cause human diseases like narcolepsy and blindness.[64]
Julian Voss-Andreae, a German-born artist specializing in "protein sculptures,"[65] created sculptures based on the structure of GFP, including the 1.70 m (5'6") tall "Green Fluorescent Protein" (2004)[66] and the 1.40 m (4'7") tall "Steel Jellyfish" (2006). The latter sculpture is located at the place of GFP's discovery by Shimomura in 1962, the University of Washington's Friday Harbor Laboratories.[67]
See also
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- ^ Pouwels LJ, Zhang L, Chan NH, Dorrestein PC, Wachter RM (Sep 2008). "Kinetic isotope effect studies on the de novo rate of chromophore formation in fast- and slow-maturing GFP variants". Biochemistry. 47 (38): 10111–22. doi:10.1021/bi8007164. PMC 2643082 . PMID 18759496.
- ^ Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA (Jul 2010). "Fluorescent proteins and their applications in imaging living cells and tissues". Physiological Reviews. 90 (3): 1103–63. doi:10.1152/physrev.00038.2009. PMID 20664080.
- ^ Stepaneko, O. V.; Verkhusha, V. V.; Shavlovsky, M. M.; Kuznetsova, I. M.; Uversky, V. N.; Turoverov, K. K. Understanding the role of Arg96 in structure and stability of green fluorescent protein. Proteins: Struct., Funct., Bioinf. 1999, 73 (3), 539-551.
- ^ Jugder, Bat-Erdene; Welch, Jeffrey; Braidy, Nady; Marquis, Christopher P. (2016-07-26). "Construction and use of aCupriavidus necatorH16 soluble hydrogenase promoter (PSH) fusion togfp(green fluorescent protein)". PeerJ. 4: e2269. doi:10.7717/peerj.2269. ISSN 2167-8359. PMC 4974937 . PMID 27547572.
- ^ Arun KH, Kaul CL, Ramarao P (2005). "Green fluorescent proteins in receptor research: an emerging tool for drug discovery". J Pharmacol Toxicol Methods. 51 (1): 1–23. doi:10.1016/j.vascn.2004.07.006. PMID 15596111.
- ^ Song, Y. H.; Kim, C. S.; Seo, J. H. Noninvasive monitoring of environmental toxicity through green fluorescent protein expressing Escherichia coli. Korean J. Chem. Eng. 2016, 33 (4), 1331-1336.
- ^ Pan, Y.; Leifert, A.; Graf, M.; Schiefer, F.; Thoröe-Boveleth, S.; Broda, J.; Halloran, M. C.; Hollert, H.; Laaf, D.; Simon, U.; Jahnen-Dechent, W. High-Sensitivity Real-Time Analysis of Nanoparticle Toxicity in Green Fluorescent Protein-Expressing Zebrafish. Small. 2013, 9 (6), 863-869.
- ^ Chalfie M (Jun 2009). "GFP: Lighting up life". Proceedings of the National Academy of Sciences of the United States of America. 106 (25): 10073–10080. Bibcode:2009PNAS..10610073C. doi:10.1073/pnas.0904061106. PMC 2700921 . PMID 19553219.
- ^ Yuste R (Dec 2005). "Fluorescence microscopy today". Nature Methods. 2 (12): 902–4. doi:10.1038/nmeth1205-902. PMID 16299474.
- ^ Komorowski M, Finkenstädt B, Rand D (Jun 2010). "Using a single fluorescent reporter gene to infer half-life of extrinsic noise and other parameters of gene expression". Biophysical Journal. 98 (12): 2759–2769. Bibcode:2010BpJ....98.2759K. doi:10.1016/j.bpj.2010.03.032. PMC 2884236 . PMID 20550887.
- ^ Gunkel M, Erdel F, Rippe K, Lemmer P, Kaufmann R, Hörmann C, Amberger R, Cremer C (Jun 2009). "Dual color localization microscopy of cellular nanostructures". Biotechnology Journal. 4 (6): 927–38. doi:10.1002/biot.200900005. PMID 19548231.
- ^ Chudakov DM, Lukyanov S, Lukyanov KA (Dec 2005). "Fluorescent proteins as a toolkit for in vivo imaging". Trends in Biotechnology. 23 (12): 605–13. doi:10.1016/j.tibtech.2005.10.005. PMID 16269193.
- ^ Livet J, Weissman TA, Kang H, Draft RW, Lu J, Bennis RA, Sanes JR, Lichtman JW (Nov 2007). "Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system". Nature. 450 (7166): 56–62. Bibcode:2007Natur.450...56L. doi:10.1038/nature06293. PMID 17972876.
- ^ Baker BJ, Mutoh H, Dimitrov D, Akemann W, Perron A, Iwamoto Y, Jin L, Cohen LB, Isacoff EY, Pieribone VA, Hughes T, Knöpfel T (Aug 2008). "Genetically encoded fluorescent sensors of membrane potential". Brain Cell Biology. 36 (1–4): 53–67. doi:10.1007/s11068-008-9026-7. PMC 2775812 . PMID 18679801.
- ^ Adesnik H, Nicoll RA, England PM (Dec 2005). "Photoinactivation of native AMPA receptors reveals their real-time trafficking". Neuron. 48 (6): 977–85. doi:10.1016/j.neuron.2005.11.030. PMID 16364901.
- ^ Lakadamyali M, Rust MJ, Babcock HP, Zhuang X (Aug 2003). "Visualizing infection of individual influenza viruses". Proceedings of the National Academy of Sciences of the United States of America. 100 (16): 9280–5. Bibcode:2003PNAS..100.9280L. doi:10.1073/pnas.0832269100. PMC 170909 . PMID 12883000.
- ^ Joo KI, Wang P (Oct 2008). "Visualization of targeted transduction by engineered lentiviral vectors". Gene Therapy. 15 (20): 1384–96. doi:10.1038/gt.2008.87. PMC 2575058 . PMID 18480844.
- ^ Remy S, Tesson L, Usal C, Menoret S, Bonnamain V, Nerriere-Daguin V, Rossignol J, Boyer C, Nguyen TH, Naveilhan P, Lescaudron L, Anegon I (Oct 2010). "New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy". Transgenic Research. 19 (5): 745–63. doi:10.1007/s11248-009-9352-2. PMID 20094912.
- ^ Elliott G, McGrath J, Crockett-Torabi E (Jun 2000). "Green fluorescent protein: A novel viability assay for cryobiological applications". Cryobiology. 40 (4): 360–369. doi:10.1006/cryo.2000.2258. PMID 10924267.
- ^ Fakhrudin N, Ladurner A, Atanasov AG, Heiss EH, Baumgartner L, Markt P, Schuster D, Ellmerer EP, Wolber G, Rollinger JM, Stuppner H, Dirsch VM (Apr 2010). "Computer-aided discovery, validation, and mechanistic characterization of novel neolignan activators of peroxisome proliferator-activated receptor gamma". Molecular Pharmacology. 77 (4): 559–66. doi:10.1124/mol.109.062141. PMC 3523390 . PMID 20064974.
- ^ Gather MC, Yun SH (2011). "Single-cell biological lasers". Nature Photonics. 5 (7): 406–410. Bibcode:2011NaPho...5..406G. doi:10.1038/nphoton.2011.99.
- ^ Matson J (2011). "Green Fluorescent Protein Makes for Living Lasers". Scientific American. Retrieved 2011-06-13.
- ^ Kouros-Mehr H, Bechis SK, Slorach EM, Littlepage LE, Egeblad M, Ewald AJ, Pai SY, Ho IC, Werb Z (Feb 2008). "GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model". Cancer Cell. 13 (2): 141–52. doi:10.1016/j.ccr.2008.01.011. PMC 2262951 . PMID 18242514.
- ^ Rodman MK, Yadav NS, Artus NN (2002-09-01). "Progression of geminivirus-induced transgene silencing is associated with transgene methylation". New Phytologist. 155 (3): 461–468. doi:10.1046/j.1469-8137.2002.00467.x.
- ^ Zhu YJ, Agbayani R, Moore PH (Apr 2004). "Green fluorescent protein as a visual selection marker for papaya (Carica papaya L.) transformation". Plant Cell Reports. 22 (9): 660–7. doi:10.1007/s00299-004-0755-5. PMID 14749892.
- ^ Niwa Y, Hirano T, Yoshimoto K, Shimizu M, Kobayashi H (1999). "Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants". The Plant Journal. 18 (4): 455–63. doi:10.1046/j.1365-313X.1999.00464.x. PMID 10406127.
- ^ Baker SS, Vidican CB, Cameron DS, Greib HG, Jarocki CC, Setaputri AW, Spicuzza CH, Burr AA, Waqas MA, Tolbert DA (2012-01-01). "An epifluorescent attachment improves whole-plant digital photography of Arabidopsis thaliana expressing red-shifted green fluorescent protein". AoB PLANTS. 2012: pls003. doi:10.1093/aobpla/pls003. PMC 3296078 . PMID 22479674.
- ^ "PlantEdDL - Using SRL digital cameras in quantitative investigations of plants expressing green fluorescent protein (GFP)". planted.botany.org. Retrieved 2016-03-23.
- ^ Eduardo Kac. "GFP Bunny".
- ^ "Glow-In-The Dark NeonMice". Retrieved August 30, 2016.[dead link]
- ^ Scientists in Taiwan breed fluorescent green pigs
- ^ Wongsrikeao P, Saenz D, Rinkoski T, Otoi T, Poeschla E (2011). "Antiviral restriction factor transgenesis in the domestic cat". Nature Methods. 8 (10): 853–9. doi:10.1038/nmeth.1703. PMC 4006694 . PMID 21909101.
- ^ [1]
- ^ Voss-Andreae J (2005). "Protein Sculptures: Life's Building Blocks Inspire Art". Leonardo. 38: 41–45. doi:10.1162/leon.2005.38.1.41.
- ^ Pawlak A (2005). "Inspirierende Proteine". Physik Journal. 4: 12.
- ^ "Julian Voss-Andreae Sculpture". Retrieved 2007-06-14.
Further reading
- Pieribone V, Gruber D (2006). Aglow in the Dark: The Revolutionary Science of Biofluorescence. Cambridge: Belknap Press. ISBN 0-674-01921-0. OCLC 60321612. Popular science book describing history and discovery of GFP
- Zimmer M (2005). Glowing Genes: A Revolution In Biotechnology. Buffalo, NY: Prometheus Books. ISBN 1-59102-253-3. OCLC 56614624.
External links
|Library resources about
Green fluorescent protein
|Wikimedia Commons has media related to Green fluorescent proteins.
- A comprehensive article on fluorescent proteins at Scholarpedia
- Brief summary of landmark GFP papers
- Interactive Java applet demonstrating the chemistry behind the formation of the GFP chromophore
- Video of 2008 Nobel Prize lecture of Roger Tsien on fluorescent proteins
- Excitation and emission spectra for various fluorescent proteins
- Green Fluorescent Protein Chem Soc Rev themed issue dedicated to the 2008 Nobel Prize winners in Chemistry, Professors Osamu Shimomura, Martin Chalfie and Roger Y. Tsien
- Molecule of the Month, June 2003: an illustrated overview of GFP by David Goodsell.
- Molecule of the Month, June 2014: an illustrated overview of GFP-like variants by David Goodsell.
This page is based on a Wikipedia article. The text is available under the Creative Commons Attribution/Share-Alike License.
This tab holds the annotation information that is stored in the Pfam database. As we move to using Wikipedia as our main source of annotation, the contents of this tab will be gradually replaced by the Wikipedia tab.
Green fluorescent protein Provide feedback
No Pfam abstract.
Literature references
Tsien RY , Annu Rev Biochem 1998;67:509-544.: The green fluorescent protein. PUBMED:9759496 EPMC:9759496
External database links
This tab holds annotation information from the InterPro database.
InterPro entry IPR011584
The green fluorescent protein (GFP) is found in the jellyfish (Aequorea victoria), and functions as an energy-transfer acceptor. It fluoresces in vivo upon receiving energy from the Ca2+-activated photoprotein aequorin. The protein absorbs light maximally at 395 nm and exhibits a smaller absorbance peak at 470 nm. The fluorescence emission spectrum peaks at 509 nm with a shoulder at 540 nm. The protein is produced in the photocytes and contains a chromophore, which is composed of modified amino acid residues. The chromophore is formed upon cyclisation of the residues ser-dehydrotyr-gly. There are several other members of the GFP family, which are able to fluoresce different colours, several of which are non-fluorescent [PUBMED:10852900]. These proteins are all essentially encoded by single genes, since both the substrate and the catalytic enzyme for pigment biosynthesis are provided within a single polypeptide chain [PUBMED:12325128].
Gene Ontology
The mapping between Pfam and Gene Ontology is provided by InterPro. If you use this data please cite InterPro.
|Biological process
|bioluminescence (GO:0008218)
Domain organisation
Below is a listing of the unique domain organisations or architectures in which this domain is found. More...
The graphic that is shown by default represents the longest sequence with a given architecture. Each row contains the following information:
- the number of sequences which exhibit this architecture
a textual description of the architecture, e.g. Gla, EGF x 2, Trypsin.
This example describes an architecture with one
Gladomain, followed by two consecutive
EGFdomains, and finally a single
- a link to the page in the Pfam site showing information about the sequence that the graphic describes
- the UniProt description of the protein sequence
- the number of residues in the sequence
- the Pfam graphic itself.
Note that you can see the family page for a particular domain by clicking on the graphic. You can also choose to see all sequences which have a given architecture by clicking on the Show link in each row.
Finally, because some families can be found in a very large number of architectures, we load only the first fifty architectures by default. If you want to see more architectures, click the button at the bottom of the page to load the next set.
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Pfam Clan
This family is a member of clan GFP (CL0069), which has the following description:
This superfamily has an unusual fold of an 11 stranded beta barrel enclosing an alpha-helix. This superfamily includes green fluorescent protein as well as a domain from nidogen.
The clan contains the following 2 members:G2F GFP
We store a range of different sequence alignments for families. As well as the seed alignment from which the family is built, we provide the full alignment, generated by searching the sequence database (reference proteomes) using the family HMM. We also generate alignments using four representative proteomes (RP) sets, the UniProtKB sequence database, the NCBI sequence database, and our metagenomics sequence database. More...
There are various ways to view or download the sequence alignments that we store. We provide several sequence viewers and a plain-text Stockholm-format file for download.
Alignment types
We make a range of alignments for each Pfam-A family:
- seed
- the curated alignment from which the HMM for the family is built
- full
- the alignment generated by searching the sequence database using the HMM
- Representative Proteomes (RPs) at 15%, 35%, 55% and 75% co-membership thresholds
- UniProt
- alignment generated by searching the UniProtKB sequence database using the family HMM
- alignment generated by searching the NCBI sequence database using the family HMM
- meta
- alignment generated by searching the metagenomics sequence database using the family HMM
You can see the alignments as HTML or in three different sequence viewers:
- jalview
- a Java applet developed at the University of Dundee. You will need Java installed before running jalview
- an HTML page showing the whole alignment.Please note: full Pfam alignments can be very large. These HTML views are extremely large and often cause problems for browsers. Please use either jalview or the Pfam viewer if you have trouble viewing the HTML version
- PP/Heatmap
- an HTML-based representation of the alignment, coloured according to the posterior-probability (PP) values from the HMM. As for the standard HTML view, heatmap alignments can also be very large and slow to render.
You can download (or view in your browser) a text representation of a Pfam alignment in various formats:
- Selex
- Stockholm
You can also change the order in which sequences are listed in the alignment, change how insertions are represented, alter the characters that are used to represent gaps in sequences and, finally, choose whether to download the alignment or to view it in your browser directly.
You may find that large alignments cause problems for the viewers and the reformatting tool, so we also provide all alignments in Stockholm format. You can download either the plain text alignment, or a gzipped version of it.
View options
We make a range of alignments for each Pfam-A family. You can see a description of each above. You can view these alignments in various ways but please note that some types of alignment are never generated while others may not be available for all families, most commonly because the alignments are too large to handle.
|Representative proteomes
1Cannot generate PP/Heatmap alignments for seeds; no PP data available
Key: available, not generated, — not available.
Format an alignment
Download options
We make all of our alignments available in Stockholm format. You can download them here as raw, plain text files or as gzip-compressed files.
|Representative proteomes
|Raw Stockholm
You can also download a FASTA format file containing the full-length sequences for all sequences in the full alignment.
HMM logo
HMM logos is one way of visualising profile HMMs. Logos provide a quick overview of the properties of an HMM in a graphical form. You can see a more detailed description of HMM logos and find out how you can interpret them here. More...
If you find these logos useful in your own work, please consider citing the following article:
This page displays the phylogenetic tree for this family's seed alignment. We use FastTree to calculate neighbour join trees with a local bootstrap based on 100 resamples (shown next to the tree nodes). FastTree calculates approximately-maximum-likelihood phylogenetic trees from our seed alignment.
Note: You can also download the data file for the tree.
Curation and family details
This section shows the detailed information about the Pfam family. You can see the definitions of many of the terms in this section in the glossary and a fuller explanation of the scoring system that we use in the scores section of the help pages.
|Seed source:
|Previous IDs:
|Sequence Ontology:
|Bateman A
|Number in seed:
|Number in full:
|Average length of the domain:
|186.20 aa
|Average identity of full alignment:
|34 %
|Average coverage of the sequence by the domain:
|81.01 %
HMM information
|HMM build commands:
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 45638612 -E 1000 --cpu 4 HMM pfamseq
|Model details:
|Model length:
|Family (HMM) version:
|download the raw HMM for this family
Species distribution
Sunburst controlsHide
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Colour assignments
|Other sequences
|Unclassified sequence
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This visualisation provides a simple graphical representation of the distribution of this family across species. You can find the original interactive tree in the adjacent tab. More...
This chart is a modified "sunburst" visualisation of the species tree for this family. It shows each node in the tree as a separate arc, arranged radially with the superkingdoms at the centre and the species arrayed around the outermost ring.
How the sunburst is generated
The tree is built by considering the taxonomic lineage of each sequence that has a match to this family. For each node in the resulting tree, we draw an arc in the sunburst. The radius of the arc, its distance from the root node at the centre of the sunburst, shows the taxonomic level ("superkingdom", "kingdom", etc). The length of the arc represents either the number of sequences represented at a given level, or the number of species that are found beneath the node in the tree. The weighting scheme can be changed using the sunburst controls.
In order to reduce the complexity of the representation, we reduce the number of taxonomic levels that we show. We consider only the following eight major taxonomic levels:
- superkingdom
- kingdom
- phylum
- class
- order
- family
- genus
- species
Colouring and labels
Segments of the tree are coloured approximately according to their superkingdom. For example, archeal branches are coloured with shades of orange, eukaryotes in shades of purple, etc. The colour assignments are shown under the sunburst controls. Where space allows, the name of the taxonomic level will be written on the arc itself.
As you move your mouse across the sunburst, the current node will be highlighted. In the top section of the controls panel we show a summary of the lineage of the currently highlighed node. If you pause over an arc, a tooltip will be shown, giving the name of the taxonomic level in the title and a summary of the number of sequences and species below that node in the tree.
Anomalies in the taxonomy tree
There are some situations that the sunburst tree cannot easily handle and for which we have work-arounds in place.
Missing taxonomic levels
Some species in the taxonomic tree may not have one or more of the main eight levels that we display. For example, Bos taurus is not assigned an order in the NCBI taxonomic tree. In such cases we mark the omitted level with, for example, "No order", in both the tooltip and the lineage summary.
Unmapped species names
The tree is built by looking at each sequence in the full alignment for the family. We take the name of the species given by UniProt and try to map that to the full taxonomic tree from NCBI. In some cases, the name chosen by UniProt does not map to any node in the NCBI tree, perhaps because the chosen name is listed as a synonym or a misspelling in the NCBI taxonomy.
So that these nodes are not simply omitted from the sunburst tree, we group them together in a separate branch (or segment of the sunburst tree). Since we cannot determine the lineage for these unmapped species, we show all levels between the superkingdom and the species as "uncategorised".
Since we reduce the species tree to only the eight main taxonomic levels, sequences that are mapped to the sub-species level in the tree would not normally be shown. Rather than leave out these species, we map them instead to their parent species. So, for example, for sequences belonging to one of the Vibrio cholerae sub-species in the NCBI taxonomy, we show them instead as belonging to the species Vibrio cholerae.
Too many species/sequences
For large species trees, you may see blank regions in the outer layers of the sunburst. These occur when there are large numbers of arcs to be drawn in a small space. If an arc is less than approximately one pixel wide, it will not be drawn and the space will be left blank. You may still be able to get some information about the species in that region by moving your mouse across the area, but since each arc will be very small, it will be difficult to accurately locate a particular species.
Tree controlsHide
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Selected sequences
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- graphically
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The tree shows the occurrence of this domain across different species. More...
Species trees
We show the species tree in one of two ways. For smaller trees we try to show an interactive representation, which allows you to select specific nodes in the tree and view them as an alignment or as a set of Pfam domain graphics.
Unfortunately we have found that there are problems viewing the interactive tree when the it becomes larger than a certain limit. Furthermore, we have found that Internet Explorer can become unresponsive when viewing some trees, regardless of their size. We therefore show a text representation of the species tree when the size is above a certain limit or if you are using Internet Explorer to view the site.
If you are using IE you can still load the interactive tree by clicking the "Generate interactive tree" button, but please be aware of the potential problems that the interactive species tree can cause.
Interactive tree
For all of the domain matches in a full alignment, we count the number that are found on all sequences in the alignment. This total is shown in the purple box.
We also count the number of unique sequences on which each domain is found, which is shown in green. Note that a domain may appear multiple times on the same sequence, leading to the difference between these two numbers.
Finally, we group sequences from the same organism according to the NCBI code that is assigned by UniProt, allowing us to count the number of distinct sequences on which the domain is found. This value is shown in the pink boxes.
We use the NCBI species tree to group organisms according to their taxonomy and this forms the structure of the displayed tree. Note that in some cases the trees are too large (have too many nodes) to allow us to build an interactive tree, but in most cases you can still view the tree in a plain text, non-interactive representation. Those species which are represented in the seed alignment for this domain are highlighted.
You can use the tree controls to manipulate how the interactive tree is displayed:
- show/hide the summary boxes
- highlight species that are represented in the seed alignment
- expand/collapse the tree or expand it to a given depth
- select a sub-tree or a set of species within the tree and view them graphically or as an alignment
- save a plain text representation of the tree
Please note: for large trees this can take some time. While the tree is loading, you can safely switch away from this tab but if you browse away from the family page entirely, the tree will not be loaded.
There are 5 interactions for this family. More...
We determine these interactions using iPfam, which considers the interactions between residues in three-dimensional protein structures and maps those interactions back to Pfam families. You can find more information about the iPfam algorithm in the journal article that accompanies the website.
For those sequences which have a structure in the Protein DataBank, we use the mapping between UniProt, PDB and Pfam coordinate systems from the PDBe group, to allow us to map Pfam domains onto UniProt sequences and three-dimensional protein structures. The table below shows the structures on which the GFP domain has been found. There are 1212 instances of this domain found in the PDB. Note that there may be multiple copies of the domain in a single PDB structure, since many structures contain multiple copies of the same protein sequence.
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text:
Although her death in December 1949 prevented the completion of this book, enough of the author's life story emerges to show the stages in her journey from Christian evangelism to mastery of the science of esotericism, author, lecturer and teacher.
The Unfinished Autobiography
Alice Ann Bailey somewhat
reluctantly agreed to attempt her own autobiography. What finally
decided her to write about her life was a letter from a friend who, she
says, felt deeply that "I would really render a service if I could show
people how I became what I am from what I was. It might be useful to
know how a rabid orthodox Christian worker could become a well-known
occult teacher."
From her conservative British background, Alice A. Bailey's life led her in many directions, but always in one direction--towards the time when through drastic personal experience of many kinds she had acquired a synthesis of outlook and understanding, and an absolute conviction that one divine life pervades and animates the one humanity; that the Plan for man requires the cooperation and service of trained and dedicated human beings intelligently informed about world affairs, in collaboration with those who form the spiritual hierarchy, the miner government of the planet. Her life work became an initegral part of this synthesis and this realisation. Without in the least losing any of her very human qualities and involvement, her soul took up its commitment to her Master, and her personality provided full cooperation in the field of her accepted service.
Basically the work developed as a duality--her discipleship service to her own Master, Koot Humi, which included the establislinnent of the Arcane School; and her initially reluctant agreement to work with the Tibetan, the Master Djwhal Khul, in the writing of a series of books presenting the next phase in the continuity of the Ancient Wisdom teaching for the present and the immediate future.
This work with the Tibetan Master started in 1919. In his introduction to "A Treatise on Cosmic Fire", Foster Bailey remarks: "The story of many years of telepathic work by the Tibetan with Alice A. Bailey is revealed in her Unfinished Autobiography published in 1951. This includes the circumstances of the first contact with Him on the physical plane which took place in California in November 1919. Thirty years work was planned. When this had been accomplished and within 30 days after that period (in December 1949) Mrs. Bailey gained her release from tIne limitations of the physical vehicle."
This quick release accounts also for the fact that the autobiography is unfinished.
As her work with the Tibetan developed, and as her own writing, lecturing and early Arcane School work began to demonstrate their usefulness, the Tibetan took advantage of his close telepathic link with AAB to establish certain activities required by the work of Hierarchy for humanity. The Arcane School, although never controlled or supervised by the Tibetan, was used to anchor such service activitjes as Triangles, World Goodwill and the distribution of the Great Invocation. Certain types of meditation work were also suggested by the Tibetan as a planetary service, particularly the Redemptive Meditations on Preparation for the Reappearance of the Christ and on Attracting Money for Hierarchical purposes; and the Arcane School in 1947 began a serious study of the basic problems of humanitv based on his book of that title.
The Arcane School, therefore, as an increasingly useful and useable channel between hierarchy and humanity, was used by the Master DK with AAB's consent and cooperation to anchor certain needed esoteric principles and realities firmly in human consciousness and to demonstrate the fact that esotericism is a way of life, and not an escape into vague, mystical abstractions. Her whole life work, although devoted to two distinct services, demonstrated that synthesis of outlook and attitude characteristic of every world disciple.
In the words of Foster Bailey, included in the introduction to the Autobiogaphy: "By far the greater part of her life work has always been subjective. We have seen the outer effects, watched the outer comings and goings, helped her and loved her, sometimes criticised, sometimes complained, but always gone on with her, and because of her yet a little higher and a little better than would otherwise have been the case. . . . Her chosen path is on the line of the World Saviours. She has gone back to her own Master KH for yet more daring work with Him and for the Christ."

subset: commoncrawl


text:
Behind The Scenes: Miguel (Feat. Kendrick Lamar) - How Many Drinks? Remix
Miguel enlists Kendrick Lamar for the remix to his 'Kaleidoscope Dream' track, "How Many Drinks?" The "How Many Drinks" video was shot in Los Angeles and directed by Constellation Jones. Go behind the scenes of the video shoot with "the coming kings of rap and R&B, the trailblazers to carry the baton for the next generation as VIBE calls them, in our latest cover story on the two." - vibemedia
Posted by Jay
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text:
Under the Lily Banners is a tactical battle game featuring five battles from the Thirty years War. This is the third volume from the award-winning Musket & Pike Battles Series detailing 17th Century warfare, often known as the Dawn of Modern Warfare. This volume features the rise of the French Army after their entry into the Thirty Years War in 1635. Two of the greatest captains of French history - Louis de Bourbon, the Grand Conde and Henri de la Tour, the vicomte de Turenne - take the field to set the stage for Le Grande Siecle - the Golden Age of France under Louis XIV, the Sun King
After taking on the English Civil War in This Accursed Civil War and the combined arms tactics of the Swedes and Imperials in Sweden Fights On, the Musket & Pike Battle Series looks at the French, Spanish, and Bavarian armies. The Spanish tercios face the superb French gendarmes. The stalwart Bavarians with their excellent cavalry and love of the entrenchment battle the veteran Weimarian cavalry corps.
The Musket & Pike Battle Series emphasizes Command and Control. Armies are divided into several wings. Each has a commander and an order that limits the types of actions that units of that wing may perfrom. Victory goes to the player that can coordinate the actions of his wings in the heat of battle. Units are infantry battalions, cavalry regiments and artillery batteries. Rules include cavalry charges, cavalry pistols for skirmishing or for close combat, and artillery grazing fire. Play is highly interactive and most battles can be played through in one sitting. The five battles in Under the Lily Banners are:
3 full-color counter sheets
Four maps (two sheets backprinted)


text:
March_ 12_ 2012_ Poster_ FINAL
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Who will be the West's new boss?
One of the great parlor games of the West is to guess who the next president will choose to be secretary of the Interior Department.
The man or woman succeeding Idaho's Dirk Kempthorne will be the nation's top wildlife manager and federal landlord of more than 507 million acres of national parks, rangeland and wildlife refuges.
He or she will be the water-master for more than 600 dams that bring water to 31 million Westerners and irrigate 60 percent of all the vegetables grown in the United States. The new secretary will also be responsible for 68 percent of the nation's oil and gas reserves and millions of acres of federal mining lands.
First, look at McCain. Would he keep Dirk Kempthorne? Kempthorne has helped repair the reputation of the Bush administration after Interior's lowest moments since the Teapot Dome scandal of Warren G. Harding's day. The convictions of Deputy Secretary Steve Griles for his dealings with corrupt lobbyist Jack Abramoff, the recent scandal uncovered in the Minerals Management Service, and the blatant meddling in scientific decisions on listing endangered species have undercut the integrity of the agency that has so much control over the West. This all happened on the watch of Interior Secretary Gale Norton.
Kempthorne has helped restore and grow funding for national parks, listed the polar bear as endangered, acknowledged the existence of climate change, taken responsibility to clean up the agency and improved employees' morale. And he has made the case that keeping him on through the transition would allow McCain to focus on the economy and other challenges.
Other contenders include Colorado Republican Sen. Wayne Allard, a states' rights advocate who is stepping down from the Senate, and Rep. Steve Pearce, a New Mexico Republican likely to lose to Democratic Rep. Tom Udall in a race for the Senate.
Neither one of these picks will make environmentalists happy, but Allard has supported some environmental projects in Colorado. If McCain wanted environmental support, he might step out of the West and pick GOP Sen. Lamar Alexander of Tennessee, who has a great environmental record in the Senate. Or he might tap John Turner, the former assistant secretary of state for environmental affairs under Bush and the director of the U.S. Fish and Wildlife Service under Bush's father.
Another possibility is Marc Racicot, the former Montana governor. He led George Bush's transition team and the Republican Party until 2003. As governor, he was a peacemaker with environmentalists, advocating reintroduction of grizzly bears into the Selway Bitterroots.
One woman I would include in my list is current Deputy Interior Secretary Lynn Scarlett. She was unscathed by the mismanagement under Norton and shares McCain's environmental, libertarian philosophy.
For Obama, California Democratic Rep. George Miller is a strong candidate. The former House Resources Committee chairman is an expert on Interior and has strong support among environmental groups. He buys into the new collaborative approach to public lands management.
Another possible contender is John Leshy, the former solicitor under Bruce Babbitt and Bill Clinton and a veteran of the Interior Department going back to the days of Cecil Andrus. Leshy, a professor of law at the Hastings College of the Law in San Francisco, served as chief counsel under Miller on the Resources Committee.
Montana Democratic Gov. Brian Schweitzer would be the obvious choice, since he has become one of the West's leading voices on interior and energy issues. But Schweitzer has said he wants to stay in Montana, and he has a Republican lieutenant governor.
John Kitzhaber, the former Oregon governor, might be a long shot. He is the only major political figure in the Pacific Northwest to advocate breaching the four lower Snake dams in Washington to restore endangered salmon and steelhead.
Other names thrown out are Gov. Bill Richardson of New Mexico, though he's likely to be up for several Cabinet posts, including secretary of State. There's also Rep. Jay Inslee of Washington and Colorado Rep. Mark Udall, though Udall is unlikely to lose to Republican Bob Schaffer for Allard's Senate seat.
Also on the list is Arizona Gov. Janet Napolitano. She's an expert on water issues and worked with other Western governors to seek collaborative positions on fire policy and climate change. If Obama wants to go outside of government, he might choose Sally Jewell, CEO of Recreational Equipment Inc. and a leader in the Initiative for Global Development, a group of business leaders seeking to end world poverty.
Yes, it's a fun parlor game, but it has a serious purpose, because for the West, no other appointment is as important as secretary of the Interior.
Rocky Barker is a contributor to Writers on the Range, a service of High Country News (hcn.org). He is the environmental writer for IdahoStatesman.com in Boise, Idaho.
Note: the opinions expressed in this column are those of the writer and do not necessarily reflect those of High Country News, its board or staff. If you'd like to share an opinion piece of your own, please write Betsy Marston at [email protected].


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In the debate over who should be the next mayor of Los Angeles, who would you suppose argues for elimination of a business tax to kick-start economic growth?
Not the one-time investment banker who dropped out of the race early and says killing the business tax would leave a huge hole in the city treasury. Not the lone Republican in the field, who wants more modest business tax reform. Not a City Council fiscal hawk, also a candidate for mayor, who says cutting the tax could leave the city with a $400-million shortfall.
Instead it's two liberal Democrats — City Controller Wendy Greuel and Councilman Eric Garcetti — pushing what sounds a lot like Ronald Reagan- or Mitt Romney-style "supply side" fiscal dogma. Cut taxes for business, Garcetti and Greuel contend, and the overall economic pie will grow, with benefits trickling down in the form of more jobs for Angelenos and more money in the city's general fund.
-- James Rainey, Los Angeles Times


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Charles Kesler appeared last night on the News Hour on (the soon to be defunded) PBS. Here's the 8-minute segment:
And if you haven't yet read I Am the Change: Barack Obama and the Crisis of Liberalism, there's still time to get it before Tuesday if you use Amazon Prime. And even if you don't get to it by Tuesday and Romney wins, the book will not be obsolete; its argument about the pedigree of Obama's ideology will be useful for a long while to come.


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Remember the amazing Jacqueline/Jackie Nicole Brown [ read more on JNB here ]…? Welp, we have an super exciting announcement:
We're launching a YouTube channel together {!!!!!!!!! }.
Think skinny meets fashion in a va va voom, sex kitten-kinda way. Our style will be similar to the above video, which features Jackie as herself rocking her handmade arm candy.
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Hope you're all enjoying your weekend, xx.


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Modified Mustangs & FordsHow To Tech Qa
Garage Q&A - Tech Answers, September 2013
Keep Those Classic Ford and Mustang Tech Questions Coming!
Braking the Bird
I have a 1964 Falcon convertible, and would like to add power brakes with a dual-circuit master cylinder for enhanced safety without having to modify the firewall or shock tower. I found a unit from a company named Nelcohotrods online. I contacted the company and the guy I spoke to said it should fit. It is 12 inches long, but he didn't say if one had been installed on a Falcon before. Do you have any info that might help?
Jerry Larsen
Lakeville, MN
I took a look at Nelcohotrods products, and it looks like the company is selling the Master Power Brakes 7-inch diaphragm booster with a GM-style master cylinder. It takes some work, but this system will fit the Falcon. However, I have something else in mind.
First, to our other readers, Falcons have been difficult to upgrade to a power dual-circuit master cylinder because of the minimum distance between the firewall and the shock tower. Add to this the massive welded shock-tower braces and an all-Ford solution will simply not work.
You have three choices right now: the first being the type that Nelcohotrods sells, which is the small single-diaphragm booster and GM dual-master cylinder. This system does fit and it doesn't require a huge amount of effort, but because of the large shock-tower brace directly over the master cylinder filling the front reservoir requires a tube and syringe. The second option is a homebuilt setup from a Geo Metro. It requires a little more fabrication to make it work, but it uses an easy to access fluid reservoir.
I'm going to recommend the Scott Drake conversion unit, which uses a similar small diaphragm booster and dual-reservoir GM master cylinder as the Nelcohotrods product. The part number is PBC-F-D and it is made to fit your Falcon with a minimum of effort using a specially designed mounting bracket. The pushrod is threaded as well for pedal height adjustments. You'll need a new distribution block as well, and you'll have to bend up a couple of brake lines, but the Scott Drake website states it offers these items as well for installation. This should solve most of your fit problems on the early Falcon chassis. The unit is available from most Scott Drake dealers.
Swedish Mod Swap
I have a '69 Mustang SportsRoof that is going to get a '05 4.6L Three-Valve modular engine swap. I will modify the shock towers for it to fit, of course. I wonder if you know what oil pan to use for this swap and maybe the front coilover shocks?
Hans (last name withheld)
Canton Racing www.cantonracing.com sells the oil pan you will need to run the original rear-mounted steering system in your car after you've trimmed back the shock towers to fit the engine. For the Three-Valve 4.6L modular, use pan PN 15-790. The pickup tube for the Three-Valve should be 15-791. Canton also sells a dipstick tube for the application under PN 20-850.
As far as the coilovers, the two major players for stock-style suspension coilover conversions would be Total Control Products (www.totalcontrolproducts.com) and Global West Suspension (www.globalwest.net). If you are looking at a coilover system, look at one that connects the shock as close to the lower knuckle as possible (the point where the spindle attaches to the lower control arm)—this makes the shock work more efficiently. As you know, using a coilover allows for the shock tower sheetmetal to be trimmed for fitment of some modular engines without having to go to the expense of a Mustang II-style front suspension.
8.8 Axle Upgrade
OK, you got me, where's the 8.8 rearend upgrade for early Mustangs? I've got a '66 coupe sitting in my garage and an 8.8-inch axle with a 3.73 geared Posi sitting on the floor next to it. I really would have liked to have seen that article before I lit up my torch. What's up? Could you email it to me? And while we're on the subject, my 8-inch has the big Galaxie drum brakes, the 8.8-inch axle has discs; any advice on how to safely connect the new system with the old? Will the original master cylinder be OK or do I need something later? What bolts in? Did you address this in your article? You can see my dilemma and disappointment. Thanks.
Tom Gunshannon
Larksville, PA
The 8.8-inch as a cheap alternative to the 9-inch or aftermarket debate is kind of like someone giving you a free aquarium: sure it's cheap, but the fish are expensive. There are a lot of 8.8-inch rears floating around out there, most with Traction-Lok differentials and disc brakes. The problem is that most times it costs as much to make these pieces fit as it would be to buy a nicely made-to-fit aftermarket 9-inch unit. In the case of your '66 coupe, it's a little harder since the only 9-inch units available come from Hi-Po cars.
So which is better: the 8.8-inch or the 9-inch? Opinions are pretty much split down the middle. Both are good, strong units with a myriad of gear selections available. The 8.8-inch has less reciprocating weight and less friction, which means more horsepower transfer. But the 9-inch still remains king when it comes to overall strength, and top competitors use it no matter what your emblems say on the side of their car. The 8.8 suffers at the bearing ends over the 9-inch, and a lot of built 8.8s use the 9-inch housing ends, bearings, and axles. Also, the 8.8-inch axle tubes are pressed in and not welded, and if you hit something hard enough you can knock them out of place. The only real factory 9-inch leaf spring rear axle with disc brakes was the Lincoln Versailles from the 1970s, and those were heavy units and very rare now in scrap yards. Some people have tried the leaf spring Bronco 8.8-inch with discs, but because they are four-wheel-drives, the center section is way too far offset for practical use. The common Explorer 8.8-inch requires shortening one side to fit properly and relocating the spring perches.
As for your '66, your original 8-inch axle is 57.25-inches wide. Fox body 8.8-inch rears are 59.25, so you're already 2 inches too wide to start. You can make this up with wheel offset, but you have already started limiting yourself. Welding on a set of spring perches isn't terribly difficult, but you need to make sure you get it straight. By the time you start adding in the pieces to make it fit, it starts looking like an aftermarket disc setup for your 8-inch will start looking pretty good. Take a look at the article concerning the remaining issues. It was published in the June '13 issue. In my opinion, it's still a toss up on whether to improve the axle you have vs. installing the newer one and taking on the fabrication effort, however, the bolt-in suspension kit used in the June 8.8-inch story does make the process much less painful.
Boss 351 Hose ID
Dave, I have more of a restoration type question for you. We're restoring a '71 Boss 351 Mustang for a customer. There is a pair of holes in the side of the Ram-Air air cleaner that were missing their OE hardware/hoses. I assume that they are for the PCV system. Do you know what parts/fittings are supposed to fit in those holes? Any info would be a huge help.
Bob Roeder
KR Motorsports, LLC
There are two holes on the passenger side of the Boss 351 Ram-Air base. The rear one attaches to the passenger-side valve cover breather cap to the base. It uses a clip and there is a filter mounted to the inside of the base where it is used to soak up oil fumes. The front hole is the port to the evaporative emissions charcoal canister mounted on the passenger-side inner fender. This system takes excess gas fumes from the tank and sends them to the engine to keep emissions down. The front hose is a cardboard like or corrugated paper tubing and is no longer available from Ford. Hope this helps.
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Expected longevity is established by statistically characterizing the power consumed by the device and the power available from the device battery. This characterization is applied to a number of parameter configurations. The statistical mean value minus three standard deviations is used as the expected longevity for determining if a battery depleted normally.
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Hell Rell & Big Joe Are About To Expose Someone! "We Got Paperwork On Everybody's Deals. You Know What You Did Last Summer Nicca. You Wasn't Even With Brandy"
Hell Rell & Big Joe behind the scenes of Dipsexxx
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text: This book isn't about ghosts exactly, but Alton's uncle, Trapp, is haunted by his past. Even Toni seems to be haunted. Perhaps she hears ghosts or perhaps there is something wrong with her mind. But Alton, in the ending of the novel, seems to feel something too. Could it be the presence of his uncle still telling him what cards to play to win the game? Did you feel a touch of the supernatural in this story?


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Amyraldism is an off-shoot of Calvinistic theology (sometimes called "4-point Calvinism" or "moderate Calvinism"). It is named after its creator, Moses Amyraut, a 16th-century French theologian, who was influential in the development of the doctrine of "hypothetical redemption" or "hypothetical universalism." This doctrine, in essence, softens the Calvinistic doctrine of limited atonement. In order to better understand Amyraldism, it might be beneficial to recap what Calvinism is.
Classic Calvinism centers on the so-called five points of Calvinism. It should be noted that Calvinism isn't limited to just five points, nor was it the invention of John Calvin. Rather, Calvin put together his system of five points from the clear doctrines of Scripture. The five points of Calvinism were responses by the Dutch Reformed Church to Arminianism, another five-point system which was developed by Jacob Arminius (1560–1609), a Dutch theologian. The five points of Calvinism are summarized below:
1. Total Depravity—Man, in his fallen state, is completely incapable of doing any good that is acceptable to God.
2. Unconditional Election—As a result of man's total depravity, he is unable (and unwilling) to come to God for salvation. Therefore, God must sovereignly choose those who will be saved. His decision to elect individuals for salvation is unconditional. It is not based on anything in man, but solely on God's grace.
3. Limited Atonement—In order to save those whom God has unconditionally elected, atonement for their sin must be made. God the Father sends His Son, Jesus Christ, to atone for the sins of the elect and secure their pardon by His death on the cross.
4. Irresistible Grace—The Holy Spirit applies the finished work of salvation to the elect in "space and time" by irresistibly drawing them to faith and repentance. This saving call of the Holy Spirit (not to be confused with the general call to preach the gospel to all people) cannot be resisted and is referred to as an efficacious call.
5. Perseverance of the Saints—Those whom God has elected, atoned for and efficaciously called are preserved in faith until the last day. They will never fall away because God has secured them with the seal of the Holy Spirit. The saints persevere because God preserves them.
As mentioned earlier, the particular point that Amyraldism takes issue with is the third point, limited atonement. Amyraldism replaces it with the concept of "hypothetical universalism," which in essence asserts that Christ died for the sins of all people, but because God knew that not all would respond (due to man's total depravity, to which Amyraldians do hold), He elected some to whom He would impart saving faith. By doing this, Amyraldism avoids some of the problems that limited atonement raises, while at the same time, preserves the doctrine of unconditional election.
This places Amyraldism somewhere between Calvinism and Arminianism when it comes to the extent of the atonement. Calvinism teaches that the extent of the atonement is limited to the elect only; Christ's death on the cross makes salvation a reality for the elect. Arminianism teaches that the extent of the atonement is unlimited and available to all; Christ's death on the cross makes salvation possible to all and man must exercise faith to make salvation actual. Amyraldism says that Christ died for all men, but God only applies this salvation to those whom He has chosen. This is very similar to a view that is circulating in some Calvinistic circles called unlimited/limited atonement.
However, in attempting to resolve some of the apparent problems that limited atonement presents—namely, the biblical passages that teach Christ died for all men (John 3:16; 2 Peter 3:9; 1 John 2:2 to name a few)—the Amyraldians create larger problems that require resolution. The same objection that can be applied to Arminianism regarding the extent of the atonement can also be applied to Amyraldism. If Christ died for all men as it says in 1 John 2:2, then we have a problem; namely, there are people in hell right now who have had their sins atoned for. The Arminian would respond by saying that they didn't activate their salvation by believing in Christ, and the Amyraldian would respond by saying that God didn't elect them. Yet, that does nothing to resolve the dilemma. Whether or not they responded in faith or God elected them, their sins have been atoned for and they should not be in hell! If Christ died for my sins, then my believing it or not believing it doesn't make it any less true. If my sin debt has been paid, then I should go to heaven, not hell where I will pay for the same debt for eternity. In fact, Amyraldism makes things worse than Arminianism because it posits God passing over people for whom Christ died.
Without going into a detailed exegesis of 1 John 2:2, the apparent contradiction between what some think that verse means and the doctrine of limited atonement can be easily resolved by looking at the context of the verse. John writes:
"My little children, I am writing these things to you so that you may not sin. But if anyone does sin, we have an advocate with the Father, Jesus Christ the righteous. He is the propitiation for our sins, and not for ours only but also for the sins of the whole world" (1 John 2:1–2 ESV).
It is clear from the context that John is writing to a group of believers ("My little children"). John is telling his believing readers that a life of continual sin is incompatible with being a Christian. Yet if someone does sin, we have an Advocate who stands in our defense before the bar of God's justice. He was the atoning sacrifice for our sin, but not ours only but also the whole world. In other words, the extent of Christ's saving work isn't only applicable to John's readers, but is a message for the whole world! Christ not only died to save God's remnant amongst the Jews, but His elect throughout the whole world. God's elect people are chosen "from every nation, tribe, people and language" (Revelation 7:9). As Jesus said, "I have other sheep that are not of this sheep pen. I must bring them also. They too will listen to my voice, and there shall be one flock and one shepherd" (John 10:16).
Another thing to note is that John says that Christ is the propitiation for our sins. In other words, Christ actually atoned for the sins of John and his readers; it is a statement of fact. John then applies this actual atonement not only for his readers, but the whole world. The Arminians say that the atonement didn't actually save anyone, but makes all men savable. Yet that's not what John is saying. He is referring to an actual atonement, not a potential atonement. It would be hermeneutically incorrect to go from an actual atonement to a potential atonement in the same sentence. On the other hand, as noted earlier, the Amyraldian claims that God actually atoned for the whole world, but only applies it to the elect, to which we would argue that God is sending people to hell whose sins have been atoned for.
In conclusion, Amyraldism seeks to modify some of the "harsh" conclusions of the Calvinist position, but as we have seen, it raises much more serious questions than it attempts to answer. The five points of Calvinism are links in a chain that put forth a strong biblical case that God is completely sovereign in salvation. The act of saving a people unto Himself is a testament to God's grace and mercy and displays His glory in a unique way.[1]
[1] Got Questions Ministries. (2010). Got Questions? Bible Questions Answered. Bellingham, WA: Logos Bible Software.


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Software Wedge for Windows
Software Wedge for Windows
The Software Wedge for Windows is an application that enables a serial device to be interfaced to a PC. The Software Wedge acts as a keystroke simulator; it takes data from the serial device and makes the computer act as though it were typed in at the keyboard. The Software Wedge collects data from any serial device such as digital micrometers, scales, or barcode readers into any Windows application. Required for connecting the above serial devices to Partner for automated specimen dimension entry.
When the RS232 interfacing options have been purchased, the Software Wedge is also suitable for collecting data from the SATEC DI-300, MARK III, and MARK III Smart C indicators and sending the data to MSExcel or a comma delimited text file.
Includes RS232 serial interface cable, 9 to 25 Pin Serial Adapter Cable and Software Wedge manual.
|Software Wedge for Windows


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SUNRISE, Fla. (AP) — Peter Holland and the Toronto Maple Leafs took advantage after both Panthers goalies were injured Tuesday night and beat Florida 3-2 to snap a franchise-worst 16-game, road-losing streak.
Holland netted the winning goal 5:28 into the third period against Al Montoya, who had replaced injured starter Roberto Luongo after the first period. It was the Maple Leafs' first road win in 17 games (0-14-2) since they beat Boston on Dec. 31
The Panthers changed goalies twice, and even had goalie coach Robb Tallas get dressed on an emergency basis, but the 41-year-old Tallas never entered the game. He hasn't played in an NHL game since 2001.
Montoya was injured...


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Miia wore a truly darling mint green 30s vintage number, which she found on a relative's attic. I'm in love with the dress and think it looks truly spectacular on her! I wore my Mary dress from Heyday! Vintage Style. I paired the peacock blue frock with my beloved red bow shoes, a red belt and a hair orchid.
Dress - Heyday! Vintage Style
Shoes - Office
Hair flower - Olivia Rouge


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