text:
|Publication number
|Publication type
|Application number
|Publication date
|Jul 19, 1983
|Filing date
|Oct 7, 1981
|Priority date
|Mar 2, 1977
|Fee status
|Also published as
|Publication number
|Werner Muller, Hans Bunemann
|Original Assignee
|Boehringer Mannheim Gmbh
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (6), Non-Patent Citations (2), Referenced by (1), Classifications (50), Legal Events (3)
|External Links: USPTO, USPTO Assignment, Espacenet
This is a division of application Ser. No. 880,914, filed Feb. 24, 1978 now U.S. Pat. No. 4,335,226.
The present invention relates to the separation of macromolecular materials, particularly of biopolymers. More specifically, the invention relates to an adsorbent for the affinity-specific separation of such macromolecular materials. In a still more specific aspect, the invention relates to the separation of mixtures of one- and/or two-chained nucleic acids, especially desoxyribonucleic acids, the preparation and utilization of such adsorbent.
The isolation of individual genes or of groups of identical genes in repetitive arrangement from the genome of eukaryotic cells is of interest not only from a purely scientific point of view but is also of great practical importance from the point of view of gene technology. Such isolations were only possible when the base composition of the gene or of the gene group with their "spacers" differed by at least 6 to 7% from the average base composition of the total genome. Separation processes which have mostly been used include caesium ion density gradient centrifuging with DNA base-specific additions, such as silver, mercury or platinum ions, or actinomycin, netropsin or the dyestuff "Hoechst 33258". These processes have only a limited capacity and are expensive and time consuming.
The development of materials for the affinity chromatography of biopolymers has, in years past, considerably simplified the isolation of a large number of biopolymers or has even made the pure preparation thereof possible for the first time. In the cases of these processes, it is usual to start from a carrier material which, after chemical activation, is reacted with a substance which binds as specifically as possible with the bipolymer to be isolated. On the basis of this specificity, the desired biopolymer can, in the ideal case, be selectively adsorbed from a mixture of similar compounds on to the chromatographic material and subsequently, under appropriate conditions, can be desorbed in a pure state (see P. Cutarecasas and C. B. Anfinsen, Ann. Rev. Biochem., 40, 259-278/1971).
In spite of the considerable number of examples of the successful use of these methods, for a number of biopolymers hitherto no chromatographic material has been developed which also makes possible a similar selective and programmed separation of mixtures of nucleic acids. A high resolving fractionation of mixtures of nucleic acids on a gramme scale is, however, a prerequisite for such an isolation of individual genes or of groups of identical genes.
In the case of the available processes for the separation of nucleic acids of low molecular weight, for example of transfer ribonucleic acids, the separation is achieved in various species of adsorbents which combine ion exchange properties with lipophilic exchange actions (see M. Kothari and V. Shankar, J. Chromatography, 98, 449-475/1974). The exchange action possibilities of the carrier with the rare bases of the nucleic acids, which are present in varying amounts in the various transfer ribonucleic acids, are thereby utilised.
Since, as a rule, the high molecular weight ribonucleic acids and desoxyribonucleic acids do not contain any rare bases, methods for the separation thereof can only be built upon the following characteristic differences:
(a) ratio of single to double chain
(b) differences in the base composition
(c) differences in the base sequence
(d) differences in the molecular weights
(e) differences in the tertiary structures.
All these characteristic features are, in fact, utilized in conventional methods of fractionation (see R. M. Kothari, Chromatog. Rev., 12, 127-155/1970).
In the case of the most effective methods, differences according to b, c and e lead, in the fractionation in the salt gradients, to differences in the suspension density for the individual DNA species which can be still further increased by the addition of base specific substances. The sharpness of separation decreases with decreasing molecular weight and the capacity with increasing molecular weight. In the case of adsorption chromatography on hydroxyapatite, fractionation takes place essentially according to a and only to a limited extent according to b, guanine-cytosine-rich nucleic acids already being desorbed at somewhat lower salt concentrations than the adenine-thymine-rich components (see. W. Pakroppa and W. Muller, Proc. Nat. Acad. Sci. USA, 71(3), 699-703/1974).
Analogously, double chain nucleic acids can also be fractionated on specific protein-keiselguhr adsorbents (for example methyl serum albumin-keisulguhr) according to differences of the base composition, the guanine-cytosine-rich DNA species being eluted first (see J. D. Mandell and A. D. Hershey, Analytical Biochemistry, 1, 66-77/1960; N. Sueoka and Ts'ai-Ying Cheng, J. Mol. Biol., 4,161-172/1962). The actual manner of working of these rather accidentally discovered adsorbents is not known. Therefore, the low sharpnesses of separation of these materials have hitherto not been fundamentally improved, in spite of all attempts to do so.
Only in recent years have there been found, in the case of the systematic investigation of numerous substances which form complexes with nucleic acids, compounds which appear to be suitable for a directed synthesis of materials for affinity chromatography (see W. Muller and D. M. Crothers, Eur. J. Biochem., 54, 267-277/1975; W. Muller, H. Bunemann and N. Dattagupta, Eur. J. Biochem., 54, 279-291/1975; and W. Muller and F. Gautier, Eur. J. Biochem., 54, 385-394/1975). The advantages provided by using such well investigated substances for the separation of mixtures of nucleic acids has already been demonstrated by the combined use of hydroxyapatite and ethidium bromide as base specific addition for the separation of superhelical and helical DNA (see W. Pakroppa, W. Goebel and W. Muller, Analytical Biochemistry, 67, 372-383/1975) and of hydroxyapatite in combination with phenyl neutral red derivatives as base specific complex formers for the separation of double chained DNA species (see W. Pakroppa and W. Muller, Proc. Nat. Acad. Sci. USA, 71(3), 699-703/1974).
The resolving properties of these last-mentioned methods is comparable with that of a preparative caesium chloride density gradient method, i.e. DNA fractions with differences in the (G+C) content (G stands for guanine and C for cytosine) can be separated from one another. In spite of the high capacity, this process has the disadvantages that DNA mixtures with an average molecular weight of the components of more than 20�106 can no longer be readily dealt with and that the special hydroxyapatite employed as adsorbent must itself be prepared.
It has been known for a long time that under certain conditions, nucleic acid mixtures can be separated in the polyethylene glycol-dextran system into RNA and single chained DNA, on the one hand, and into double chained DNA, on the other hand. The double chained DNA thereby always becomes enriched in the lighter polyethylene glycol phase, i.e. it possesses a higher partition coefficient than the single chained nucleic acids. The absolute values of the partition coefficients can admittedly be varied by about 3-4 factors of 10 by the addition of potassium and lithium salts but a fractionation according to the base composition is not successful in these systems.
It is an object of the present invention to provide adsorbents with which can be achieved a base- and/or structure specific separation of biopolymers and especially of mixtures of single chained and/or double chained and/or supercoiled and/or linear nucleic acids, especially of DNA mixtures, with a high capacity.
Affinity specificity is here to be understood to mean, in particular, structural specificity and base specificity.
We have found that this object can be achieved with an adsorbent which comprises a polymeric carrier material to which is covalently bound, directly or via comparatively long intermediate groups (spacers), an affine residue for the biopolymer or a base and/or structure-specific group.
Therefore, the present invention provides an adsorbent for the affinity specific separation of macromolecular materials, especially of biopolymers, such as nucleic acids, which comprises a polymeric carrier material to which is covalently bound, directly or via a polymeric "spacer", an affine residue for the biopolymers.
According to an especially preferred embodiment of the present invention, there is provided an adsorbent of the defined type which comprises a small-pored, low compressable, polymeric carrier material on to which is grafted a copolymer of at least two co-polymerizable monomers, one of which carries an affine residue for the biopolymer.
As affine residues for the biopolymer, there can be used base- and/or structure-specific complex formers, which are described in the above-mentioned literature references. In particular, there can be used residues of dyestuffs of the following general formulae (I) and (II): ##STR1## in which X is a CH group or a nitrogen atom, Y is an oxygen or sulphur atom or an NH group or a group of the general formula: ##STR2## each R1, independently of one another, is a hydrogen atom or a methyl radical, R2 is a hydrogen atom or a methyl radical, R3 is a hydrogen atom or a methyl radical, R4 is a hydrogen atom or a methyl radical and A.sup.⊖ is an anion, such as a chlorine anion, a perchlorate anion or an oxalate anion.
Examples of base- and/or structure-specific complex formers which, according to the present invention, are especially preferably bound on to the polymeric carrier material, include the residues of the following dyestuffs: ##STR3##
However, according to the present invention, the most preferred base- and/or structure-specific complex formers are phenyl neutral red of the formula: ##STR4## and malachite green of the formula: ##STR5##
These dyestuffs are known and are commercially available or can be prepared by processes which are well known to the art skilled and are described, for example, in the above-mentioned publications of W. Muller and D. M. Crothers (Eur. J. Biochem., 54, 267-277/1975), W. Muller, H. Bunemann and N. Dattagupta (Eur. J. Biochem., 54, 279-291/1975) and W. Muller and F. Gautier (Eur. J. Biochem., 54, 385-394/1975).
These base- and/or structure-specific complex formers or dyestuff residues can be covalently bound to the carrier material by methods which are well known to the art skilled, for example by esterification with hydroxyl groups present on the carrier material via carboxyl groups introduced into the dyestuffs molecule, by amide formation, by urethane formation or also by copolymerization in the absence or preferably in the presence of other copolymerizable monomers via copolymerizable double bonds which are introduced into the dyestuff molecule, for example via an acrylamide group, such as is the case with acrylic-phenyl neutral red and acrylic-malachite green of the following formulae which, according to the present invention, are preferred as base- and/or structure-specific complex formers. These designations are trivial names, the exact designations being acryloylaminophenyl neutral red and acryloylaminomalachite green: ##STR6##
These base- and/or structure-specific complex formers can be prepared by the art skilled in known manner by the introduction of the acrylic radical into the dye-stuff molecules. This also applies to the other dyestuff molecules mentioned hereinbefore.
The term acrylic radical has, in the present cases, the same meaning as the term acryloyl radical.
The preparation is preferably carried out by the processes described in the following Examples 3 and 4. The acryloyl dyestuffs are new.
According to the present invention, as polymeric carrier material there is preferably used a small pored, low compressability polymeric carrier material, such as poly-bisacrylamide, the small pored and low compressibility polymeric carrier materials having proved to be very advantageous for the affinity chromatography of highly viscous nucleic acid solutions. Furthermore, these polymeric carrier materials do not exhibit any strong exchange actions with the affine residues, especially with the base- and/or structure-specific complex formers for nucleic acids which are bound to the polymeric carrier material either directly or via polymeric spacers.
It has proved to be advantageous to introduce, between the polymeric carrier material and the base- and/or structure-specific complex former, a comparatively long spacer, i.e. a comparatively long molecular chain without functional groups, since complex formation of the base- and/or structure-specific complex former with the helical nucleic acid is hereby not impaired by steric hindrance.
Therefore, it is especially advantageous when, in the adsorbent according to the present invention, the affine residue for the biopolymer or the base- and/or structure-specific complex former is bound to the polymeric carrier material via a copolymer which preferably has a degree of polymerization of from 200 to 300. For the formation of this copolymer, it is preferable to use a further monomer which is copolymerizable with the group introduced for the polymerization into the base- and/or structure-specific complex former and with the copolymerizable groups present on the polymeric carrier material.
This further monomer is preferably acrylamide and the above-mentioned acrylic radical-containing dyestuffs are grafted on to a poly-bisacrylamide which still has free, copolymerizable double bonds.
The present invention is also concerned with a process for the preparation of the above-mentioned adsorbents, wherein there is first formed the polymeric material by polymerization or by polycondensation of at least one monomer which, in addition to the functional group necessary for the polymerization or polycondensation, has a further functional group via which the affine residue for the bipolymer can be bound directly or via a polymeric spacer, this polymeric carrier material is optionally comminuted to the desired particle size and then the affine residue for the biopolymer is grafted on either directly or by copolymerization in the presence of at least one further monomer as comonomer.
Grafting on can take place in any desired manner, for example by esterification, amide formation or urethane formation, using conventional reagents and conventional reaction conditions or there are introduced into the polymeric carrier material the groups suitable for these reactions or there are introduced the affine residues for the biopolymer.
However, it is also possible to graft the affine residues for the biopolymer, into which has been introduced a copolymerizable double bond, in the presence of an excess of a further copolymerizable monomer as comonomer, on to poly-bisacrylamide since poly-bisacrylamide still contains free, copolymerizable double bonds. The graft copolymerization can be carried out in water or in an inert organic solvent and in the presence of a conventional polymerization catalyst.
The copolymerizable affine residue for the biopolymer is preferably acrylic-phenyl neutral red or acrylicmalachite green and the further copolymerizable monomer is preferably acrylamide, a copolymer of these components being grafted on to poly-bisacrylamide. According to a preferred method of working, a copolymer of acrylamide and acrylic-phenyl neutral red or acrylic-malachite green with a degree of polymerization of 200 to 300 is grafted on.
In the case of this graft copolymerization, it is preferable to work with a ratio of the affine residue for the biopolymer having a copolymerizable double bond to the further copolymerizable monomer of 1:100 to about 1:500, this reaction more preferably being carried out with a ratio of about 1:300. We have also found that the molar ratio of the further monomer to the affine residue for the biopolymer having a copolymerizable double bond is also retained in the copolymer. In the case of an average degree of polymerization of the copolymer of 200 to 300, it is possible for the grafted on copolymer chains each to bear only one affine residue for the biopolymer. The copolymers thus form spacers of differing length between the affine residue for the biopolymer and the polymeric carrier material, especially the poly-bisacrylamide particles.
We have found that the adsorbents according to the present invention fulfilled the desired requirements and can be obtained synthetically without difficulty and in a simple manner. For example, the polymeric carrier material can be obtained by the polymerization of bisacrylamide in a highly concentrated solution. The polymerization product is a btittle, colorless solid body which can be very easily comminuted to give readily sedimenting particles which, by several sievings, can be homogenized to give the desired particle size. The swelling behavior in aqueous solution is constant, even in the case of extreme salt concentrations, and is practically independent of the flowthrough rate in the case of chromatography. Since these particles still contain a number of unreacted polymerizable double bonds, they can, as already explained above, be used for the further graft polymerization.
Thus, with the adenine-thymine-specific acrylicmalachite green and with the guanine-cytosine-specific acrylic-phenyl neutral red, there are obtained with acrylamide in the presence of these poly-bisacrylamide particles, by copolymerization in aqueous solution, the adsorbents which are preferred according to the present invention. 40 to 50% of the formed copolymer with an average degree of polymerization of about 200 are thereby bound under the usual reaction conditions in the form of graft copolymers on to the carrier particles of the polybisacrylamide. After thorough rinsing, the strongly colored adsorbent particles no longer give off any dyestuff and they can then be used directly for the affinity chromatography of nucleic acids. The mixture of nucleic acids is usually adsorbed in 0.01 M phosphate buffer on to the material and desorbed with a linear salt gradient, the nature of the salt depending upon the nature of the nucleic acids. Whereas for the desorption of ribonucleic acids, including transfer-ribonucleic acids, it is sufficient to use sodium chloride, for the base-specific elution of DNA from acrylic-malachite green-containing adsorbents, perchlorate is necessary.
We have found that for the preparation of the carrier material, it is also possible to start from insoluble materials containing hydroxyl or amino groups which are present in pearl form, for example aminoalkyl-Sepharose, microcrystalline cellulose, cross-linked hydroxyethyl methacrylate or epoxypropyl acrylate. In the case of these carriers, the acrylic groups necessary for subsequent grafting are introduced by reaction with acryloyl chloride. Thus, there can be achieved a high loading with dyestuff similar to that in the case of the grafting from acrylamide fragments.
Thus, the present invention is also concerned with the use of the said adsorbents for the separation of biopolymers by two-phase affinity partitioning, by two-phase partition chromatography, by gel electrophoresis and especially by affinity chromatography. These adsorbents can also be advantageously used for the separation of mixtures of one-chain and/or two-chain nucleic acids and especially of DNA mixtures.
Since the adsorbents according to the present invention, which carry base- and/or structure-specific complex formers on the basis of 5-phenyl-phenazonium salts, act specifically for guanine-cytosine-rich DNA and the adsorbents according to the present invention which act as base- and/or structure-specific complex formers on the basis of triphenylmethane dyestuffs act specifically for adenine-thymine-rich DNA, the desired base- and/or structure-specific or sequence specific separations of mixtures of nucleic acids can be carried out. Predominantly struture-specific complex formers can be compounds capable of intercalation. By intercalation, there is to be understood, in particular, a special exchange of dyestuff molecules with DNA, the dyestuff molecule thereby slipping between two neighboring base pairs of the DNA double helix. Which substances are capable, due to their structure, of intercalation is known to the skilled artisan. They can be planar, electron-rich chromophors and, in particular, can be the residues of planar, polycylic ring systems, such as bi-, tri- or tetracyclic, condensed ring systems, which can contain heteroatoms, such as nitrogen, oxygen or sulfur.
The effect can be increased when, for example, in the case of ethidium bromide or of 9-(acryloylaminoethylamino)-2-methoxy-6-chloroacridine, an amino group is present on the ring system which makes possible a binding on to the phosphate groups of the DNA. It is a simple matter for one skilled in the art to use correspondingly useful compounds, for example, acridines, benzacridines, phenanthrenes, phenantridines, pyridoindoles, naphthalenes, naphthothiophenes, benzothiophenes, thianthrenes, xanthines, phenoxanthines, quinolines, quinoxalines, phenanthrolines, phenothiazines, phenoxazines, phthalazines and the like.
Acridine derivatives, for example 9-(acryloylaminoethylamino)-2-methoxy-6-chloroacridine, or phenanthridine derivatives, for example ethidium bromide (5-ethyl-3,8-diamino-6-phenyl-phenanthridium bromide), separate, after coupling thereof to bis-acrylamide particles, base-nonspecific double-chained DNA from supercoiled DNA (see elution diagram 7 of the accompanying drawings). This is very important for a simple separation of plasmid and viral DNAs from cell digests, a column replacing the laborious ethidium bromide-caesium chloride gradients. This separation can also be carried out with an acryloylaminophenyl-neutral red column since both dyestuffs form a complex-like structure (intercalation complex) with the DNA. Acryloylamino-phenyl neutral red separates not only structurally-specifically but also base-specifically. Therefore, depending upon the particular separation problem, a series of variations is possible with regard to the composition of the adsorbent.
Since the base- and/or structure-specific complex formers are bound via a stable covalent carbon-carbon bond, the adsorbents according to the present invention can also be used at more extreme pH values, washed with detergents and regenerated with salt solutions. Furthermore, it is also possible to incorporate several different base- and/or structure-specific complex formers or monomers so that adsorbents can be formed which can be used for the separation of transfer ribonucleic acids. The chain length of the grafted-on copolymers and thus the length of the chain between the polymeric carrier material and the base- and/or structure-specific complex former can be controlled in the usual manner by the use of mercaptoethanol
In the following, the present invention is explained in more detail with reference to specific Examples and to the accompanying drawings, in which:
FIG. 1 is a scheme for the preparation of adsorbents which are especially preferred according to the present invention by a two-stage polymerization;
FIG. 2 shows the elution diagram of a mixture of three sheared, bacterial desoxyribonucleic acids with the use of an adsorbent according to the present invention;
FIG. 3 shows the elution diagram of a mixture of three sheared, bacterial desoxyribonucleic acids which have been obtained with the use of another absorbent according to the present invention;
FIG. 4 shows the elution diagram of an EcoRI hydrolysate of λ-phase desoxyribonucleic acid which was obtained with the use of an adsorbent according to the present invention;
FIG. 5 shows the elution diagram which was obtained with the use of an adsorbent according to the present invention for the separation of a DNA-RNA mixture obtained by the digestion of M. luteus:
FIG. 6 shows the elution diagram of a mixture of four different transfer-ribonucleic acids from yeast which has been obtained with an adsorbent according to the present invention; and
FIG. 7 shows the elution diagram for the separation of supercoiled and linear DNA.
The following Examples are given for the purpose of illustrating the present invention:
50 g. N,N'-Methylene-bisacrylamide are suspended in 100 ml. methanol in a tall 1 liter polyethylene beaker and mixed with 200 ml. boiling double distilled water. Upon stirring the mixture with a magretic stirrer, the bisacrylamide goes completely into solution. The solution is cooled to 60� C. and, while stirring intensively, there is first pipetted in 1 ml. N,N,N',N'-tetramethylethylenediamine and then, all at once, a solution of 0.2 g. ammonium peroxide disulphate in 5 ml. water. Immediately after mixing up, the stirrer is switched off. After a few seconds, the solution becomes turbid and solidifies to a colorless block upon heating. Polymerization is stopped after about 1 minute by coarsely comminuting the block with a large spatula. The pieces obtained are suspended in 1 liter methanol in a 2 liter polyethylene beaker and comminuted to a granular slurry. For the further homogenisation of the particles, the slurry is successively rubbed through sieves of decreasing mesh size (1 mm.�1 mm.; 0.5 mm.�0.5 mm.; and 0.25 mm.�0.25 mm.). The resulting pieces are allowed to sediment, whereupon the milky-turbid supernatant is decanted off. This procedure is repeated several times, first with methanol, then with a methanol/water mixture, subsequently with a 1% acetic acid solution and finally with distilled water. The polybisacrylamide particles obtained are stored in aqueous solution and can be used at any time for the graft polymerization described in the following.
From 300 ml. of the polymerization solution, there are obtained about 300 ml. of deposited poly-bisacrylamide particles.
60 ml. of the deposited poly-bisacrylamide particles obtained in Stage A are suspended in 20 ml. distilled water in a flask provided with a screw closure. In this suspension there are dissolved 5 g. acrylamide and 10.3 mg. acrylic-phenyl neutral red chloride, for which purpose the suspension in the flask is carefully shaken until the dyestuff has dissolved completely. The copolymerization is then initiated by the addition of 0.050 ml. mercaptoethanol for the adjustment of the degree of polymerization and 1 ml. of a sodium peroxide solution (0.8 g. sodium peroxide in 100 ml. 1 M sodium acetate buffer with a pH value of 5.5) as catalyst. The suspension formed is immediately gassed for 5 minutes with nitrogen and then maintained at ambient temperature with the exclusion of air. After about 30 minutes, the reaction mixture warms up noticeably and after about 2 hours the polymerization is substantially finished. The reaction mixture is left to stand for several hours before transferring the viscous suspension to a suction filter. The viscous, deep colored solution is filtered with suction and the dark colored particles remaining behind on the filter are thoroughly washed with water until the wash solution, even when using a 1% acetic acid solution, remains colorless. After a subsequent continuous rinsing overnight with 0.01 M sodium phosphate buffer with a pH value of 6.0, the material obtained can be used for the affinity chromatography of nucleic acids.
The determination of the yield is carried out, in the case of acrylamide, by means of a 14 C marking and, in the case of the dyestuff, by means of the optical density. The yields are as follows:
______________________________________dyestuff: incorporation into the copolymer (bound and unbound): 60.5%acrylamide: incorporation into the copolymer (bound and unbound): 49.2%dyestuff: incorporation into the bound copolymer, referred to the total: 51.0%acrylamide: incorporation into the bound copolymer, referred to the total: 57.0%______________________________________
It can be ascertained that the molar starting ratio of dyestuff to acrylamide (1:3000) is also retained in the copolymer obtained. Since the average degree of polymerization of the copolymer is determined as being 200 to 300, it can be deduced therefrom that the copolymer chains each carry only one dyestuff molecule per chain.
The above-described process is illustrated schematically in FIG. 1 of the accompanying drawings.
As is shown in the elution diagrams in FIGS. 2 to 6 of the accompanying drawings, the possibilities of use of the adsorbents according to the present invention in the field of nucleic acid fractionation is very versatile. Depending upon the base specificity of the bound dyestuff, mixtures of double-chained nucleic acids can be separated according to their base composition.
Thus, FIG. 2 of the accompanying drawings shows the elution diagram of a mixture of three sheared bacterial desoxyribonucleic acids with an average molecular weight of about 700,000 D with differing base composition. For this purpose, use is made of a column with the dimensions 16 cm.�1.5 cm. which, as adsorbent, contains acrylicmalachite green-acrylamide copolymer grafted on to polybisacrylamide particles, the amount of desoxyribonucleic acid used being about 1 mg.
FIG. 3 of the accompanying drawings shows the elution diagram of a mixture of three sheared bacterial desoxyribonucleic acids with an average molecular weight of about 700,000 D which have a differing base composition. For the elution of 1 mg. of the nucleic acid mixture, use is made of a column with the dimensions 15.2 cm.�1.5 cm. which, as adsorbent, contains bis-acrylamide particles on to which have been grafted the acrylic-phenyl neutral red acrylamide copolymer.
With the use of the adsorbents according to the present invention, it is also possible to fractionate DNA fragments which have been obtained by the action of restriction endonucleases. This is shown in FIG. 4 of the accompanying drawings which illustrates the elution diagram of an EcoRI hydrolysate of λ-phage desoxyribonucleic acid which is separated in an amount of 0.5 mg. with the use of a column with the dimensions 15.8 cm.�1.5 cm. which, as adsorbent, contains poly-bisacrylamide particles on to which have been grafted acrylic-malachite green-acrylamide copolymer. The characteristics of the DNA fragments set forth in FIG. 4 hereof, which shows the results of a gel-electrophoretic separation for the individual fractions, in comparisons with the starting material corresponds to the characteristics set forth in the publication of Thomas and Davis in J. Mol. Biol. 91, 315-328 (1975).
The separation of ribonucleic acids from desoxyribonucleic acids of the same organism also takes place smoothly with the use of the adsorbents according to the present invention, as can be seen from FIG. 5 of the accompanying drawings. This FIG. 5 shows the elution diagram of a DNA-RNA mixture from a digest of M. luteus (72% G+C), the mixture being separated in an amount of 1 mg. with the help of a column with the dimensions 16.2 cm�1.5 cm. which is packaged with poly-bisacrylamide particles on to which have been grafted the acrylic-malachite green copolymers.
FIG. 6 of the accompanying drawings shows the elution diagram of a mixture of four different transfer ribonucleic acids obtained from yeast. The mixture, which is used in an amount of 1.5 mg., is a mixture of t-RNAPhe, T-RNALys, T-RNAGlu and t-RNAGly. For the affinity chromatography, use is made of a column with the dimensions 16 cm.�1.5 cm. which is packed with poly-bisacrylamide particles on to which have been grafted the acrylic-phenyl neutral redacrylamide copolymers.
FIG. 7 of the accompanying drawings shows the elution diagram for the separation of supercoiled and linear DNA. For the affinity chromatography, use is made of polybisacrylamide particles on to which 9-(acryloylaminoethylamino)-2-methoxy-6-chloroacridine particles have been grafted.
When using adsorbents according to the present invention in the manner described above for the separation of mixtures of nucleic acids by affinity chromatography, as elution agents there can be used aqueous salt concentration gradients, for which purpose the salt preferably used is an alkali metal salt, such as sodium chloride, sodium perchlorate, lithium perchlorate or the like. In certain cases, buffers can also be used, for example phosphate buffer: for adsorption agents which, as basespecific complex former, contain the residue of malachite green, there is used a weakly acidic buffer with a pH value of 5.5 to 6, for example a 0.01 molar phosphate buffer with a pH value of 5.5 to 6.
The requirements for the achievement of optimum salt gradients, their components, concentrations and pH values and the buffers necessary therefor can be determined without difficulty by the art skilled.
Thus, to summarize, the adsorbents according to the present invention are outstandingly suitable for the affinity-specific separation of macromolecular materials and especially of biopolymers, such as mixtures of nucleic acids. These adsorbents can be prepared in a simple manner and can be readily adjusted with regard to their base specificity.
(a) 15.1 g. (0.1 mol) 4-Nitrobenzaldehyde, 36.3 g. (38 ml.) (0.3 mol) N,N-dimethylaniline and 40.5 g. anhydrous zinc chloride are mixed and heated to a bath temperature of 100� C. The initially readily stirrable mixture becomes, during the course of the reaction, a solid green mass which can no longer be stirred. After a reaction time of 5 hours, the reaction mixture is allowed to cool. The product is taken up in 150 ml. acetone and, after stirring for some time, the product dissolves and the zinc salt separates out. The insoluble salt is filtered off with suction and washed with acetone and the filtrate is mixed with such a quantity of water that crystallization commences. The crystals are filtered off with suction and washed with water, isopropanol and diethyl ether. The yield is 28.7 g. (76.6% of theory); m.p. 168� C. The substance is sensitive to light.
(b) 3.75 g. (0.01 mol) of the nitro compound obtained according to (a) are dissolved in 200 ml. glacial acetic acid (99%) and 20 ml. water and mixed with 4 g. zinc chloride. Subsequently, 20 g. zinc dust are added portionwise at ambient temperature (20�-30� C.), with stirring and cooling. The reaction mixture is stirred for 30 minutes at ambient temperature and thereafter excess zinc is filtered off with suction and washed with glacial acetic acid. The filtrate is evaporated in a vacuum at 50� C., the residue is dissolved in 150 ml. chloroform and 100 ml. water and separated and subsequently the chloroform phase is shaken with 80 ml. 2 N aqueous sodium carbonate solution and 100 ml. water. The chloroform phase is separated off and dried over anhydrous sodium sulphate and then filtered, whereafter the filtrate is concentrated in a vacuum at 50� C. to about 100 ml. and further reacted directly (pale violet solution).
(c) The 100 ml. of chloroform solution of the amino compound thus obtained is mixed with 50 ml. methanol. 10 g. anhydrous sodium carbonate and a spatula tip of 1,3-dinitrobenzene are added thereto and subsequently, with stirring and ice cooling (0�-5� C.), 1.81 g. (1.65 ml.) acrylol chloride (double the molar amount) are added thereto dropwise. The suspension is stirred overnight at ambient temperature, thereafter the sodium carbonate is filtered off with suction and the filtrate is evaporated in a vacuum at 35�-40� C. The residue is taken up in 100 ml. chloroform and 50 ml. water and shaken and the chloroform phase is separated off, subsequently extracted with 50 ml. water, dried with anhydrous sodium sulphate and evaporated in a vacuum at 35�-40� C. The oily, greenish residue obtained is triturated with 20 ml. isopropanol and placed in the cold to crystallize. The crystals obtained are filtered off with suction, washed with isopropanol and diethyl ether and dried in a desiccator. The yield over stages 1b and 1c is 2.9 g. (72.7% of theory); m.p. 175� C.
______________________________________calc.: C 73.2%; H 7.27%; N 10.52%found: 75.9%; 7.21%; 9.999%______________________________________
NMR spectrum (hexadeuterodimethyl sulphoxide): CH3 -(N-methyl) S 2.83 ppm, -CH (acrylic) Q 5.7 ppm, CH2 = (acrylic) P 6.3 ppm, aromatic protons between 6.5 and 7.5 ppm.
(d) 250 mg. (0.001 mol) chloranil are dissolved in 15 ml. tetrahydrofuran and mixed with 0.4 g. (0.001 mol) of the acryloyl compound obtained in (c). The solution immediately becomes blue and, after standing for about 2 hours at ambient temperature, crystallization commences. The reaction mixture is left to stand overnight at ambient temperature, whereafter the crystals are filtered off with suction, washed with tetrahydrofuran and diethyl ether and dried in a desiccator. For further purification, the product is taken up in water, neutralized with hydrochloric acid and then shaken out with ethyl acetate. The pure product is precipitated out from the aqueous solution by the addition of sodium chloride. The yield is 0.335 g. (84.2% of theory). In the NMR spectrum of (d), in comparison with (c), there is observed a displacement of the n-methyl proton signal to 3.3 ppm.
(a) 6.9 g. (0.05 mol) 4-Nitroaniline are dissolved in 150 ml. tetrahydrofuran/chloroform (1:1 v/v) and mixed with a spatula tip of 1,3-dinitrobenzene and with 8 ml. triethylamine. 9 ml. (0.1 mol) acryloyl chloride are slowly added dropwise at 10�-20� C., while stirring. The solution is stirred overnight at ambient temperature, triethylammonium chloride thereby crystallizing out. The tetrahydrofuran/chloroform mixture is then evaporated off in a vacuum at 50� C. and the residue is triturated with water, filtered off with suction and washed with water, isopropanol and diethyl ether. The product is dried in a vacuum at 50� C.
(b) 2.5 g. of the acryloyl compound obtained in (a) are dissolved in 60 ml. tetrahydrofuran at 50� C., while stirring, and diluted with 60 ml. glacial acetic acid (99%) and cooled to 30� C. Subsequently, 10 g. zinc dust are added portionwise, the temperature, even with ice cooling, thereby increasing to 35�-40� C. The reaction mixture is thereafter stirred for 10 minutes at ambient temperature and excess zinc is filtered off with suction and washed with glacial acetic acid. The filtrate is evaporated in a vacuum at 45� C. The oily evaporation residue is chromatographically practically pure and is thus used for the next reaction stage.
(c) A solution of 4.2 g. (0.02 mol) N,N-dimethyl-p-phenylene diammonium dichloride and 2.88 g. (0.02 mol) o-toluidinium chloride in 400 ml. water are slowly mixed, while stirring, at ambient temperature, with a solution of 12 g. (0.04 mol) sodium chromate in 100 ml. water. The green product which separates out is, after 15 minutes, filtered off with suction and washed three times with water, the wash solution thereby remaining green. The precipitate is then immediately further worked up by suspending it in a comparatively small amount of water and homogenizing. The homogeneous suspension is diluted with water to 1.6 liters. After the addition of 1.15�0.02 mol N-acryloyl-p-phenylene diammonium acetate in 100 ml. water, the reaction mixture is adjusted to pH 4.9 with 130 ml. 3 M sodium acetate solution. Subsequently, the mixture is heated to the boil, while stirring. The solution thereby first becomes deep blue and then, upon boiling, dark violet (for completion of the reaction, it is boiled for 5 minutes). Subsequently, the boiling hot solution is suction filtered through a filter funnel and the filtrate (about 2 liters) is adjusted with 240 g. sodium chloride to a 2 M solution and left to stand overnight in a refrigerator. The precipitated crystals are filtered off with suction and dried in a desiccator (it must not be washed with water since the substance is readily soluble in water). 2.4 g. of crude product are obtained.
For further purification, 1.3 g. of the crude product are dissolved in 25 ml. methanol and 0.1 M sodium chloride solution (4:1 v/v) and applied to a silica gel 60 column (4�95 cm.). The column is then eluted with the same solvent (fraction volumes 2 ml.). Fractions 22-55 are combined and evaporated in a vacuum to about 15 ml. The crystals obtained are filtered off with suction, washed with a little 0.1 M sodium chloride solution and dried in a desiccator. There is obtained 0.665 g. of chromatographically pure dyestuff.
854 mg. 3,9-Dichloro-6-methoxyacridine are heated in an oilbath at 60� C. for 30 minutes with 1 ml. ethylenediamine and 7.7 g. phenol. The melt is taken up in 200 ml. chloroform and 150 ml. water, adjusted to pH 4 with acetic acid and equilibrated. The organic phase is subsequently extracted 3 or 4 times with 0.1 M sodium acetate buffer and then discarded.
The aqueous extracts are adjusted to pH 9.5 with an aqueous solution of sodium carbonate, extracted with chloroform-n-butanol (20:1 v/v) and the organic phase filtered over silicone paper and evaporated in a vacuum. The residue is dissolved in 0.1 M sodium acetate buffer, residues of dimerised product are filtered off and the solution is adjusted to pH 10. After 1 hour at ambient temperature, the precipitate is filtered off, washed with a little dilute ammonia solution at 4� C. and dried. The yield is 50-80% of theory.
206 mg. of the amino compound obtained are dissolved in 25 ml. chloroform with warming and, after cooling, mixed with 0.3 ml. acrylic chloride, the solution immediately becoming darker in colour. After a few minutes, the acryloyl derivative precipitates out. After filtering off with suction and washing with some chloroform, there are obtained 122 mg. (50% of theory) 9-(acryloylaminoethylamino)-3-chloro-7-methoxyacridine in chromatographically pure form.
It will be understood that the specification and examples are illustrative, but not limitative of the present invention, and that other embodiments within the spirit and scope of the invention will suggest themselves to those skilled in the art.
|Cited Patent
|Filing date
|Publication date
|Nov 9, 1966
|Feb 8, 1972
|Shojiro Horiguchi
|Method of making coupler-bonded-polymers and chromogen-bonded-polymers nd polymers made thereby
|Mar 15, 1974
|Apr 12, 1977
|Mobil Oil Corporation
|Preparation of finely divided styrene-divinylbenzene organic pigments
|Nov 24, 1975
|Jul 12, 1977
|California Institute Of Technology
|Cell specific, variable density, polymer microspheres
|Sep 30, 1974
|Sep 6, 1977
|California Institute Of Technology
|Ionene modified small polymeric beads
|Nov 28, 1977
|Mar 25, 1980
|United States Of America
|Dye-containing polymer composition
|Jun 29, 1978
|Jul 22, 1980
|Board of Regents, State of Florida for and on behalf of the University of Florida
|Affinity chromatography and substrate useful therefor
|Irman et al., Biochemistry, 1969, 8 (No. 10), pp. 4074-4082.
|Khym, Analytical Biochemistry, 1976, 71 pp. 231-242.
|Citing Patent
|Filing date
|Publication date
|Oct 26, 1993
|Aug 30, 1994
|Tanabe Seiyaku Co., Ltd.
|Method for determining pyrogen content
|U.S. Classification
|525/281, 525/279, 525/374, 525/296, 525/349, 525/348, 525/284, 525/378, 525/293, 525/375
|International Classification
|Cooperative Classification
|European Classification
|Jan 5, 1987
|Fee payment
Year of fee payment: 4
|Jan 11, 1991
|Fee payment
Year of fee payment: 8
|Jan 3, 1995
|Fee payment
Year of fee payment: 12


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|Too small
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|Fits as expected
|Somewhat large
|Too large
Polo Ralph Lauren Men's Bienne II Boat Shoe
|$69.99 - $104.99
|Lower price available on select options
- Leather
- Imported
- Rubber sole
- Heel measures approximately 0.5"
- Polo Ralph Lauren creates the quintessential boat shoe with 360-degree lacing and contrast moc-toe stitching.
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Product Description
Polo Ralph Lauren creates the quintessential boat shoe with 360-degree lacing and contrast moc-toe stitching.
Classic and authentic, Polo is the foundation of the world of Ralph Lauren menswear, combining the time-honored aesthetic of East Coast Ivy League casual style with proper English refinement. Often imitated but never matched, Polo is a true symbol of the preppy lifestyle. The iconic polo player logo is recognized worldwide as a symbol of heritage and authenticity.
- Product Dimensions: 12 x 8 x 4 inches
- Shipping Weight: 2 pounds (View shipping rates and policies)
- Item model number: Bienne Ii
- Date first available at Amazon.com: November 21, 2013
- Domestic Shipping: This item is also available for shipping to select countries outside the U.S.
- International Shipping: This item is not eligible for international shipping. Learn More
- #2033 in Shoes > Men's > Loafers & Slip-Ons
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Shoes were returned.
The material is hard at first but like most shoes, it gets better after a period of time.
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absolutely loive these shoes, always look good even when they show a little wear. have had them for a year almost and still wear them regularly.Published 9 months ago by christian michener
Both the shoes are very tight on me and I cannot wear them. Although I mentioned to you my exact size. Please rectify to me the reasons of this problem. Waiting for your reply.Published 10 months ago by Dr. Khalil Omar
I normally wear a 9 these flit 0.5 a size too large. Other than that I really like the shoe and the feel. Don't really expect a break in period.Published 12 months ago by Singa17


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Venue: The Dome, Nairobi, Kenya
Dates: 04th ŌĆō 06th June, 2016
Timing: 10.00 am to 06.00 pm
Business Visitors Only
Kenya has a well-developed building and construction industry with quality engineering, building and architectural design services being readily available. This industry is currently on an upward trend following re-habilitation and reconstruction of roads and bridges etc.
With increase in population, and growing demand for affordable housing, opportunities exist in the construction of residential, commercial and industrial buildings including prefabricated low-cost housing. Investors can also manufacture and supply construction materials and components for the sector.
Chinese firm Wu Yi to carry out major road expansion in Kenya
China Wu Yi, a Chinese based firm is now expected to carry out a major road expansion in Kenya in the next few weeks.
China Wu Yi won the U$158Million project to expand the a 25km stretch of Waiyaki Way starting from James Gichuru Road junction to Mai Mahiu turnoff on the Nairobi-Nakuru highway.
The road expansion in Kenya which is expected to reduce the frequent traffic jams experienced in KenyaŌĆÖs capital Nairobi is part of the World Bank-funded mega roads expansion project.
The other project that is in the World Bank mission is to converse of the 45.2km road between the Jomo Kenyatta International Airport (JKIA) and Rironi into a superhighway.
Kenya and Ethiopia agree to construct joint crude oil pipeline
Kenya and Ethiopia have agreed on a deal that will see them construct a joint crude oil pipeline linking the two countries, this is after Uganda chose to export her oil through TanzaniaŌĆÖs Tanga port. The oil pipeline will run from KenyaŌĆÖs coastal town of Lamu to EthiopiaŌĆÖs capital city, Addis Ababa.
The deal was signed following billateral talks between President Uhuru Kenyatta EthiopiaŌĆÖs Prime Minister Hailemariam Desalegn during his three day state visit to Nairobi. The deal is part of a joint infrastructure project to incorporate the region under the Lamu Port- Southern Sudan -Ethiopia Transport corridor (Lapsset).
Airtel Kenya plans 4G launch by year-end
Airtel Kenya, the local unit of Indian telecoms group Bharti Airtel, is aiming to launch 4G┬ĀLTE┬Āservices by the end of the year, to enable it to better compete in the high speed mobile data market, reports Capital FM. ŌĆśData is very essential in our industry and we are investing heavily to ensure that our capabilities in data and internet are substantially improved,ŌĆÖ the cellcoŌĆÖs┬ĀCEO┬ĀAdil El Youssefi was quoted as saying, adding: ŌĆśWe are working closely with the stakeholders, regulators and the government to ensure that we [launch] the 4G network as soon as possible, hopefully this year, to Kenya.ŌĆÖ Youssefi also revealed that the coverage of AirtelŌĆÖs 3G network, which was launched in February 2012, has been increased to 75% of the population.
Another 40MW solar power plant in Kenya to be constructed
Another 40MW solar power plant in Kenya is set to be constructed in┬ĀNandi County,┬ĀKenya this is afterKenya Solar Energy Ltd showed interest in constructing a 40MW plant in┬ĀLamu County┬Āsoon.
Kopere Solar Park Limited which is the company behind the project is currently seeking regulatory approval for them to commence on the construction. The solar plant is projected to cost US$ 10m and will be financed by┬ĀClimate Investment Funds (CIF) and Martifer Solar which is a Portugal based firm will be involved as the technical partners to develop, build and operate the solar plant during its lifetime.
KenyaŌĆÖs economy expands by 5.6pc
The economy grew by 5.6 percent last year mainly driven by agriculture that performed well due to good rains received during the year.
The growth is higher than the 5.3 percent registered in 2014. Agriculture grew 5.6 percent in 2015 from 3.5 percent the previous year, driven by abundant rainfall.
Most economic sectors also expanded except tourism, which continued to bear the brunt of travel advisories by key source markets and worries about insecurity.
Dangote to nearly double production capacity across Africa in next three years
Dangote Cement, Nigeria's biggest listed company, said it aims to nearly double its production capacity over the next three years and half of its output by the end of 2019 will come from African plants outside its home market.
The company, majority owned by billionaire Aliko Dangote and Africa's biggest cement producer, plans new plants in Nigeria which will add between nine-million and 12-million tonnes to its existing capacity and will also expand production into Ivory Coast, Liberia, Mali, Niger, Kenya and Zimbabwe, it said in a presentation on Tuesday.
Kenya among few countries that weathered 2015 economic storm
Kenya is among the few Sub-Saharan African countries that weathered the 2015 economic storm to record an impressive growth, according to a new survey.
The International Monetary Fund (IMF) in its latest Regional Economic Outlook Survey for Sub-Saharan Africa, showed that while the continent recorded its lowest growth in 15 years in 2015, Kenya and a host of other African countries shrugged off the turbulence to record impressive growth.


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19 Stars Who Have Spent Time in the Slammer (One for 3 Whole Hours!)
Jail. Prison. The clink. The slammer. Lockup. The brig.
Up the river. The pen. The pound. The black hole.
Jail. Prison. The clink. The slammer. Lockup. The brig.
Up the river. The pen. The pound. The black hole.
Monday on T.I. and Tiny: The Family Hustle Season 4 Episode 16, Tiny and her posse took center stage as the focus shifted to their life on the road.
Monday night, on the unforgettable VH1 reality show installment also known as T.I. and Tiny: The Family Hustle Season 4 Episode 15, the Messiah himself sought advice from T.I.
Really, shouldn't we all be so lucky?
It's sort of official: there's serious bad blood brewing between Taylor Swift and Katy Perry.
It all started when Swift told Rolling Stone that she penned a song on hew new album about an enemy in the world of music, someone who did "something so horrible" when she tried to "sabotage an entire arena tour."
Aussie rapper Iggy Azalea seemed to come out of nowhere this summer with two chart-topping hits in "Problem" and "Fancy."
Iggy's rapid ascent has caused onlookers to cry foul, understandably confused with the way Azalea went from virtual unknown to hottest rapper in the game seemingly overnight.
Floyd Mayweather apparently admits that, well, T.I. had a pretty good reason for coming at him this spring, as Floyd says he was doing the rapper's wife.
It's still not entirely clear what caused the brawl, or at least it wasn't before Mayweather, promoting his upcoming fight, shed some light on it Saturday.
Last month T.I. and Floyd Mayweather fought outside a Vegas fast food restaurant, and the feud-happy rapper remarkably lived to tell the tale.
The beef reportedly stemmed from Mayweather's advances toward T.I.'s wife, Tameka "Tiny" Cottle.
The 2014 World Cup isn't the only thing going on in Brazil right now. As the video for T.I.'s new single "No Mediocre" shows us, Iggy Azalea is in Rio gettin' her twerk on!
The feud between Azealia Banks and T.I. got so ugly yesterday that Tip threatened Banks' life on Instagram.
As expected, Banks quickly shot back back with a veiled warning of her own.
Azealia Banks is an "up and coming" rapper who released a popular single in 2012 and followed it up with nothing but bizarre shots at more popular celebs on Twitter.
Banks' latest target is T.I. and his wife Tameka "Tiny" Harris - and not surprisingly, Tip isn't taking it lying down. After all, this is a man willing to fight Floyd Mayweather over his family.
Seriously... is there ANYthing Hugh Jackman cannot do?!?
He's portrayed a superhero on the big screen. He's played a gay man on Broadway.
Looks like T.I. was already operating with a pretty short fuse just hours before he got into a fight with Floyd Mayweather in Las Vegas last weekend.
When security at a crowded pool party denied the rapper entry, things got heated to say the least. Check out the TMZ video of that skirmish below:


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[Watch] Here's What Happens When Your Side Chick Crashes Your Wedding
It's a scene out of a Tyler Perry movie.
Nene Leakes is a lucky birthday girl, and she doesn't care who knows it. The Real Housewives of Atlanta star shared her birthday bounty with the Twitterverse by tweeting pics of her over-the-top birthday gifts.
"Yes it's my Birthday & I'm working n Hollywood #blessed so I'll celebrate later! Heres my first gift of the day," she wrote, attaching a pic of a luxe gray handbag.
Later: "My hotel room is full of birthday gifts because I'm workin all day. here's another cute gift! Cork Louboutins."
She didn't disclose who sent her the expensive swag, but it's probably safe to assume they didn't come from her RHOA castmates.
BET.com is your #1 source for Black celebrity news, photos, exclusive videos and all the latest in the world of hip hop and R&B music.
(Photo: Frazer Harrison/Getty Images for IMG)
It's a scene out of a Tyler Perry movie.
A few fellow students have taken to Instagram displaying an appalling lack of remorse


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We present a computational study of the sensitivity of SAW chemical sensors. Such sensors detect chemical vapors by showing resonant frequency shifts caused by the mass variations of chemoselective films coated on the pass way of surface waves. The sensitivity of this kind of sensors is significantly influenced by the film properties. The optimal design requires the quantitative understanding of the effects of film geometry, stiffness and conductivity on the sensor sensitivity. Previous analytical models for the study of the effects of film properties on the sensitivity of SAW chemical sensors assume that the film thickness is negligible compared to the central wave length of surface waves. As the central frequency of a SAW chemical sensors increases, the central wavelength decreases and the effects of film thickness may become significant. We have studied these effects using a finite element model [Xu, 2001]. The model has been developed based on the Galerkin method by discretizing the equations of wave propagation in piezoelectric materials and can be used for direct simulation of electromechanical wave propagation in SAW chemical sensors. Our results show linear response of frequency shift to film density variation and indicate better sensitivity for thicker and softer films within the range of these parameters we have studied. The computational results correlate well with the experimental results obtained by I. D Armonv, et al [Avramov, 2002]. In addition, our simulation shows that films with relative low stiffness can significantly delay the surface waves, and an increase in density can prolong this delay. The implications of these findings on the optimal design of SAW chemical sensors are discussed
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Showing 1-4 of 4 Health Resorts
Hawks Cay Resort
Head to a tropical island to reinvent yourself. Spend the day hiking or splash in the waters, or even try your hand at diving! After a long, eventful day, head to the on-site spa at Hawks Cay Resort, voted 2009 Best Hotel Spa in the U.S. by the readers of Travel + Leisure. You'll feel brand new after your vacation for Duck Key Florida. ... more
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iTrip - Islamorada
You're sure to stay in shape with a getaway at iTrip Islamorada! With a tropical paradise awaiting outside your door, you'll never want to stay still. Try diving at John Pennekamp Coral Reef Park and soak in the marine scenery. Onsite, you'll find a fitness center at one of the rental properties and hot tubs and tennis courts at numerous lodgings! ... more
The Moorings Village
Restore your health and begin a journey to self-improvement at the onsite fitness center, or with an incredible Island Body & Sol Spa treatment. Your comfort is #1 and The Moorings Village will remind you with its modern day stress relief gemstone facials, restoring massages, and hands and feet harmony treatments.... more
Pier House Resort & Spa
At the Pier House Resort & Spa, you won't have to give up on your fitness routine. Enjoy the onsite fitness study before you head out into the Florida Keys. After a full day, treat yourself to a treatment at the spa. Enjoy a massage or a facial. You'll never want to leave! ... more
Showing 1-4 of 4 Health Resorts


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I have a grid with the row editor plugin enabled. I am using the autoSync: true directive since I am still learning this framework. After update,...
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xtype: 'timefield',
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A colleague of mine and were discussing starting unit testing our ExtJs code. In the download for 4.0 I see a Build folder...
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spket need it! thx!
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Bypass the box office line at many theaters with guaranteed advance tickets. If something comes up, you can return or exchange them up until the posted showtime.
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Looking for movie tickets? Enter your location to see which movie theaters are playing Le Rouge et le Noir near you.
Stendhal's brilliant but difficult novel Le Rouge et le Noir all but defies transfer to film, but adaptor/director Claude Autant-Lara comes within shouting distance of full success. Stripped to essentials, the plot concerns Julien Sorel (Gerard Philipe), a carpenter's son who becomes a tutor. While his duties do not include the seduction of his employer's wife (Danielle Darrieux), Sorel offers this service free of charge. After this episode, Sorel becomes a priest...and the story isn't over yet. Though the director is too doggedly literal in his adaptation at times, and despite the fact that Gerard Philipe was about ten years too old for the part of Jean Sorel, Le Rouge et le Noir manages to hold the audience in its thrall for 2 hours and 50 minutes (most American prints, retitled The Red and the Black, run only 140 minutes). ~ Hal Erickson, Rovi


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Paul Frederick Herman Fitzke
Position: WB
5-10, 195lb (178cm, 88kg)
July 30, 1900
in La Crosse, WI
June 30, 1950
in Sacramento, CA
Wyoming ,
Weighted Career AV (100-95-...): 0 (15060th overall since 1960)
High School:
La Crosse (WI)
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Certificate IV in Business
Australian Institute of Commerce and Technology (AICT)
Certificate IV
|Mid Year Intake?
|Study Mode
|Entry Requirements
|Full-time internal, Part-time internal
No minimum education
Course Description
This qualification is part of the Business Services Training Package. It is suited to those working as administrators and project officers. In this role, individuals use well-developed skills and a broad knowledge base to apply solutions to a defined range of unpredictable problems and analyse information from a variety of sources. They may provide leadership and guidance to others with some limited responsibility for the output of others.
Subjects you can Study
Core: Implement and monitor WHS policies, procedures and programs to meet legislative requirements. Electives: Coordinate implementation of customer service strategies; Work effectively in a business continuity context; Review and maintain a website; Report on financial activity; Organise meetings; Coordinate business resources; Use and respect copyright; Protect and use new inventions and innovations; Make a presentation; Develop and use complex spreadsheets; Undertake marketing activities; Provide information from and about records; Analyse and present research information; plus more.
About Australian Institute of Commerce and Technology (AICT)Established in 1988, the Australian Institute of Commerce and Technology is a Registered Training Organisation that delivers an innovative style of training in Information Technology, Business and English. Their practical hands-on approach, combined with industry certifications and utilisation of their network of industry partners contributes to their success. They hold affiliations and partnerships with over 100 companies throughout the Perth area. It is through these partnerships that AICT can successfully offer work experience and job placements through a variety of different programs. This means that by the time students graduate, they can hold multiple certifications, professional work experience and local work references. Centrally located in the heart of the Perth CBD, commuting to and from AICT is simple. With vibrant shopping and extra curricular activities surrounding the college, AICT is strategically placed for optimising educational experiences.
Provider CRICOS: 02540M RTO code: 2058
Certificate IVCourse code: BSB40215
- Full-time internal = 0.5 year
- Part-time internal
10 units: 1 core; 9 elective
Entry Requirements
No minimum education
Life after Study
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Administrative assistants perform a range of administrative tasks in an organisation.
Office Administrator
Office administrators undertake a range of functions to make sure the administration activities within an organisation run smoothly. They may be responsible for the management of human resources, budgets, accommodation and property facilities and records. These functions can be performed at various levels ranging from junior through to upper management.
Receptionists act as the first point of contact in an organisation, greeting people and attending to enquiries made by phone or in person.
Secretaries assist professionals, managers and executives by performing clerical and secretarial duties.


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Posted: 1/25/2009Provided by Citysearch -
This is a small restaurant with a friendly attitude. The food is very good especially the carne guisada. The sea food plates are delicious. The part I like the most is the extreme friendliness of all the waitresses. They go beyond and give it that extra effort to make you feel at home. My favorite restaurant.


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Add some variety to your fitness routine with these upper and lower leg exercises from The American Council on Exercise. We have a large selection of leg exercises that will will help you strengthen and build muscle throughout your legs including your hamstrings, thighs, shin and calf muscles. Start taking advantage of this free information today! If you are looking for full workout routines that incorporate these leg exercises, check out our Workouts section.
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It can be challenging to eat healthy on vacation, especially if your adventure includes a lot of time spent on the road. But with a little pre-planning and some mindfulness, you can eat healthier on your trip, even when your choices are limited!
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ADO.NET 2.0 Provider for SQLite
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Peter Mullan - Peter Mullan, London, England - at the London Evening Standard British Film Awards 2011 at the Marriot Hotel - Arrivals Monday 7th February 2011 (11 Pictures)
Photo credit: WENN.com2
Mike Leigh
Photo credit: WENN.com2
Harold Tillman,
Photo credit: WENN.com2
David Thewlis
Photo credit: WENN.com2
Harry Treadaway
Photo credit: WENN.com2
Lizzie Cundy
Photo credit: WENN.com2
Anthony Daniels
Photo credit: WENN.com2
Luke Treadaway
Photo credit: WENN.com2
Jim Broadbent
Photo credit: WENN.com2
Emily Watson
Photo credit: WENN.com2
Ed Stoppard
Photo credit: WENN.com2
View All: Peter Mullan - Peter Mullan, London, England...
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Bjork Digital comes to London's Somerset House in September, along with a single live show at the Royal Albert Hall.
Kim Kardashian released an audio excerpt from a phone call between Kanye and Taylor Swift over the lyrics of 'Famous' - but if it was recorded...
There's very much a strength of conviction in remaining what you were, but arguably more so in becoming what you want to be.
The BBC drama starring Aidan Turner returns to BBC One on September 4th.
Guns N' Roses were detained at the Canadian border last week for gun possession but they're adamant the weapon didn't belong to them.
This isn't your usual period movie. A powerfully emotional depiction of rural Scottish life at...
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Widower Joseph spends his days walking the streets and frequenting various pubs in an industrial...
In rural England during the First World War, a horse named Joey befriends a young...


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Release Date: 2016
Duration: 226 minutes (HD version : 226 minutes)
cast: Nishikawa Yui
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Series: –
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Release Date: 2016-05-01
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2/2 Indigo Reef w/ 32' Complimentary Boat Slip
From $350 / night
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Description from the owner
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Type: Villa
Book this 2 bedroom, 3 bathroom villa in Marathon today! A total of 4 people can sleep here comfortably.
Guests can enjoy a pool, air conditioning, and internet access among other amenities offered by this rental.
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Costs vary, with the average weekly rate as low as $2,100.00, depending on travel dates.
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IHG Opens Hotel Indigo Düsseldorf - Victoriaplatz
Monday December 2nd, 2013 - 9:13AM
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DUSSELFDORF, GERMANY—IHG has opened Hotel Indigo Düsseldorf - Victoriaplatz located here. The hotel will be the third Hotel Indigo property in Germany, alongside Hotel Indigo Berlin – Alexanderplatz and Hotel Indigo Berlin – Ku'Damm.
Hotel Indigo Düsseldorf-Victoriaplatz will operate under a franchise agreement with LFPI Hotels Management Germany GmbH.
Located in the city's fashion district, Hotel Indigo Düsseldorf – Victoriaplatz's 126 individually designed bedrooms take inspiration from the local area with furniture, bedding and colors. Each floor pays tribute to a different decade of fashion from the 50s through to the 2000s, illustrated through the fabrics and patterns used.
The hotel is situated near the city's fashion exhibitions including the NRW Forum, where the first Igedo fashion fair was launched in the 50s, just a short walk away from two of the biggest fashion trade shows and The Gallery which showcases the latest fashion-related products and designs.
Katja Schnabel, General Manager, Hotel Indigo Dusseldorf - Victoriaplatz said in a statement, "Set in the heart of the city, Hotel Indigo Düsseldorf - Victoriaplatz offers the perfect setting for exploring this stylish location and the iconic exhibitions. Steeped in our local history and charm, the uniquely designed hotel sits in the core of the fashion district, on the same street as 800 designer showrooms, providing an ideal location for fashion lovers. We look forward to welcoming guests to the Hotel Indigo Düsseldorf to show them what our glamorous city is all about."
Tags: IHG • Hotel Indigo • Hospitality • Brands • Openings •
Well, it's halfway through 2016, and not much has changed since the beginning of the year. Despite the terrorism acts in Orlando, FL and Turkey, and the Brexit referendum, little has happened to affect the travel industry. Overall, things might have slowed a bit, but a majority of the fundamentals are holding steady, including occupancy, ADR and RevPAR. I've been asked why this cycle has lasted longer than those preceding it. As far as I am concerned, it's due to a single phrase: Never Again.
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Photos from The Tall Men
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13 January 2012
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Titles: The Tall Men
Names: Clark Gable
The Tall Men (11)
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Sunday, July 24
Known for arranging abstract forms to create figurative scenes, Farah Atassi presents two new painting series. Using her signature vocabulary of geometric shapes and patterns, she deconstructs Cubism and, separately, examines the chicly psychedelic aesthetics of the 1970s.
Farah Atassi
108, rue Vieille du Temple / +33140270555 / xippas.com
Tue - Sat 10am to 7pm
DeFeo's first solo show in Paris spans the late American artist's career with collages, photographs, drawings, and paintings made between the 1950s and 1980s, including two early tempera and charcoal works made in Paris in 1951. Rarely shown black-and-white photos from the 1970s include landscapes, still lifes, and cropped close-ups of DeFeo's Tuxedo Junction, 1965/1974, a large-scale oil-on-paper work mounted on three Masonite panels, which is concurrently on view in the Centre Pompidou's "Beat Generation" exhibition (June 21–October 3).
Jay DeFeo Jay DeFeo
66 rue de Turenne / +33148875004 / galeriefrankelbaz.com
Tue - Sat 11am to 7pm, Tue - Sat 11am to 7pm
Concurrent with her solo show at the Palais de Tokyo, which features films made between 2008 and 2016—including the oddly sensuous No Nose Knows, which debuted at last year's Venice Biennale—Rottenberg's exhibition draws attention to her work in other media. A new series of drawings shown in the company of sculptures from the "Textures" series echo themes familiar from Rottenberg's films: feminine corporeality, work, and byzantine systems of production.
Galerie Laurent Godin | Rue du Grenier Saint-Lazare
5 rue du Grenier Saint-Lazare / +33142711066 / laurentgodin.com
Tue - Sat 11am to 7pm
The first contemporary artist invited to show at the Picasso museum's newly renovated Hôtel Salé, Spanish-born Miquel Barceló presents paintings, sculptures, ceramics, and works on paper made between the 1990s and today. In dialogue with certain motifs found in Picasso's oeuvre, Barceló shows off his own diverse and prolific creative process. In conjunction, the Bibliothèque Nationale de France will present an exhibition of Barceló's prints (March 22–August 28).
Miquel Barceló Sol y sombra
5 rue de Thorigny / +33142712521 / musee-picasso.fr
Tue - Fri 11:30am to 6pm, Sat - Sun 9:30am to 6pm
Turning the spotlight on Paul Klee's ironic humor, this retrospective is France's first large-scale show dedicated the to the Swiss-German artist in over fifty years. Some 250 works (paintings, sculptures, drawings, and paintings on glass) track Klee's stylistic evolution from Cubism and Dada to Surrealism and Constructivism. Highlights include 1920s illustrations for Voltaire's "Candide," and the artist's large-scale 1938 masterpiece, Insula dulcamara.
Paul Klee L'Ironie à l'œuvre
Drawing heavily from its Arte Povera–rich permanent collection, the Centre Pompidou shows off its Fontanas, Penones, Manzonis, Pistolettos, and Burrises. But the exhibition, which focuses on the decade between 1964 and 1974, also broadens our understanding of the Italian art movement by including work by less celebrated but equally essential adherents such as Piero Gilardi and Mario Ceroli.
Un art pauvre
The Icelandic artist's first solo show in France was inspired by an epic novel by Nobel laureate and fellow Icelander Halldór Laxness. Among the new works created specifically for the Palais de Tokyo: a repeating performance of a fleeting encounter, a video installation of idealized portraits, and a theatrical freestanding painting depicting an icy mountain.
Ragnar Kjartansson SEUL CELUI QUI CONNAÎT LE DÉSIR
13, Avenue du Président Wilson / +33147235401 / palaisdetokyo.com
Tue - Sun 12pm to 9pm


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