text:
|Publication number
|Publication type
|Application number
|Publication date
|Jan 30, 2001
|Filing date
|Jul 30, 1998
|Priority date
|Aug 1, 1997
|Fee status
|Publication number
|Roger Y. Tsien, Stephen R. Adams
|Original Assignee
|The Regents Of The University Of California
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (16), Non-Patent Citations (5), Referenced by (4), Classifications (21), Legal Events (5)
|External Links: USPTO, USPTO Assignment, Espacenet
This application claims priority under 35 U.S.C. �119(e) to U.S. Provisional Patent Application Ser. No. 60/054,441 filed Aug. 1, 1997.
The present invention relates generally to methods of determining analyte concentrations, and more particularly, to photolabile chelators.
Although Ca2+ is a well established intracellular messenger, there are still many unanswered questions concerning the kinetics and spatial localization of its effects. Because of the importance of calcium as an intracellular messenger, a variety of methods have been developed for measuring intracellular Ca2+ levels. The most successful of these use dye-linked chelators which change absorbance or fluorescence upon binding Ca2+ ions. Existing indicators for physiological cations such as Ca2+, as well as, Mg2+, H+, and Na+ change their optical properties reversibly in response to changes in cation concentrations. See, for example, U.S. Pat. No. 4,603,209, and EP 314,480. Chelating compounds have been developed that facilitate changing the intracellular calcium concentration. See, for example, U.S. Pat. Nos. 4,689,432, 4,806,604, and 5,141,627; and EP 177,202, in which a photochemical reaction causes the release or uptake of Ca2+ and thereby alters the ambient Ca2+.
In general, the invention features optical indicators which memorize the spatial location and/or concentration of an analyte at a particular chosen point in time. This can allow for time-resolved analysis of analyte concentration. Analysis of the temporal and spatial resolution of the intracellular concentrations of physiologically significant analytes, such as calcium and other cations, can be facilitated by freezing a snapshot of the analyte concentration in the intracellular matrix. The snapshot can allow one to postpone the analysis of the analyte concentration at that particular moment to a later time.
In one aspect, the invention features a compound including a chromophore carrying a photolabile group capable of undergoing an irreversible and detectable chemical transformation upon irradiation by light, the chromophore being linked to or including a binding or chelating site for an analyte. Binding of the analyte to the binding site alters an optical property of the chromophore, thus altering the ability of the photolabile group to undergo the chemical transformation upon irradiation of the compound.
The analyte can be a cation, such as calcium and the chelator can be a BAPTA or BAPTA-like derivative. The chromophore can be any chemical grouping, such as an aromatic, heteroaromatic, or polyene group which absorbs light. One type of photolabile group that photolyzes with high efficiency to an isoxazole is a β-azido, α,β-unsaturated carbonyl group.
In another aspect, the invention relates to a method of memorizing an intracellular concentration of an analyte. The method includes the steps of: (a) loading a cell with an effective amount of a compound as described above under conditions which do not alter the chromophore; (b) irradiating the cell with a wavelength of light which selectively photolyzes the compound to yield a photolyzed product; (c) detecting the presence of the photolyzed product and/or unphotolyzed compound; and (d) relating the presence of the photolyzed product and/or unphotolyzed compound to the intracellular concentration of the analyte. The cell is briefly irradiated. The compound can be bound to the analyte when photolyzed to form the product. Only one form of the compound (i.e., bound or not bound) forms the photolyzed product.
In another aspect, the invention features kits for practicing the above method.
The term "hydrocarbyl" refers to an organic radical comprised of carbon chains to which hydrogen and other elements are attached. The term includes alkyl, alkenyl, alkynyl and aryl groups, groups which have a mixture of saturated and unsaturated bonds, carbocyclic rings and includes combinations of such groups. Hydrocarbyl can refer to straight chain, branched-chain, cyclic structures or combinations thereof.
The term "alkyl" refers to a branched or straight chain acyclic, monovalent saturated hydrocarbon radical of one to twenty carbon atoms.
The term "lower-alkyl" refers to an alkyl radical of one to six carbon atoms. This term is further exemplified by such radicals as methyl, ethyl, n-propyl, isopropyl, isobutyl, sec-butyl, n-butyl and tert-butyl, n-hexyl and 3-methylpentyl.
The term "alkenyl" refers to an unsaturated hydrocarbon radical which contains at least one carbon—carbon double bond and includes straight chain, branched chain and cyclic radicals.
The term "alkynyl" refers to an unsaturated hydrocarbon radical which contains at least one carbon-carbon triple bond and includes straight chain, branched chain and cyclic radicals.
The term "acyl" refers to the group R—C(O)—, wherein R represents a hydrocarbyl group or a heterocyclic or homocyclic aromatic group.
The term "lower" referred to herein in connection with organic radicals or compounds respectively defines such with up to and including six, preferably up to and including four carbon atoms. Such groups may be straight chain or branched.
The term "aryl" refers to an aromatic carbocyclic radical having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl, anthracenyl). Aryl radicals can be optionally substituted with substituents exemplified, but not limited, to those listed below.
The term "heterocyclic aromatic" refers to an aromatic mono- or poly-cyclic radical having at least one heteroatom within a ring, e.g., nitrogen, oxygen, or sulfur. For example, typical heteroaryl groups with one or more nitrogen atoms are tetrazoyl, pyrrolyl, pyridyl (e.g., 4-pyridyl, 3-pyridyl, 2-pyridyl), pyridazinyl, indolyl, quinolyl (e.g., 2-quinolyl, 3-quinolyl, etc.), imidazolyl, isoquinolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridonyl or pyridazinonyl; typical oxygen heteroaryl radicals with an oxygen atom are 2-furyl, 3-furyl or benzofuranyl; typical sulfur heteroaryl radicals are thienyl, and benzothienyl; typical mixed heteroatom heteroaryl radicals are furazanyl, oxazolyl, isoxazolyl, thiazolyl, and phenothiazinyl. Further the term also includes instances where a heteroatom within the ring has been oxidized, such as, for example, to form an N-oxide or sulfone. Heterocyclic aromatic radicals can be optionally substituted with substituents exemplified, but not limited, to those listed below.
The term "optionally substituted" refers to the presence or lack thereof of a substituent on the group being defined. When substitution is present, the group may be mono-, di- or tri-substituted, independently, with alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl, aminolower-alkyl, hydroxyl, thiol, amino, halo, nitro, lower-alkylthio, lower-alkoxy, mono-lower-alkylamino, di-lower-alkylamino, acyl, hydroxycarbonyl, lower-alkoxycarbonyl, hydroxysulfonyl, lower-alkoxysulfonyl, lower-alkylsulfonyl, lower-alkylsulfinyl, trifluoromethyl, cyano, tetrazoyl, carbamoyl, lower-alkylcarbamoyl, and di-lower-alkylcarbamoyl. The substituent can be a carboxylic acid, ester, sulfonamides, phosphono or phosphoryl group, or arsono group.
The term "pharmaceutically acceptable salt" refers to those salts which retain the biological effectiveness and properties of the free bases or free acids and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, benzoic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid and the like; or inorganic or organic bases.
The term "pharmaceutically acceptable ester" refers to those nontoxic readily hydrolyzable esters conventionally used in the pharmaceutical industry, especially α-acyloxyalkyl esters, such as acetoxymethyl esters. The invention contemplates those compounds of which are esters as described herein and at the same time are the pharmaceutically acceptable acid addition salts thereof. It is contemplated that pharmaceutically unacceptable salts and esters could be used if pharmaceutical acceptability were not desired, such as in certain in vitro studies.
The term "effective amount" refers to the amount which is necessary to create a desired effect. In the case when the desired effect is to determine accurately the intracellular concentration of an analyte by its binding to a photolyzable compound of this invention, the effective amount of the photolyzable compound (or its precursor) is that amount which allows that determination.
The term "[Ca2+]i" refers to the intracellular calcium ion concentration.
The term "chromophore" refers to a chemical grouping which when present in a compound (the chromogen) results in the displacement of, or appearance of, absorbent bands in the optical spectrum of the compound. Typically, the presence of the chromophore gives color to the compound by causing shifts in the visible or ultra-violet spectrum.
The term "fluorophore" refers to a chemical grouping which when present in a compound (the fluorogen) which is irradiated with light results in the compound emitting light of a wavelength different to the irradiated light. Typically, the wavelength of the emitted light is of longer wavelength than that of the irradiating light.
The term "photolyzable" or "photolabile" refers to a compound or chemical grouping, which when irradiated with light of an appropriate wavelength, undergoes a chemical transformation. Such chemical transformations can involve the making and/or breaking of covalent bonds in the compound.
The term "quantum efficiency" refers to the yield of product in a photochemical reaction compared to the amount of light applied.
As used herein, MOPS means 3-N-morpholino)-propanesulfonic acid.
As used herein, EGTA means ethylene glycol bis(-beta-aminoethyl ether)-N,N,N′,N′-tetracetic acid.
As used herein, BAPTA means 1,2-bis(2-aminophenoxy)-ethane-N,N,N′N′-tetraacetic acid.
As used herein, BAPTA-like means substituted derivatives of BAPTA which retain the essential characteristic of two bis(carboxymethyl)amino-substituted phenyl rings, the rings being linked at the positions ortho to the amines through a four atom bridge wherein the atom adjacent to each phenyl ring is N or O and the two center atoms are each C. By this definition, it is meant that "BAPTA-like" can include compounds like quin-1 and quin-2.
As used herein, quin-1 means 2-[[2-[bis(carboxymethyl)amino]-5-methylphenoxy]methyl]-8-[bis(carboxymethyl)amino]-quinoline.
As used herein, quin-2 means 2-[[2-[bis(carboxymethyl)amino]-5-methylphenoxy]-methyl]-6-methoxy-8-[bis(carboxymethyl)amino]-quinoline.
As used herein, nitr-7 means cis-1-(2-bis(carboxymethyl)amino-5-(1-hydroxy-1-(2-nitro-4,5-methylenedioxyphenyl)methyl)phenoxy)-2-(2-bis(carboxymethyl)amino-5-methylphenoxy)cyclopentane.
BAPTA and BAPTA-like compounds that can be used as starting materials can be obtained, for example, from Molecular Probes, Eugene, Oreg.
FIG. 1 shows the photochemistry of the mem-1 Ca2+ binding compound.
FIG. 2 shows the absorbance spectra of mem-1 bound or unbound to Ca2+.
FIG. 3 shows the snapshot memorization of intracelular Ca2+ using mem-1 by irradiating at >450 nm, reducing unphotolyzed mem-1 to an amine with dithiothreitol (DTT) and differentially detecting the photolyzed product, the isoxazole and the unphotolyzed product, the amine by fluorescence spectroscopy.
FIG. 4 shows fluorescence excitation spectra of the photolysis product, the isoxazole, and unphotolyzed product, the amine.
FIG. 5 shows fluorescence emission spectra of the photolysis product, the isoxazole, and unphotolyzed product, the amine.
FIG. 6 shows memory indicators for other physiologically significant cations.
FIG. 7 shows structures of aromatic azido-BAPTA linked memory indicators.
FIG. 8 shows a synthetic approach to memory indicators.
The invention features molecules that can be triggered by appropriate wavelengths of light to memorize the instantaneous spatial pattern of an ambient analyte as a stable snapshot that can be read out later, even after the analyte concentration has changed or the biological tissue has been fixed. The advantages of such analyte-memorizing molecules are that analyte concentrations can be simultaneously and instantly recorded in much larger numbers of cells or volumes of tissue and at much higher spatial and temporal resolution than presently possible. This improvement is because the final readout or imaging can be performed at leisure by optical cytometry or electron microscopy, after specimen fixation if desired, rather than in real time by imaging of the living tissue.
The molecules are composed of a binding site which is capable of binding or coordinating an analyte, a chromophore, and a photolabile group. The photolabile group undergoes an irreversible and detectable chemical transformation when the chromophore absorbs light. The binding site and the chromophore are linked in such a manner that the absorbance spectrum of the chromophore shifts when analyte binds to the binding site. Therefore, analyte binding renders the photolabile group more or less likely to undergo the photochemical transformation by a defined wavelength of light. For example, if analyte binding shifts the absorbance spectrum to shorter wavelengths, photolysis at a longer, defined wavelength converts molecules which are unbound by analyte to a different species, whereas molecules which were bound by analyte remain unchanged. Either or both of the original compound and reaction product from the photolysis can be detected subsequently. Since the reaction ceases when irradiation is stopped, the formation of the photolysis reaction product provides an imprint which freezes into place the analyte concentration which existed at the moment of irradiation, in both a spatial and temporal sense, preserving it for subsequent detection.
These compounds include a chromophore incorporating a photolabile group capable of undergoing an irreversible and detectable chemical transformation upon irradiation by light, the chromophore being linked to a binding site capable of binding an analyte, wherein binding of the analyte to the site alters an optical property of the chromophore, thus altering the ability of the photolabile group to undergo the chemical transformation upon irradiation at a defined wavelength of light. Such compounds can be represented by the generic formula I:
where P represents a photolabile group, C represents a chromophore and B represents a binding site which is capable of binding an analyte.
Photolabile groups are those groups which undergo an irreversible chemical transformation when irradiated with light. In this context, an "irreversible chemical transformation" refers to any transformation which results in the making or breaking of one or more covalent bonds. Thus, groups which do no more than undergo cis-trans isomerization of a carbon-carbon double bond are not categorized as photolabile groups in the context of this invention. Representative, but non-limiting examples of photolabile groups can be found in "Photolytic Deprotection and Activation of Functional Groups", by V. N. Pillai in Organic Photochemistry, A. Padwa (eds.), 9:225-323 (1987) (Marcel Dekker). Preferred photolabile groups for use in biological systems are those which undergo photolysis at wavelengths above 300 nm, preferably above 350 nm, since at these wavelengths most proteins, nucleic acids and other cellular constituents will not strongly absorb light and thus are less likely to undergo degradative or competitive reactions. The photolabile group can be attached to or partially or wholly embedded into a chromophore.
Azides, particularly aromatic azides, which undergo photolysis with relatively high efficiency are favored photosensitive groups (Reiser, A., Wagner, H. M. (1971) "Photochemistry of the azido group", in The Chemistry of the Azido Group (S. Patai, ed.), pp. 441-501. New York: Wiley). An example of a photolabile group disclosed herein is a β-azido, α,β-unsaturated carbonyl group. Upon irradiation with light of a suitable wavelength, the azido group extrudes nitrogen and the resulting nitrene cyclizes to an isoxazole. An azide can insert into a number of other chemical groups such as, but not limited to, nitro (to form a furoxan), azo (to form a triazole), carbon—carbon double bond (to form an indole), carbon-nitrogen double bond (to form a pyrazole or imidazole). Such groups can be incorporated into adjacent heterocyclic (e.g., pyridines or isoxazole) or aryl (e.g. phenyl, substituted phenyl rings). In addition, azides which are not adjacent to a suitable functionality will produce characteristic and defined products through well-known ring expansions (e.g., to form benzazirines) or addition reactions to intermediate reactive species such as nitrenium ions formed from nitrenes in aqueous reactions. Such products are distinctive and can be differentiated from the unphotolyzed precursor. Azides incorporated into carbon-carbon double bonds (e.g. vinyl azides) can also be used as they form distinctive azirine products upon photolysis.
Other photolabile groups which can be utilized include diazoketones that produce carboxylic acids on photolysis. Suitably attached nitrobenzyl groups photolyze to a nitrosobenzaldehyde or nitrosoacetophenone derivatives often with release of a carboxylic acid, alcohol, phosphate, amine, or amide.
As a result of the photolytic transformation, the photolytic reaction product will be different from and thereby distinguishable from the precursor unphotolyzed compound. Most conveniently, this distinction will be made based on some optical property of the compound, e.g., absorbance or fluorescence. However, as described below, the presence of the photolyzed reaction product can also be detected with specific binding reagents such as antibodies.
Typically, the photolabile group undergoes an irreversible reaction when exposed to light of an appropriate wavelength.
The chromophore can be any chemical grouping which absorbs light. Generally, chromophores will have at least one double bond, preferably a series of conjugated double bonds. Typical chromophores include aromatic and heteroaromatic groups, optionally substituted with electron donating or electron withdrawing groups. Many chromophores are known to those of skill in the art and this invention is not intended to be limited to particular groups of chromophores.
The chromophore is attached to an analyte binding site in a manner such that binding of analyte by the binding site alters an optical property of the chromophore thus allowing detection of binding of analyte to the compound. Generally, this is accomplished by a linkage between chromophore and binding site which allows for electronic coupling of the chromophore to the binding site. Typically, this electronic coupling is maintained by π-electron conjugation via carbon-carbon double and triple bonds, aromatic rings and lone pair electrons of heteroatoms. If linked by carbon-carbon bonds, as long as there is no more than one sigma bond in a row between the chromophore and the binding site, this electronic coupling will be maintained. The binding site may be linked to the chromophore via a single point of attachment or multiple points of attachment as is the case with binding sites which are fused onto the chromophore. The link between the chromophore and the binding site of the compound may also be a complex aromatic group or aromatic heterocycle, which in turn can be part of the chromophore or part of the binder or both.
In one embodiment of this invention, compounds disclosed herein have a structure represented by an azido-substituted aromatic ring A with additional substituents Y and Z and are represented by the formula:
and the pharmaceutically acceptable salts and esters thereof, wherein:
X is independently O, NH, S or CH═CH;
Y is an aromatic or heteroaromatic group carrying the binding site capable of binding an analyte and is fused or attached to a single atom of A;
Z is a substituent attached to A ortho to the azido group and is represented by R—C(O)—, wherein R is independently a hydrocarbyl radical or a heterocyclic aromatic radical. Examples include X=O, and Y=BAPTA where the furan ring is fused to one of the benzene rings of BAPTA. The compounds can be made by one of skill in the art.
Particular compounds are represented by the formula:
and the pharmaceutically acceptable salts and esters thereof, wherein
R1 is independently selected from the group consisting of acyl, carboxy, sulfonamido, phosphono, hydrocarbyl, cyano, and nitro. Structures of suitable compounds are shown in FIG. 7.
Other particular compounds are represented by the formula:
and the pharmaceutically acceptable salts and esters thereof, wherein:
W=O, S, NH, NR2, with R2 being aryl, heterocyclic aromatic, alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl, aminolower-alkyl, hydroxyl, thiol, amino, halo, nitro, lower-alkylthio, lower-alkoxy, mono-lower-alkylamino, di-lower-alkylamino, acyl, hydroxycarbonyl, lower-alkoxycarbonyl, hydroxysulfonyl, lower-alkoxysulfonyl, lower-alkylsulfonyl, lower-alkylsulfinyl, trifluoromethyl, cyano, tetrazoyl, carbamoyl, lower-alkylcarbamoyl, di-lower-alkylcarbamoyl, carboxylic acid, ester, sulfonamide, phosphono, phosphoryl group, or arsono, and wherein R1 is independently selected from the group consisting of acyl, carboxy, sulfonamido, phosphono, hydrocarbyl, cyano, and nitro.
The compounds may be additionally derivatized to facilitate loading into a biological cell. Frequently, the compounds will be membrane-permeant, readily hydrolyzable esters, such as α-acyloxyalkyl esters (e.g., acetoxymethyl) as are known in the pharmaceutical industry.
Binding of the analyte to the binding site alters an optical property of the chromophore such that the photolabile group is selectively protected from undergoing the photolytic chemical transformation. Typically, this is accomplished by a shift in the absorption spectrum of the chromophore when analyte binds to the binding site. For example, if analyte binding shifts the absorbance of the chromophore to a shorter wavelength, irradiation at a longer wavelength will not affect the photolabile group on the chromophore on analyte-bound compounds because they will not absorb the longer wavelength light energy. However, compounds which are not bound to analyte will absorb light energy at the longer wavelength and thus photolytic chemical transformation of the photolabile group will occur. The converse will hold for compounds in which analyte binding shifts the absorbance spectrum of the compound to longer wavelength. Irradiation at shorter wavelengths will result in selective photolysis of compound not bound to analyte. Because many chelating optical indicators shift their absorbance maxima to a substantial extent upon analyte binding, it generally will be possible to attach any of a wide variety of photolabile groups to an existing optical indicator and find a band of wavelengths at which there is a large difference in absorbance between the free and analyte-bound forms of the indicator. Irradiation at a selected wavelength as described above will result in photolysis and transformation of the photolabile group on the unprotected (i.e., analyte-unbound) indicator, thus allowing the detection of analyte-bound compound. One skilled in the art will recognize that one can also detect remaining unphotolyzed compound and derive similar information about analyte concentrations. In certain systems, some of which are exemplified below, it is desirable to detect both the photolyzed product and the unphotolyzed compound.
Alternatively, binding of the analyte can result in an increase (or decrease) in the absorbance intensity of the indicator, thus rendering the indicator more or less likely to photolyze. In compounds of this type, the absorption maxima can remain unchanged, but the intensity of the absorption can be altered. When a change in an optical property (e.g., absorbance or fluorescence) between photolyzed and unphotolyzed product is being used to derive an instantaneous snapshot of an analyte concentration, the change is ideally large enough to allow for good discrinmination between them. The shift in absorbance maxima or fluorescence excitation or emission maxima is typically at least about 25 nm, preferably about 50 nm and more preferably about 100 nm. If the shift in wavelength is not sufficiently large and a high degree of spectral overlap exists between the compounds, one can still achieve good discrimination by irradiating/detecting with selected band pass/cut off filters or using a laser to irradiate at a specific wavelength.
The binding sites that are present in the compounds of this invention are determined by the analyte that is being detected. In many cases the binding site will be a multidentate ligand which is capable of chelating the analyte. Crown ethers and cryptands can be used when the analyte being detected is a cation. The precise structural requirements which provide selectivity for one cation over another are known to those of skill in the art and will not be elaborated here. A particularly useful class of ligands is based on a octacoordinate tetracarboxylate chelating unit, such as, for example, is contained in an ethylene glycol bis(-beta-aminoethyl ether)-N,N,N′,N′-tetracetic acid unit (EGTA). BAPTA and BAPTA-like derivatives can also be used as chelating units in memory indicators.
The compounds described above can be used to "memorize" the intracellular concentration of an analyte. Biologically significant analytes include cations such as Ca2+, as well as, Mg2+, H+, and Na+. The methods include the steps of: (a) loading a cell with an effective amount of a memory indicator described earlier under conditions which do not alter the chromophore; (b) irradiating the cell with a wavelength of light which selectively photolyzes indicator which is not bound to the analyte, to form a chemically transformed product; (c) detecting the presence of the transformed product and/or unphotolyzed indicator; and (d) relating the presence of the transformed product and/or unphotolyzed indicator to the intracellular concentration of the analyte.
Most of the memory indicators disclosed by this invention are light sensitive, particularly to visible light and will photodegrade under ambient light conditions. Therefore, they typically can be loaded into the cell in the dark and then exposed to light of the appropriate wavelength. When carboxylic acid groups are present in the memory indicators, the compounds can be used as their α-acyloxyalkylester derivatives (e.g., acetoxymethylesters). Alternatively, the compounds can be loaded by direct microinjection, scrape loading, patch clamp techniques and the like.
It will frequently be necessary to destroy unphotolyzed indicator prior to detecting the presence and location of the photolyzed product. This is particularly true when the method of detection involves another irradiation step, albeit at a different wavelength, such as when one uses absorbance or fluorescence. Any convenient method can be used to destroy unphotolyzed indicator and the method will usually be determined by the functionality present in the photolabile group. For example, when the photolabile group is an azide, a convenient biologically compatible method which will not destroy the cellular integrity of the sample is reduction, e.g. with a thiol or dithiol such as for example, but not limited to, dithiothreitol (DTT), dithioerythritol (DTT), or a mild inorganic reductant (e.g., a hydride reductant such as for example, sodium cyanoborohydride).
The photolyzed and/or unphotolyzed indicators can be conveniently detected by fluorescence ratio imaging techniques as described in more detail below. Alternatively, the indicators can be detecting by staining with labelled specific binding reagents, such as monoclonal or polyclonal antibodies. The reagents can be used as labelled derivatives and their binding detected directly or any of a variety of labelled secondary reagents can be used to detect the presence of the first binding reagent.
Antibodies that can be used include any immunoglobulin having an area on its surface or in a cavity that specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of another molecule. The antibody can be polyclonal or monoclonal. Antibodies may include a complete immunoglobulin or fragments thereof, which immunoglobulins include the various classes and isotypes, such as IgA (IgA1 and IgA2), IgD, IgE, IgM, and IgG (IgG1, IgG2, IgG3, and IgG4) etc. Fragments thereof may include Fab, Fv and F(ab′)2, Fab′, and the like.
Useful labels that can be used to detect binding of a primary or secondary reagent in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others along with their precipitating substrates commonly used in histochemical staining), colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads and chemiluminescent labels. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
Another aspect of the invention provides a kit including a compound capable of memorizing the concentration of an analyte as described above, detection reagents and instructions for their use.
A suitable class of molecules whose photochemistry is controlled by Ca2+ is shown in FIG. 1 as "mem-1". When the molecules are Ca2+-free, they absorb light at >420 nm and undergo a ready photochemical reaction whose probable course is shown in FIG. 1. This reaction proceeds by extrusion of N2 from the azide group to give a nitrene, which attacks the adjacent carbonyl group to give an isoxazole ring. Mem-1 when Ca2+-free has an absorbance peak at 425 nm (ε=20,000 M−1cm−1), which shifts to 375 nm (ε=23,000 M−1cm−1) when Ca2+ binds (FIG. 2). For wavelengths >420 nm, the Ca2+-free form absorbs much more strongly than the Ca2+ complex, so that the former photolyzes about 20-fold faster than the latter. Thus, near 420-440 nm, the sensitivity to light or product of the photolysis quantum efficiency and extinction coefficient is 2700 M−1cm−1 for the free dye compared to 134 M−1cm−1 for its Ca2+ complex. High [Ca2+]i can protect mem-1 from visible-light photolysis by shifling the absorbance peak into the ultraviolet. Because many optical indicators shift their absorbance maxima to a substantial extent upon cation binding, and because azides attached to chromophores generally photolyze with high efficiency, it should be possible in most cases to attach an azide moiety to such an existing cation indicator and find a band of wavelengths at which there is a large difference in absorbance and photolytic sensitivity between the free and cation-bound forms of the indicator. In the case of mem-1, the Ca2+ dissociation constant is about 530 nM, so that in resting unstimulated cells where [Ca2+]i is <100 nM, mem-1 will be largely Ca2+-free and photolyzable at wavelengths >420 nb, whereas in activated cells or regions of cells where [Ca2+]i>1 μM, the mem-1 will be resistant to such wavelengths. Restriction to wavelengths >450 nm will give the highest discriminations between the free and Ca2+-bound forms at the cost of requiring somewhat higher light intensities.
If subcellular resolution is desired, attachment to cell macromolecules is necessary to prevent difflusion of the probe after flashing and during fixation. Mem-1 has been prepared with R=NCS (isothiocyanate) and other amine-reactive groups such as N-hydroxysuccinimide esters. Other functionalities which can be used to label macromolecules can be found in the Handbook of Protein Conjugation (CRC Press, 1991). It has been verified that such molecules label amino acids or proteins such as serum albumin within a few minutes, and that the conjugates retain the relevant properties desired (the above spectral and binding data were obtained on the glycine conjugate, (R=NHCSNHCH2COO−). The isothiocyanate also seems to attach to proteins in real cells, because within a few minutes after injection into cells such as fibroblasts, some dye was retained upon cell permeabilization.
Mem-1 would be used by ester-loading or injection or perfusion into cells, then waiting briefly for covalent attachment, all the while avoiding illumination <500 nm. In other words, it would be handled only in the dark or under yellow or red safelight illumination. At the appropriate moment for [Ca2+]i to be recorded, at a desired interval after physiological or pharmacological stimulation of the tissue, a flashlamp with appropriate wavelength filters or a laser would be fired to illuminate the tissue at wavelengths of 450-500 nm. Spatially uniform illumination will trigger a photochemical change in predominantly those molecules sitting in regions of low [Ca2+]i, whereas where [Ca2+]i is high, the molecules will be mostly unaffected because they had bound Ca2+ and could not absorb the triggering photons. Finally, the tissue will be frozen and/or fixed by conventional means and prepared for cytometry, fluorescence microscopy, or immunohistochemistry to map the extent of photolysis.
Direct fluorescence cytometry or microscopy would rely on the fluorescence of the isoxazole photolysis product of mem-1, whose excitation maximum is 354-385 nm depending on Ca2+, and emission maximum is 490 nm. In order to monitor this fluorescence without photolyzing previously unphotolyzed mem-1, the azide groups are removed by reduction to amino groups, for example using a dithiol such as dithiothreitol or 1,3-propanedithiol (Bayley, H., Standring, D. N., Knowles, J. R. (1978) Tetrahedron Lett. 36, 33-3634). This step does not affect the isoxazole, and is analogous to the need to put photographic film in fixer to remove unreacted silver halide before turning on the room lights. The chemical reduction of the azide takes a few minutes at room temperature and produces an amine that is fairly photostable and also fluorescent. Its excitation and emission are at longer wavelengths than those of the isoxazole photoproduct, so fluorescence ratio imaging can distinguish isoxazole photoproduct from unphotolyzed mem-1 reduced to amine.
FIG. 3 summarizes the chemistry of photolysis vs. reduction: mem-1 in low Ca2+ takes the left-hand path to the isoxazole, whereas in high Ca2+ it takes the right-hand path to the amine. FIG. 4 compares the normalized excitation spectra of the isoxazole photoproduct (solid line, peaking at 376 nm) and the amine reduction product (dashed line, peaking at 408 nm), each separately coupled to bovine serum albumin (R=NHCSNH . . . protein), fixed with dimethyl suberimidate, and measured in Ca2+-free conditions (excess EGTA). FIG. 5 shows the corresponding emission spectra, peaking at 498 nm (isoxazole) and 520 nm (amine) respectively. The excitation and emission wavelengths that maximize the distinguishability of the isoxazole from the amine are not the above peaks but are displaced to either side, namely 380 nm excitation, 460 nm emission vs. 440 nm excitation, 550 nm emission. The ratio of fluorescences at these two wavelength sets changes from 0.21 for pure isoxazole to 1.74 for the pure amine. Intermediate [Ca2+]i concentrations giving rise to mixtures of isoxazole and amine would give intermediate fluorescence ratios that could be calibrated back to the [Ca2+]i values.
An alternative method for detecting the extent of photolysis would be to expose the tissue sections to specific antibodies (polyclonals or monoclonals) previously raised against protein conjugates of the photolyzed or unphotolyzed indicator. Both are the right size to be haptens, are separately available as pure chemical species, and are already conveniently flurnished with reactive groups for conjugate to carrier proteins or to affinity columns for antibody purification by adsorption of cross-reacting species. Antibodies are known to make structural discriminations much subtler than the differences between the isoxazole photoproduct and the starting azide or the amine reduction product (Pressman, D., Grossberg, A. L. (1968) The Structural Basis of Antibody Specificity. (New York: Benjamin); Karush, F. (1962) Adv. Immunol. 2:1-40), so raising discriminating antibodies should be possible. If necessary, monoclonal antibodies would be generated. The antibodies can be labelled with any of a variety of conventional labels and thereby visualized. For example, the antibodies would then be labelled and visualized by conventional fluorescein or rhodamine, that are excited or that emit at wavelengths different from the mem-1 products. Alternatively fluorescently-labeled antibodies against the first antibodies would be applied as in conventional indirect "sandwich" immunofluorescence. If visualization by electron microscopy is needed, this would simply require substitution of electron-dense labels such as ferritin, colloidal gold, iron-dextran complexes, or peroxidase-antiperoxidase complexes (Stemberger, L. A. (1986) Immunocytochemisty, 3rd ed. New York: Wiley). Ideally one would stain simultaneously and independently for both the photolyzed and unphotolyzed indicator species, for example with a fluorescein-labeled antibody to the photolyzed and rhodamine-labeled antibody to the unphotolyzed. The ratio of red to green immunofluorescence would then reflect the [Ca2+]i during the flash. If one stained for only one species, say the photolyzed, an absence of stain could mean either that [Ca2+]i had been very high or that insufficient indicator had penetrated into that region of cytoplasm. Ratio recording removes that ambiguity. Ratio recording in electron microscopy is possible with a pair of distinguishable labels such as ferritin and iron dextran (Dutton, A. H.,Tokuyasu, K. T., Singer, S. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76:3392-3396) or large and small gold particles (DeMey, J. (1983) "Colloidal gold particles in immunocytochemistry", in Immunocytochemistry: Practical Applications in Pathology and Biology (J. M. Polak and S. Van Noorden, eds). Bristol: Wright PSG), or one could apply alternate antibodies to alternate serial sections.
Regardless of the approach used to measure photolysis, this method will sacrifice continuous recording in favor of spatial resolution during a single photochemical exposure. All other methods at present make the opposite choice. Spatial resolution would be particularly valuable in neurobiological applications, since adjacent neurons are generally expected to be doing different things while processing information. It would also be highly valuable in pharmaceutical screening of large numbers of compounds in parallel, for example arising from combinatorial libraries, because the peak response of cells to each compound could be resolved. The limit on time resolution will be set by the rapidity with which the indicator binds and releases Ca2+, which for this class of indicator is typically in the millisecond time domain (Kao, J. P. Y., Tsien, R. Y. (1988) Biophys. J 53: 635-639). Photodynamic damage should not be important because it can hardly build up significantly in a millisecond of flash illumination. This method is much more tolerant of mechanical thickness, light scattering and tissue pulsation than any method trying to measure images of live tissue. As long as the photolysis flash can penetrate the tissue, randomization of the photon paths is irrelevant. Mechanical thickness and light scattering are also unimportant during readout because the tissue will have been sectioned and cleared.
The concentration of intracellular dye should not need to exceed a few micromolar, because immunocytochemistry is known to be able to trace proteins whose concentration in cytoplasm is not greater. Such low concentrations have usually been easily attained or exceeded by intracellular hydrolysis of membrane- permeant esters. Micromolar concentrations of cytoplasmic Ca2+ chelators have generally been without effect on physiological responses because higher concentrations of Ca2+-binding proteins are already present in vivo. To average [Ca2+]i over a longer period, the illumination could be weakened and prolonged. Alternatively, if the stimulus is repetitive, the flashes could be likewise repeated at a given phase delay in order to signal-average the response.
If not all the dye molecules are securely anchored when the flash arrives, the mobile molecules should not interfere so long as they do not bind after the flash. This is because they should get washed out of the tissue section during the histology. Also, if subcellular spatial resolution is not required, covalent reaction with cellular macromolecules should not be necessary, because the indicator molecules should remain trapped in their respective cells. Therefore R could be simply H or COO−. One would merely have to avoid membrane permeabilization during the subsequent processing.
Calibration of [Ca2+]i would be easiest with detection of the ratio between the photolyzed and unphotolyzed species, since the absolute amount of indicator would not matter. Comparisons would be made with the appearance of protein gels or cells photolyzed while containing known [Ca2+]i concentrations. The method should then tolerate a certain amount of crosstalk, for example incomplete spectra separation between the Ca2+-free and Ca2+-bound species, or insufficient light intensity or quantum yield to completely photolyze all the molecules that ought to photolyze, or imperfect antibody specificity. While these types of deficiencies would reduce the maximum possible image contrast between very high and very low [Ca2+]i, they would not invalidate the [Ca2+]i reading so long as they also apply to the calibration protein gels or cells.
FIG. 6 shows how the memory indicator concept might be extended to other cations. The structure proposed as a memory indicator for H+ is a hybrid of the present azidoketone with the known fluorescent pH indicator "quene-1" (Rogers, J., Hesketh, T. R., Smith, G. A., Metcalfe, J. C. (1983) J Biol. Chem. 258:5994-5997). Likewise the Na+- and Mg2+-memorizing molecules combine the azidoketone with known binding sites for those cations respectively (Minta, A., Tsien, R. Y. (1989) J Biol. Chem. 264:19449-19457; Raju, B., Murphy, E., Levy, L. A., Hall, R. D., London, R. E. (1989) Am. J Physiol. 256:C540-C548. Ligands which selectively bind to biologically significant analytes are known to those of skill in the art and it is contemplated that indicators made from all such ligands linked to chromophores with a photolabile group as described herein can be used in this invention.
The indicator would be loaded into the tissue by standard methods such as membrane-permeant, hydrolyzable esters, microinjection, or dialysis through a patch pipet. If subcellular resolution is desired, the indicator molecules should carry chemically reactive groups to covalently attach them to random cellular macromolecules. At the moment at which [Ca2+]i is to be memorized, the specimen is illuminated with a brief flash of light of appropriate wavelengths. The local degree of progress of the photochemical reaction constitutes a latent but irreversible image of the [Ca2+]i levels existing during the flash. Subsequent changes in [Ca2+]i have no influence because the light necessary for the photochemistry is no longer being delivered. After stabilization of the tissue, for example by freezing or fixation and sectioning, and chemical removal of any remaining light-sensitive moieties, the latent image is visualized either by the difference in fluorescence of the molecules that have vs. have not undergone the photochemistry, or by standard techniques of immunocytochemistry with tagged antibodies specific for the same two sets of molecules.
The synthesis of memory indicators is generally presented in FIG. 8. The synthetic scheme was carried out as follows:
Synthesis of the starting materials:
as described for Y=H, Z=O, Hal=Br (Pascal Y., Ann. Chim. 3:245-275, 1968) and Y=H, Z=S, NH, NMe (Arcoria, A., Fisichella, E. Maccarone, and Scarlata, G. J. HeterocycL Chem. 12:215-218, 1975).
Chemicals (Aldrich; Milwaukee, Wis., U.S.A.) and solvents (HPLC-grade, Fisher; Fair Lawn, N.J,, U.S.A.) were used directly as received unless otherwise noted. Chloroform and dimethylformamide (DMF) were dried over 4 Å molecular sieve.
Proton magnetic resonance spectra (1H NMR) were recorded on a Gemini 200-MHz spectrometer (Varian; Palo Alto, Calif., U.S.A.) in CDCl3 unless otherwise noted, and the chemical shifts are given in δ values relative to tetramethylsilane. Ultra-violet spectra were recorded on a Lambda Array 3840 spectrophotometer (Perkin-Elmer; Norwalk, CT, CA) or a Cary 3E UV-Visible spectrophotometer (Varian; Palo Alto, Calif., U.S.A.) at 20� C.
Thin layer chromatography (TLC) was carried out on precoated silica gel 60F-254 or reverse-phase RP-18, F-254 plates (E. Merck, EM Separations; Gibbstown, N.J., U.S.A.). For column chromatography, silica gel 60 (230-400 mesh, E. Merck) was used. All manipulations of compounds sensitive to near ultraviolet light were performed under an orange safety lamp.
Amine (2): Cyanophenol 1 (prepared as described in U.S. Pat. No. 5,552,555) (65 mg, 0.1 mmol), phenacyl bromide (0.12 mmol, 24 mg) and anhydrous K2CO3 in dry DMF (0.25 mL) were heated at 120� C. for 30 minutes. After cooling, H2O (5 mL) and HOAc (2 drops) were added and the crude product was extracted into ethyl acetate (3�10 mL). After drying (Na2SO4), evaporation and separation by silica gel chromatography (ethyl acetate:hexane 1:1) the product, 2 was obtained as yellow solid. Yield, 51 mg.
Azide (3): Amine 2 (30 mg, 0.039 mmol) was dissolved in HOAc (0.2 mL) at room temperature and added dropwise to an ice-cold solution of nitrosylhydrogen sulfate (NOHSO4)(30 mg) in conc. H2SO4 (0.3 mL). After stirring on ice for 30 mins, the mixture was added dropwise to an ice-cold saturated a aqueous solution of NaN3 (CARE: Hydrazoic acid formed; carry out in well-ventilated fume-hood). The mixture was neutralized with sodium acetate trihydrate (2 g) and extracted with ethyl acetate (3�30 mL). After drying, the crude product was separated by silica gel chromatography (ethyl acetate:hexane 1:1) to give a yellow solid (17 mg after trituration with EtOH).
4-Trifluoroacetamido-acetophenone: 4-Aminoacetophenone (1 mmol, 135 mg) was dissolved in trifluoroacetic acid (1 mL) at −10� C. under argon. Trifluoroacetic anhydride (2 mmol, 0.28 mL) were added dropwise with stirring. The reaction mixture was allowed to warm to room temperature for 15 mins and then evaporated to dryness. The resulting solid was triturated with isopropyl ether to yield the product as a white solid (yield, 84%).
2′-Bromo-4-trifluoroacetamido-acetophenone: 4-Trifluoroacetamido-acetophenone (1.8 g, 8 mmol) were dissolved in dioxane:diethyl ether (1:1 v/v; 10 mL) and cooled to −15� C. Bromine (8.1 mmol, 0.42 mL) was added dropwise allowing the reaction mixture to decolorize between additions. After 20 minutes, the solution was poured into H2O (50 mL) and extracted into ethyl acetate (3�30 mL), dried (Na2SO4) and evaporated. The crude product was recrystallized from 95% EtOH to yield the product as white crystals (Yield, 40%).
The intracellular calcium concentration can be memorized by ratio imaging and, for example, antibody staining.
The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity and understanding. Other embodiments are within the claims. It will be obvious to one of skill in the art that changes and modifications may be practiced within the scope of the appended claims. Therefore, it is to be understood that the above description is intended to be illustrative and not restrictive. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the following appended claims, along with the full scope of equivalents to which such claims are entitled.
All patents, patent applications and publications cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual patent, patent application or publication were so individually denoted.
|Cited Patent
|Filing date
|Publication date
|Nov 6, 1972
|Jun 18, 1974
|Syva Corp
|Enzyme amplification assay
|Oct 29, 1971
|Nov 26, 1974
|Akzona Inc
|Process for the demonstration and determination of low molecular compounds and of proteins capable of binding these compounds specifically
|Apr 29, 1974
|Feb 17, 1976
|Board Of Trustees Of The Leland Stanford Junior University
|Fluorescent immunoassay employing total reflection for activation
|Jun 30, 1975
|Dec 7, 1976
|Syva Company
|Fluorescence quenching with immunological pairs in immunoassays
|Nov 24, 1978
|Jun 23, 1981
|Syva Company
|Macromolecular environment control in specific receptor assays
|Dec 10, 1979
|Jul 7, 1981
|Syva Company
|Kit for carrying out chemically induced fluorescence immunoassay
|Aug 7, 1980
|Dec 28, 1982
|Syva Company
|Concentrating zone method in heterogeneous immunoassays
|Sep 7, 1984
|Jul 29, 1986
|The Regents Of The University Of California
|Fluorescent indicator dyes for calcium ions
|Sep 7, 1984
|Aug 25, 1987
|The Regents Of The University Of California
|Chelators whose affinity for calcium is decreased by illumination
|May 13, 1987
|Feb 21, 1989
|Regents Of The University Of California
|Photosensitive calcium chelators
|Sep 25, 1989
|Aug 25, 1992
|The Regents Of The University Of California
|Chelators whose affinity for calcium ion is increased by illumination
|Feb 27, 1992
|Nov 16, 1993
|Miles Inc.
|Colorimetric methods and reagents for the assay of calcium in a test sample
|Oct 22, 1992
|May 10, 1994
|Miles Inc.
|Arylazo chromoionophores
|Dec 30, 1992
|Apr 25, 1995
|The University Of Maryland At Baltimore
|Long-wavelength fluorescent probe compounds for calcium ions and their use in ratiometrically measuring calcium ion concentrations
|Jun 6, 1995
|Sep 3, 1996
|The Regents Of University Of California
|Azide-containing chelators whose affinity for calcium ion is decreased by illumination
|Sep 9, 1985
|Apr 9, 1986
|The Regents Of The University Of California
|Chelators whose affinity for calcium is decreased by illumination
|Am. J. Physiol., Raju 1989, 256:C540-48.
|Biophysics J., 53:635-39, Kao, Tsien 1988.
|J. Biol Chem., Minta 1989, 264:19449-57.
|J. Biol. Chem., Rogers 1983, 258:5994-97.
|PNAS USA, Dutton (1979) 76:3392-3396.
|Citing Patent
|Filing date
|Publication date
|Sep 11, 2008
|Oct 23, 2012
|International Business Machines Corporation
|Techniques for recording signals
|Aug 13, 2003
|Feb 17, 2005
|International Business Machines Corporation
|Techniques for recording signals
|Sep 29, 2004
|Aug 18, 2005
|Affinium Pharmaceuticals, Inc.
|Novel purified polypeptides from bacteria
|Apr 10, 2009
|Aug 13, 2009
|Lamdagen Corporation
|Optical system including nanostructures for biological or chemical sensing
|U.S. Classification
|436/79, 549/468, 549/60, 436/519, 549/467, 436/805, 548/242, 548/525, 435/40.5, 435/7.21, 540/461, 436/537, 546/89
|International Classification
|Cooperative Classification
|European Classification
|Sep 18, 1998
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text:
I know that you can open a window in kiosk mode from the command line by using -k, but I want to do it programmatically. How do you do this from within a .asp page?
P.S. Just to save everybody a lot of typing, I know I can do something "similar" through Javascript. I'm wondering if I can do true kiosk mode from VB.
Thanks for any help you can provide!
Steve Manning

subset: commoncrawl


text:
During story time, children learn about books, how to handle them, that words read left to right on the page, that reading starts at the top of the page, and that pictures tell us more about the story. So much more happens, though, when the story being read is purposely accompanied by activities or related stories that further stretch the literacy experience! By integrating the story into other activities such as drawing, letter recognition, fine motor, matching, sorting, and sequencing, you are laying the groundwork for growing a reader in way that is interactive and fun!
Dragon's Fat Cat
Hi, Cat!
I Must Have Bobo!
If You Give a Cat a Cupcake
Kitten's First Full Moon
Kitty Cat, Kitty Cat, Are You Going to Sleep?
Millions of Cats
Skippyjon Jones
Skippyjon Jones and the Big Bones
Skippyjon Jones Class Action
Skippyjon Jones in Mummy Trouble
Skippyjon Jones in the Doghouse
Skippyjon Jones Lost in Spice
Three Little Kittens
Alexander and the Terrible, Horrible, No Good, Very Bad Day
Glad Monster, Sad Monster
I Was So Mad
The Little Old Lady Who Was Not Afraid of Anything
Lots of Feelings (non-fiction)
The Pigeon Has Feelings, Too!
Today I Feel Silly
The Very Lonely Firefly
Wemberly Worried
What Are You so Grumpy About?
When Sophie Gets Angry - Really, Really Angry...
A House is a House for Me
The Emperor's Egg
Grandfather's Journey
The House in the Night
How I Became a Pirate
If the Dinosaurs Came Back
Jamaica Louise James
Ladybugs Can't Be Tall
The Little Red Hen and the Ear of Wheat
My Little Sister Ate One Hare
Song and Dance Man
Rattletrap Car
The Seven Silly Eaters
This is a Tree
Walter Was Worried
Yesterday I Had the Blues
ABC I Like Me
Giraffes Can't Dance
I Like Myself!
I'm Gonna Like Me: Letting Off a Little Self-Esteem
January's Child the Birthday Month Book
Stand Tall Molly Lou Melon
The Eensy Weensy Spider Freaks Out!
The Giant Jellybean Jar
The Littlest Owl
The Sissy Duckling
Velma Gratch & the Way Cool Butterfly
Blueberries for Sal
Curious George
The Giving Tree
Goodnight Moon
Green Eggs and Ham
If You Give a Mouse a Cookie
The Keeping Quilt
The Little Engine that Could
The Little House
Love You Forever
Make Way for Ducklings
The Mitten
The Mysteries of Harris Burdick
No, David!
The Other Side
Owl Moon
The Polar Express
Skippyjon Jones
The Snowman
The Snowy Day
Sylvester and the Magic Pebble
The Very Hungry Caterpillar
Where the Wild Things Are
Arthur's Family Vacation
Carl's Summer Vacation
Eva's Summer Vacation
The House Takes a Vacation
How I Spent My Summer Vacation
Pictures From Our Vacation
Strega Nona Takes a Vacation
Tiffany's Weird, Totally True Summer Vacation
Vacations Over! Return of the Dinosaurs
What I Did on My Summer Vacation
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I added Proffesor Akuroma. According to CoroCoro, he's the professor in Black and White 2. The Science. 16:56, April 12, 2012 (UTC)
Professor Birch gives Johto starters in Emerald, doesn't he? It's been years since I completed that Pokédex, but I'm almost positive he does.Casus-luciferi (talk) 02:44, May 28, 2016 (UTC)


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Since 2010 Courchevel is back on the Alpine Ski World Cup circuit with a women's event.
This winter, Courchevel will also host the ski mountaineering World Cup.
Courchevel has been hosted the summer International Ski Jumping Grand Prix for 23 years.
During the 16th Winter Olympics in 1992, which took place in Albertville, Courchevel hosted ski jump trials and the Nordic Combined. The resort also accommodated the International Olympic Committee.
Courchevel is proud to have six promising athletes who might compete in the coming Olympic Games: Alexis Pinturault, Anne-Sophie Barthet, Taïna Barioz, Nicolas Mayer, Arnaud Dunand and Léa Lemare.
Courchevel has been acknowledged over the years as a prestige ski resort, ideally located in the 3 Valleys, the world's biggest ski area.
Austria's Eva-Maria Brem, who was first after the opening run, clocked a combined time of 2:01.72 to edge Norway's Nina Loeseth and Switzerland's Lara Gut, who tied for second, 0.17 seconds out.
It's Throwback Thursday today we want to revisit some of the most exciting tech races this past winter… So this is how things went down last December.


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If you have troubles since the update to WordPress 4.5 please read this post before opening a new thread.
Here's the site: http://www.jiwire.com/
if you scroll down below the fold the layersliders and enfold elements start to appear through the upper navigation of the header. Is there a hotfix for this? Was working fine prior to the update.
Thank you
Hi kbreslin!
You can add this on your custom.css or Quick CSS:
.avia-layerslider {
z-index: 0;
position: relative;
.template-page.content.twelve.alpha.units {
z-index: 0;
.fixed_header #header {
position: fixed;
z-index: 9999;
Remove browser cache then reload the page a few times.
That worked! Thanks Ismael
The topic 'Fixed Header Navigation and Menus Become Transparent After 2.3 Update' is closed to new replies.


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From Our Editors
Atlanta Workshop Players was founded in 1981 as the brainchild of seasoned Executive Director Lynn Stallings. Since its inception, the company—a creative springboard for young children, teenagers, and adults—has produced dozens of plays and original musicals. FOX 5 spotlighted the program's fun-filled curriculum, which strives to improve self-confidence in students. Stallings and her husband, Don, get into the act as well, sometimes teaching classes alongside guest artists such as William Shakespeare's ghost.


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Yes,celtics should be in the mix. East finals even possible tittle. It would be nice to see. Boston will miss Allen thought. I don't agree that he was more than replaced with terry,lee,Bradley. All good player but their no Allen . On or off the court!
- Isaiah Thomas Narrowly Avoids Suspension
- Can The Celtics Make a Run In The Playoffs?
- Isaiah Thomas Key To Celtics Capturing Atlantic Division Title
- Ainge and the Celtics Maintain "Status Quo" as Trade Deadline Passes
- Celtics Can't Battle Back, Lose to Ginobli and Spurs 108-105
- Thomas Lead Celtics Drubbing of Cousins and Kings 114-97
- Amir Johnson, Avery Bradley Lead Celtics To Win Over Heat, 105-95
- Magic and Oladipo Stop Celtics 110-91
- Celtics Dominate Wizards, Led by Thomas, 111-78
- Celtics Eek Out Victory Versus Hapless Sixers, 84-80
2012-13 Celtics Are On a Mission To Hoist Another Championship Banner
- Updated: October 27, 2012
Last season, the Celtics season shockingly ended abruptly after folding to the eventual Champion Miami Heat in the Eastern Conference playoffs. Not that they didn't make a good run. They came back from a difficult start and rolled through the playoffs looking like they were in good form. However, after some controversial contests with the Heat where there were a number of questionable calls they almost made it to the next stage…. a place that Celtics fans are more than familiar with…. the NBA Finals!!!!
The NBA finals and the subsequent Championship are well within reach and seems to be this year's Celtics' chief goal and mission. Led by experienced coaching veteran Doc Rivers, who many are pegging to be frontrunner as the next Olympic Coach, the Celtics are determined and will settle for nothing less than Title #18, maintaining their postion as the Basketball team with the most Championships!!!
In the aftermath of last year's early exit from the playoffs, during a chaotic NBA shortened season, the Celtics would add many new players and reform their team while keeping their core group of players from 2008! They have Kevin Garnett, Rajon Rondo, Paul Pierce, and would have had Ray Allen if he decided to stay! In a move that made many waves, Ray Allen went to the Miami Heat. However, we thanks him for his time in Boston and he will be more than replaced by Incredible bench players like 3-point specialist Jason Terry, Courtney Lee, and Avery Bradley(who had a breakout performance season last year)! The Celtics will more than bounce back from the loss of Allen with these three fine players.
The player who must now rise as leader is the capable Rajon Rondo. Perhaps the most talented point-guard in the league. He is also enterring his prime playing years!! Media reports are accurate in saying…."It's Rondo's team now!" We look forward to what he can do and not only will Pierce, Garnett, and Co. be there for support but a much deeper bench with newly-acquired proven players will be there for relief! Under the experienced tutelage of former point guard and now Head Coach Doc Rivers Rondo has all that he needs to showcase what he can do on the hardwood!!! The Celtics will be counting on him.
In a postive leadership sign… Rondo called and organized practice scrimmages in L.A. during the off-season inviting many Celtics players to refine their skills while learning to play together. They even squeezed in a few fun flag-football sessions in between. Garnett and Rondo joked with each other about their newly-found prowess on the football field.
Well that's the long(Garnett) and short(Rondo) of it… LOL! All in all the Celtics look to be fired up for another exciting championship run. With so many teams adding depth the NBA predictions won't be as easy this year. Boston certainly has the experience, personnel, players, etc. It won't be easy though. The fans will be there supporting their team like they always do. It's Autumn time in New England and that means the end of baseball while we transition inside the gym to Basketball. Did I mention first regular season game is a re-match against the Miami Heat on Tuesday Night October 30, 2012? I can hardly wait.
Latest posts by Khai Pham (see all)
- Isaiah Thomas Narrowly Avoids Suspension - April 25, 2016
- Can The Celtics Make a Run In The Playoffs? - February 26, 2016
- Isaiah Thomas Key To Celtics Capturing Atlantic Division Title - February 26, 2016


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Somewhere under the Atlantic, a Soviet sub commander has just made a fateful decision: the Red October is heading west. The Americans want her. The Russians want her back. And the most incredible chase in history is on....
The Hunt for Red October is the runaway bestseller that launched Tom Clancy's phenomenal career. A military thriller so accurate and convincing that the author was rumored to have been debriefed by the White House. Its theme: the greatest espionage coup in history. Its story: the chase for a runaway top secret Russian missile sub. --This text refers to an out of print or unavailable edition of this title.
"Flawless…frighteningly genuine."—The Wall Street Journal
"Remarkable…intricate and nerve tingling."—Clive Cussler
"Gripping narrative…Navy buffs and thriller adepts have been mesmerized."—Time --This text refers to the Paperback edition.
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Customer Reviews
Top Customer Reviews
Tom Clancy has churned out dozens of novels since, of descending quality; "Red October" was his first and it's by far his best. He goes into great technical detail describing the workings of a submarine but he explains it so well that any reader can understand what he's saying. His characters aren't very profound, but characterization isn't the main draw here; the hunt and the subsequent chase of Red October by the Americans, who want to keep it and the crew safe, and the Russians, who want to destroy it at any cost, keep us spellbound while turning page after page. Clancy didn't fall into the trap of venting out the political propaganda that spoiled so many of his later books; he keeps "Red October" squarely on target, concentrating on the thrill of the chase, and the actions rockets along non-stop from the first page to the last. If you read only one Clancy novel, this is definitely the one to go with. None of his other books can touch it.
This deception is made doubly daring by the Soviet Navy's claim that he is a renegade who threatens independent missile launch. Luckily, the crew believes him after his sub is fired upon by a Soviet aircraft, narrowly escaping.
He says, "if they were really shooting at us, we'd be dead." This study in the psychology of leadership presents a fascinating conclusion: though there are only 12 officers aboard, the 180+ enlisted men obey them faithfully, simply because the naval code requires it. Apparently, that's what has the navigator so worked up when he exclaims, "we could have a mutiny on our hands."
The possibility of a renegade or "rogue" launching nuclear weapons is quite real. In today's new world order, that possibility is increased dramatically, as evidenced by the recent crisis in the formerly Soviet Chechnya. Some of the new states in the Commonwealth have nuclear weapons which were strategically placed by the Soviet equivalent of the Strategic Air Command.
Thus the Soviet ploy of telling the U.S. that the missile launch was imminent was actually a shrewd move. In this way, the U.S. would have to destroy the sub or else let on that top officials knew Ramius's true intentions, alerting the Soviets that their leadership had been penetrated by CIA.
However, since Ryan acted alone and used his own instinct, the U.S. was able to simulate the destruction of the sub and take it to Norfolk, VA, where it may remain today.
Interestingly enough, President Reagan endorsed this book when it was first published, implying that the story is true.Read more ›
It is December 1986 and Captain First Rank Marko Ramius, son of a dominant Communist Party Secretary and the Soviet Union's most confident and acclaimed submarine commander, is setting out to sea with his country's newest and greatest military venture. She contains an advanced new quiet propulsion system that allows her to almost perfectly escape both radar and sonar detection, a terrifying first strike vehicle. She is also carrying one-hundred and eighty two nuclear warheads and twenty-six missile delivery systems. Her name is Krazny Oktyabr (Red October) .Once at sea, Ramius contravenes his orders and disappears into the North Atlantic after setting a course for the Eastern Seaboard of the United States. Both Soviet and American leaders are petrified, but they insist on snowballing each other with false information. Fears of either a surprise attack on the US or a possible defection abound. Both navies go on full alert, eventually resulting in a massive confrontation in the North Atlantic. And, just for kicks, Clancy tosses the British into the mix to really confuse things!
As Clancy's first, and quite possibly best, work, I feel that this is a very firmly woven story all together. The characters, even those like the C.I.A. Deputy Director (Operations), Robert Ritter who do not appear for very long are exceedingly well developed and presented. The details in the book are amazing, from the encrypted messages to the details and intricacies of the interior of the submarines and the extensive knowledge of military protocol and procedure presented. It definitely gives an "eye opening" experience to the reader.Read more ›
Most recent customer reviews
Intense action. Many twists. Riveting action and intrigue! A book I could not put down. A must read for sure!Published 1 month ago by Alfred Clarke
Great story ... and now I have it in hardcover edition !Published 14 months ago by Pierre Desjardins
Have read every Tom Clancy book and enjoyed each and everyone totally . I wish the producers of the Movie had followed the book more closely as I think it could have won an award... Read morePublished 15 months ago by Richard Arklie
Reagan called this the perfect yarn, and I concur. I was not a president however and I cannot evaluate the realism and detail of the geo-political details in the book, but they... Read morePublished on Feb. 19 2014 by elginblatherford
Had to purchase the book because it was not available for Kindle.
That seems to be happening a lot, but it's never talked about in reviews of Kindle. Read more
The book is full of action , suspense, drama and amazingly moving characters . Also recommended: Putin's Russia, Union Moujik,Kremlin RisingPublished on Aug. 28 2005 by Doris
I don't want to trash a really good book, but one aspect of this book is really off the mark. Tom Clancy's research is legendary so I don't know how this got by him. Read morePublished on July 17 2004 by jdubs55


text: 288 FXUS61 KRLX 270253 AFDRLX Area Forecast Discussion National Weather Service Charleston WV 1053 PM EDT TUE JUL 26 2016 .SYNOPSIS... A weak east to west frontal boundary will dissolve over the area overnight and Wednesday. An upper level system will tap copious moisture to likely produce heavy rainfall Thursday into Friday. && .NEAR TERM /THROUGH WEDNESDAY/... 1045 pm update... Signs of more post-rain fog and low stratus forming so updated forecast to reflect. 1000 pm update... With the area of showers and thunderstorms and its causative upper level short wave trough having moved on to the east, the flash flood watch is cancelled an hour early. However, in the very humid air mass, with a weak surface boundary across Ohio and northern WV, and lingering weak ripples in the westerly upper level flow, cannot entirely rule out the chance for another shower or thunderstorm overnight. 800 pm update... Forecast on track as main area of showers and thunderstorms, associated with an upper level trough, moves out of the area. Still allow for a slight chance overnight. 545 pm update... Hiked PoPs into this evening with thunderstorm complex associated with weak upper level short wave trough rolling through. Previous discussion... A weak but persistent east to west frontal boundary meanders back and forth across the northern portions of the area this period. The addition of upper disturbances riding eastward across the area on the southern edge of westerlies and PW`s aoa 2 inches will lend to a general unsettled pattern for showers and storms. Will tie the best chance for convection to the favored afternoon adn early night time frame. Have hoisted a flash flood watch most of northern half of area through 11 pm tonight...given one well defined vort max over southwestern Ohio and increasing area of heavy convection well ahead of it near the frontal boundary. Otherwise...as the vort moves east of the area tonight...expect convection to really diminish...leaving widely scattered showers in the north after midnight. Will do similar pops again Wednesday with little change in the pattern. It will continue hot and humid through Wednesday...but should be a touch below heat advisory criteria. && .SHORT TERM /WEDNESDAY NIGHT THROUGH FRIDAY NIGHT/... Active weather to start the short term period...with warm and humid conditions to persist. Stalled frontal boundary across northern zones will lift north of the area during the day Thursday as upper shortwave trough approaches from the west. Strong llj developing across the region...will aid in moisture transport...with pw values rising to over 2 inches Thursday. Wave of low pressure moving north through the region Thursday and Thursday night...will help to enhance precipitation...with showers and thunderstorms...with heavy downpours expected. In addition...could see strong to severe storms during the afternoon and evening hours on Thursday...with the potential for stronger winds aloft to be brought down to the surface. Will place the mention of possible water headlines for the Thursday/Thursday night time frame. Still difficult to pinpoint any area of heaviest precipitation/greatest threat...so will highlight entire cwa in the hwo for now. Additional showers and thunderstorms expected for later in the day Friday and Friday night as an upper shortwave trough crosses the area. && .LONG TERM /SATURDAY THROUGH TUESDAY/... Weather looks to remain unsettled for the first part of the extended period as additional disturbances move through the region. Drier weather possible early to mid next week as upper ridging strengthens across the region. && .AVIATION /03Z WEDNESDAY THROUGH SUNDAY/... Last of the showers and thunderstorms associated with an upper level short wave trough crossing the area were crossing the mountains as of 00Z. They will move on out by 02Z, with MVFR conditions the likeliest worst impact, with a very low chance for thunder. While a shower or thunderstorm remains possible overnight and Wednesday, the expected coverage is too low to explicitly code up in the TAFs. However, there will be some uptick in coverage in the afternoon heating on Wednesday. Otherwise, valley fog is possible overnight through dawn unless a stratocu deck forms again. Surface flow will be light and variable except for strong gusts in thunderstorms Wednesday afternoon. Flow aloft will continue light west. CONFIDENCE AND ALTERNATE SCENARIOS... FORECAST CONFIDENCE AND ALTERNATE SCENARIOS THROUGH 00Z THURSDAY... FORECAST CONFIDENCE: Medium. ALTERNATE SCENARIOS: Timing of Fog formation and intensity may vary overnight. A shower or thunderstorm could directly affect any TAF site Wednesday afternoon. EXPERIMENTAL TABLE OF FLIGHT CATEGORY OBJECTIVELY SHOWS CONSISTENCY OF WFO FORECAST TO AVAILABLE MODEL INFORMATION: H = HIGH: TAF CONSISTENT WITH ALL MODELS OR ALL BUT ONE MODEL. M = MEDIUM: TAF HAS VARYING LEVEL OF CONSISTENCY WITH MODELS. L = LOW: TAF INCONSISTENT WITH ALL MODELS OR ALL BUT ONE MODEL. DATE WED 07/27/16 UTC 1HRLY 03 04 05 06 07 08 09 10 11 12 13 14 EDT 1HRLY 23 00 01 02 03 04 05 06 07 08 09 10 CRW CONSISTENCY H H H H M M M M M M M H HTS CONSISTENCY H H H H M M L L L M M H BKW CONSISTENCY H H H H H H M M M M H H EKN CONSISTENCY H H M M M M M M M M M H PKB CONSISTENCY H H H H M M M M M L M H CKB CONSISTENCY H H H M M M M M M L M H AFTER 00Z THURSDAY... Brief IFR conditions possible in thunderstorms every afternoon and evening. Early morning IFR fog possible into mid week depending, in part, on occurrence of showers or thunderstorms the previous afternoon or night. && .RLX WATCHES/WARNINGS/ADVISORIES... WV...None. OH...None. KY...None. VA...None. && $$ SYNOPSIS...TRM/JMV/SL NEAR TERM...TRM/JMV SHORT TERM...SL LONG TERM...SL AVIATION...TRM


text:
Top Definition
The saggy flesh located under a senior womans arms
also see flab
also see flab
"when I was at rhe craps table, i watched this woman roll. She had the nastiest dice wings I have ever seen"
di fagbot 11 dicembre 2004
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text:
Perfect Girl
Advertising Agency: McCann Erickson, Prague, Czechoslovakia
Creative Director: Lars Killi
Copywriter: Jaromir Fischer
Art Director: Alla Havlickova
Photographer: Goran Tacevski
Published:January 2007
14 comments
jennywhxJan 21, 2007 - 16:21
smart, i like this.
http://jennywhx.blogspot.com jwhx:Visionary
hang-the-djJan 22, 2007 - 6:12
Yeah, I think this works well, though wouldn't her face be skinny too?
addyhoch10Jan 22, 2007 - 19:12
yeah great, now she'll have hang-ups about her face...
i agree with Adbabel, have seen this barbie idea about a thousand times before but i have to admit this one's very well executed (apart from the face perhaps, it really should be skinnier)
quodavidJan 22, 2007 - 20:36
perfect! put the barbie in a box says it all
GuestAug 04, 2009 - 9:24
it can not be skiny because girls with such problems hide their problems under laughter


text:
Police in Canada start using BlackBerry PlayBooks in their cars
A Canadian police force is set to begin using BlackBerry Playbooks inside its vehicles.
Mobile Innovations, the brains behind the integration has already fitted two cop cars with the technology, but hopes to have as many as ten by the end of the year.
Though the integration is not quite on the same scale as the BlackBerry equipped Porsche 911 Carrera S, also unveiled at BlackBerry World, officers will be able to control their lights and sirens, receive traffic alerts and access their maps and GPS as well as being alerted to the next 911 emergency.
The PlayBook can also be used to record activity in the back of the car, effectively giving the police eyes in the back of their heads.
The Canadian police already use BlackBerry smartphones, so implementing PlayBooks into their day-to-day business makes sense.
Unlike the PlayBook at the front of the Porsche 911 Carrera S, the PlayBook is removable, sitting securely into a dock, while the device is also connected to a physical keyboard and printer.
A police office from Chatham-Kent, Canada, where the technology is being tried out told Pocket-lint that since using the kit his working day had become much easier as he was able to "take his office with him wherever he goes".
Mobile Innovations says the integration is both a securer and cheaper option than integrating a laptop into a police vehicle and hopes to begin pushing the idea to other corporate vehicle fleets.
What do you think to BlackBerry PlayBooks being used by the police and other corporate fleets? Let us know your thoughts.
Apple shifts focus on Project Titan car to self-driving tech
Hyundai Ioniq Electric review: Fully charged
Renault Zoe review: Electrical engineering
Apple brought back a longtime executive to oversee its car project
- Apple shifts focus on Project Titan car to self-driving tech
- Hyundai Ioniq Electric review: Fully charged
- Renault Zoe review: Electrical engineering
- Apple brought back a longtime executive to oversee its car project
- Jaguar demos new technology to assist future drivers
- Tesla's Master Plan part two involves self-driving trucks, buses, and cars that make money for you
- 29 weird and wonderful cars from Nissan's Zama Heritage collection
- Driving with Nissan ProPILOT: Removing the legwork
- Lexus RX 450h review: An SUV-sized slice of Japanese futurism
- Honda NSX first drive: The high-tech hooligan
- Pokemon Go Gym tips: How to battle, train and win
- Pokemon Go: How to find and catch rare Pokemon like Charizard, Blastoise and Alakazam
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- Can't get Pokemon Go in your country yet? Here's how to download it now
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- Apple iPhone 7 and iPhone 7 Plus: Release date, rumours and everything you need to know
- Acer Switch Alpha 12 review: Affordability is its ace
- Best battery cases for iPhone 6S: So you can Pokemon Go and Go and Go
- 17 Facebook Messenger tips and tricks you likely didn't know


text:
Other projects made by liliane:
Quiltingadventure under the sea
Quiltingits a wild world out there !
Quiltinghong-kong - canada
Offer some kudos...
Join the conversation...
Ask a question...
Add your comment...
Very cute!
06/09/2011 Flag
nicest quilt Ive seen on this site. Beautiful! Lucky niece. I want to be your niece. :)
08/15/2011 Flag
Very cute quilt. You did a great job on making your own pattern. Very Nice! Bev B
06/26/2011 Flag


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Maximum City: Bombay Lost and Found Paperback – September 27, 2005
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From Publishers Weekly
Copyright © Reed Business Information, a division of Reed Elsevier Inc. All rights reserved. --This text refers to an out of print or unavailable edition of this title.
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It is not written as a typical travel book. The format is to take major aspects and dominant personalities of the city and give them each a detailed, richly woven chapter. You'll learn about the quirks and numerous pitfalls of Bombay housing and how the Renter's Act has made everything much much worse. You'll meet the head politician who seems to view Bombay as his personal fiefdom. You'll meet an amazing police detective who is unique on the police force in that he is the only one who won't take bribes, and you'll even sit in on a number of torture sessions of criminals. You'll meet a whole lot of people who kill people for hire, as well as members on police force who kill criminals because the courts didn't do their jobs of prosecuting them (that reality was drop jaw amazing). You'll meet some of the top women in the Bombay beer bar/sex scene, as well as an engineer who gave up a promising career to become a poet living on the Bombay footpaths. The list goes on.
As I read this book, I was amazed at the people that Metha got to agree to give him a good chunk of their time, allowing him to develop a vivid flesh and blood portrait. To top it off, he is an amazingly good writer, who has a great sense of humor (I guffawed out loud several times as I read this book) while casting an unblinking eye on filth and corruption so deep that you feel like you're going to choke on it.
Maximum City is truly a fascinating book to read. Anyone who is interested in either India or the phenomenon of the modern city can't help but love this book.
0. Context setting: I lived in Bombay for 6 years, hope to never live there again, but am fascinated by it, and can't read enough about it. I wasn't directly exposed to either the underbelly or the glamour of Bombay, but was definitely aware of it - something you can never avoid if you live there. My perspective is thus much more middle-class, which I think would be the broadest perspective on Bombay.
1. Bombay is a city begging to be written about, and despite the almost sudden rise in interest in writing about this city, there are still only a few one can really read, so Suketu's attempt is a welcome addition.
2. Suketa's heart in the right place, but his execution is confused. He clearly wants to capture the heart and soul of Bombay, but seems to be limited by his obvious journalistic and dispassionate style. At times the city gets the better of him and he lets go, but not often enough. This is in my opinion is the biggest drawback of the book - it's just stuck somewhere in between an extended non-fiction journal piece and a string of stories linked together by the theme of a city.
3. The book is way too long. Enough people have already pointed this out, so I won't belabour the point, but really, the obsession with writing about the Mafia is totally tedious. If that was what the book was intended to be about, I would have no complaints, but it wasn't, and I think it's unfair to devote half the book (directly or indirectly) to this subject.
4.Read more ›
The only problem is that Mehta doesn't know when to stop, over-covering his subjects to the point of exhaustion, and occasionally going off on unfocused tangents, such as the story of his involvement in producing a cheesy Bollywood movie. The book mostly managed to keep me interested through all its 500+ pages, as Metha would eventually introduce intriguing new people or situations. But each chapter is usually way too long in itself, and sometimes it feels like the book will never end as you long for Mehta to wrap up one story and either get to the next or just bring the book to an authoritative conclusion. Mehta has created a highly intriguing book about an overwhelming city that would scare away the weak-hearted, but his prose tends to get overwhelming too. [~doomsdayer520~]
Most Recent Customer Reviews
An interesting overview of one of the world's remarkable cities from an outsider who brings an insiders point of view. Well written, and thoughtfully entertianing.Published 24 days ago by michael gold
Amazing voyage through the many fascinating sides of Bombay. I couldn't put this book down. What a wonderful read.Published 1 month ago by Craig N. Hartman
I will never ever go to this city after reading this book, but it sure is fun to armchair travel there. Read morePublished 6 months ago by Alice Friedemann
In the tradition of the Bhagavad Gita, a no holds barred look at previously inscrutable city life on Earth today. Read morePublished 7 months ago by David A. Mccrae
Written with love. Mumbai was always on my list of destinations - now even more. Brilliant character portaits and lifelines. Plenty of lessons in how to adapt to seeming chaos. Read morePublished 8 months ago by BusyB
Fascinating look into indian cukture that yiu wont find anywhere else.Published 11 months ago by john smith
what a great read..started it on a Friday and was done on Sunday afternoon....what a great read..lots of infoPublished 13 months ago by Amazon Customer
Fair. Interesting about Mumbai but not as cultural as I'd have liked.Published 14 months ago by Felicia Demos


text:
Skip to comments.Stabbing at National Zoo During African American Family Day
Posted on 04/25/2011 6:00:57 PM PDT by Altura Ct.Edited on 04/25/2011 6:16:16 PM PDT by Admin Moderator. [history]
D.C. Police say a teenage boy was stabbed inside the National Zoo in Northwest.
Sources tell FOX 5's Wisdom Martin that a 14-year-old male was stabbed in the chest in the zoo after being jumped by another group of young people.
Gang day at the zoo?
In the chest, you silly slitch. Can't you read???
One has to wonder, the Zoo Police
well, i assume they have plenty of lock up facilities. i mean, isn't a zoo just a kind of jail anyway?
Was there a hip hop awards show at the zoo?
Dare I say it: a zoo where the caged animals are far more civilized than the visitors.
If it's a "multi-cultural tradition" why is it called African American Family Day?
Did they think it was McDonald's?
We were just talking about that on this thread:
Okay, if it's African American day, then how can it be "multicultural" at the same time?
Rumble in the jungle.
Wow, this headline and related story....it certainly invites interesting commentary.....
Was it a south african american or a north african american?
Okay, if it's African American day, then how can it also be "multicultural" at the same time?
That gives me an idea, lets turn prisons into people zoos. You can charge admission and go to the murderer house, rape house, see the pedophile exhibit etc.
"a 14-year-old male was stabbed in the chest . . . African American Family Day . . . Zoo police custody"
You just can't make this up.
Anyway, I hope the victim will be O.K. but will stay away from the "zoo" in the future.
The Zoo has it's own police department? And people wonder why our cities and States are broke.
Never bring a knife to a zoo fight.
Extend the perimiter fencing to the entire beltway.
"observe. An entire city's population gone feral."
You really can't. I mean....the headline alone is almost too much.
I saw about 50 teenagers running down the middle of Connecticut Avenue around 4:00. They were beating and kicking another teenager, whom I think was the stabbing victim. When the mob took off down the street, the victim got up and was stumbling around in the middle of the street. It was very frightening because the mob was completely out of control. They were headed straight for my car. Traffic was heavy and at a standstill. There was nothing I could do other than lock my doors. Fortunately the mob ran past me down toward Woodley Park.
I bet when their fathers get home, there will be Hell to pay
Easter Monday is traditionally African American Family Day at the National Zoo. Easter Monday has been a Washington-area multicultural tradition that spans more than 100 years.
Easter is celebrated on Sunday. So why aren't any of these people at work.
Odds are 100 to 1 that homemade names are involved.
Given what goes on up there, they need a police force. But I have never really understood why, since they are part of the Smithsonian, that US Park Police don't have the pleasure.
Take the animals out and leave the people in.
did you think about taking refuge in a McDonald's? I hear they are pretty good safe havens.
When their fathers get home - in 8 to 10.
"It wasn't clear whether any weapons were used." A stabbing with no weapon? Were sharp words used?
"Easter Monday is traditionally African American Family Day at the National Zoo. Easter Monday has been a Washington-area multicultural tradition that spans more than 100 years."
Well...which is it; African American Family Day or a multicultural tradition. These two concepts are mutually exclusive.
And, simply put, African American Family Day is racist.
That ain't sayin' much.
That ain't sayin' much.
Wonder how I can find out when Swedish-American family
day is?
True, but if they insist on having such an event I think a different venue might be a wise idea. I mean, African American Family Day at the National Zoo? Seriously?
It's a jungle out there.
I have a background in zookeeping. Zoos that celebrate a day like this have a lot of trouble with the animals those days. They are particularly aggressive by noon. They get teased and tormented and won't take it any more.
Do say more.
Because "African-American" is, by definition, multicultural.
Apparently your PC indoctrination did not take. :-)
Good God.
The wrong animals were in cages.
Does anyone have a zoo calendar? I'm curious what day "White Family Day" is.
African American Family Day
Does anyone have a zoo calendar? I'm curious what day "White Family Day" is.
Six Flags has a Muslim day. Will not visit there until they do away with it. Who in their right mind would show up on Muslim day?
What's really surprising is that Obomba and family did not make it to the day at the Zoo....his family sure seems to qualify for it....(take that however you like)
Is anybody surprised?
I haven't had that happen, but back in the old days I used to get off the highway in the South Bronx so I could take a bridge over to Manhattan. There was one really slummy block along the way, and a stop light.
I always made sure not to pull all the way up to the light if it was red, but to leave some room so I could move forward if some of the thuggish pedestrians approached the car. Which was locked, of course, but I didn't entirely trust that while stopped. And I figured if necessary I'd run the red light and take my chances with the police.
Good advice. I will keep that in mind for the future. It all happened so fast that I just didn't have time to think and was completely boxed in. I will be thanking the Good Lord tonight for his saving grace.
Tom Wolfe, is that you?
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CLEMSON, S.C. -- Two big wins and a fifth-straight bowl trip
haven't eased the pressure on Clemson coach Tommy Bowden.
Despite the program's first win over Florida State in 14 years
and a 40-7 win over Duke that put the Tigers in line for the Gator
or Peach Bowl, Bowden showed signs Tuesday that his shaky job
status had taken its toll.
Bowden, who closes his fifth regular season Saturday night
against South Carolina, was asked whether he thought he would
return to Clemson.
Several seconds elapsed and Bowden's chin began to quiver.
"Those questions you would have to direct elsewhere," Bowden
Questions continued. Had athletic director Terry Don Phillips
told him that he would not be retained?
"You're asking the wrong guy," Bowden said.
Was he planning to leave for another job?
"I'm headed full speed to beat South Carolina," said Bowden,
voice cracking and eyes moist.
Phillips, who typically meets with reporters Tuesday, went home
sick with the flu, sports information director Tim Bourret said.
Bowden is 36-24 and the only Tigers coach to qualify for a bowl
game in each of his first five seasons.
However, it wouldn't be the first time Clemson (7-4) sent a
winning coach packing -- or let one go before the annual
season-ending rivalry.
Rice coach Ken Hatfield finished an 8-3 regular season with a
16-13 win over the Gamecocks in 1993. But he and the school agreed
to separate the next week, ending a four-year stint in which
Hatfield went 33-13-1 and brought Clemson its last Atlantic Coast
Conference title.
Five years ago, Memphis coach Tommy West, then at Clemson, asked
AD Bobby Robinson to clarify his status a few days before facing
South Carolina at Death Valley. West got the news that his fifth
season, in which the Tigers went 3-8, would be his last and it
became public.
The Tigers responded with a rousing 28-19 victory over South
Carolina and West, his wife and young son were carried off the
Phillips has said no one win, such as the 26-10 victory over
then No. 3 Florida State, or one loss will determine Bowden's fate.
Phillips, hired nearly 18 months ago, has repeatedly said he and
Bowden will meet after the regular season.
Bowden's not the only one in the in-state rivalry getting heat.
Lou Holtz and South Carolina (5-6) have lost three straight, and
the Gamecocks are in danger of missing the postseason for the
second-straight year. A Web site, www.firelouholtz.com, has popped
Nonetheless, South Carolina extended Holtz's contract Tuesday by
one year through 2008.
"It was probably my name last week," Bowden said. "They slid
my name out of there and put in his."
Holtz also said Monday that critics of Bowden were off base.
"I'm starting to like him more and more," the Clemson coach
As far as Holtz's critics, Bowden says it's hard to find fault
with a former national champion who has revived South Carolina's
It's also hard to figure out why a school might fire a winning
"That's what I would think," Clemson quarterback Charlie
Whitehurst said. "But I know there are some more factors going
into it. That's really all I know."


text:
When adding a reference to a web application project in VS (2008 in this case), the "hintpath" in the csproj file is being created as a relative reference. Is there a way (using the GUI, not manually editing the file) to make this an absolute reference (i.e. C:\Temp\DllName.dll)?
The issue I am running into is when seperate build machines have a different working directories for the project. When the reference is relative, and the referenced dll is not within the project working directory, the relative reference may not point to the same location on both machines.