The agent for dyeing keratin fibers contains one or more aryl and/or benzyl alcohols, one or more oxidizing enzymes, especially vanillyl oxidase, derivatives of vanillyl oxidase or galactose oxidase, and a nucleophilic compound that forms a dye for dyeing keratin fibers in the presence of the alcohols and enzymes. A method for dyeing keratin fibers with this agent is also disclosed. Two-component kits for performing the method of dyeing keratin fibers include a first compenent (A) and a second component (B) separate from the first component. The first component (A) includes the nucleophilic compound, the alcohol and optionally the oxidizing enzyme. The second component (B) includes the oxidizing enzyme when it is not included in the first component or more alcohol.

The present invention relates to an agent and to a method for dyeing keratin fibers, especially human hair.
The reaction between ketones or aldehydes, especially aromatic aldehydes such as benzaldehyde and various substituted benzaldehydes, with compounds having an active CH group, water being split off and compounds formed, which are suitable for dyeing keratin fibers, has already been described earlier, for example, in the German Offenlegungsschrift 197 17 281 and the German utility patent 299 08 464. The possibility of sensitizing when the ketone or the aldehyde is applied directly on the hair or the scalp is a disadvantage of using this reaction between ketones or aldehydes and compounds with active CH groups. Furthermore, it is difficult to incorporate especially aldehydes into the dyeing agents and to keep such agents for longer periods, since the aldehydes tend to oxidize in air, forming carboxylic acids, which do not participate in the color-forming reaction.
The present application avoids the direct use of ketones or aldehydes by employing primary or secondary alcohols as aldehyde or ketone precursors for the aforementioned reaction, the alcohols being oxidized enzymatically in situ to the corresponding aldehydes or ketones.
The object of the present application therefore is an agent for dyeing keratin fibers, especially wool, silk or hair, especially human hair, wherein at least one compound with a nucleophilic reaction center, at least one alcohol from the group comprising aryl alcohol derivatives and benzyl alcohol derivatives and at least one oxidizing enzyme is contained.
As inventive alcohol, especially aryl alcohols or benzyl alcohols of formula (I) may be named, which can be converted by enzyme-catalyzed oxidation to the corresponding carbonyl compounds;
Arxe2x80x94(CHxe2x95x90CH)nxe2x80x94CH2OHxe2x80x83xe2x80x83(I)
in which n=0, 1 or 2;
and Ar is a group of formula
in which Y is an oxygen atom, a sulfur atom or an NRa group; R1xe2x80x2, R2xe2x80x2, R3xe2x80x2, R4xe2x80x2, R5xe2x80x2, R6xe2x80x2 and R7xe2x80x2 independently of one another are a hydrogen atom, a hydroxy group, a methoxy group, an aryl group, a halogen atom (F, Cl, Br, I), a xe2x80x94CHOxe2x80x94 group, a xe2x80x94CORa group, a xe2x80x94CO2Ra group, an NO2xe2x80x94 group, an xe2x80x94OCORa group, an xe2x80x94OCH2aryl group, an xe2x80x94NH2 group, an xe2x80x94NH3+ group, an xe2x80x94NHRa group, an xe2x80x94NH2Ra+ group, an xe2x80x94N(Ra)2 group, an xe2x80x94N(Ra)3+ group, an xe2x80x94NHCORa group, an xe2x80x94NHCOORa group, in which Ra is a hydrogen atom, a linear or branched C1 to C4 alkyl group, an optionally substituted, aromatic, carbocyclic group or heterocyclic group, or R4xe2x80x2 and R5xe2x80x2 together with the carbon atom of the aromatic ring form a 5-membered or 6-membered alicyclic or aromatic ring, which optionally may contain one or more sulfur, nitrogen or oxygen atoms.
The following compounds of formula (I) are particularly preferred: benzyl alcohol, 4-hydroxy-benzyl alcohol, 4-hydroxy-3-methoxy-benzyl alcohol (vanillyl alcohol), 3-hydroxy-4-methoxy-benzyl alcohol (isovanillyl alcohol), 3,5-dimethoxy-4-hydroxybenzyl alcohol, 3,4-dihydroxy-benzyl alcohol, 2-hydroxy-3-methoxy-benzyl alcohol, 4-ethoxy-benzyl alcohol, 4-carboxy-benzyl alcohol, 2,5-dihydroxy-benzyl alcohol, 2,4-dihydroxy-benzyl alcohol, 2-hydroxy-benzyl alcohol, 3,5-dimethoxy-4-hydroxy-benzyl alcohol, 4-hydroxy-2-methoxy-benzyl alcohol, 2,4-dimethoxy-benzyl alcohol, 2,3-dimethoxy-benzyl alcohol, 2,5-dimethoxy-benzyl alcohol, 3,5-dimethoxy-benzyl alcohol, 3,4-methylenedioxy-benzyl alcohol, 3,4-dimethoxy-benzyl alcohol, 3-ethoxy-4-hydroxy-benzyl alcohol, 3,5-dimethyl-4-hydroxy-benzyl alcohol, 3,4-dimethoxy-5-hydroxy-benzyl alcohol, 3,4,5-trimethoxy-benzyl alcohol, 2,4,6-trihydroxy-benzyl alcohol, 3,4,5-trihydroxy-benzyl alcohol, 2,3,4-trihydroxy-benzyl alcohol, 3,5-di-t-butyl-4-hydroxy-benzyl alcohol, 2-nitro-benzyl alcohol, 3-nitro-benzyl alcohol, 4-nitro-benzyl alcohol, 2-amino-benzyl alcohol, 3-amino-benzyl alcohol, 3-amino-4-methyl-benzyl alcohol, 3,5-diamino-benzyl alcohol, 4-amino-benzyl alcohol, 4-dimethylamino-benzyl alcohol, 4-diethylamino-2-hydroxy-benzyl alcohol, 4-diethylamino-3-methoxy-benzyl alcohol, 4-dimethylamino-2-methoxy-benzyl alcohol, 4-dibutyl-amino-benzyl alcohol, 3-methoxy-4-(1-pyrrolidinyl)-benzyl alcohol, (4-methoxy-naphthalene-1-yl)-methanol, (4-dimethylamino-naphthalene-1-yl)-methanol, 2-(hydroxymethyl)-1-naphthol, 1-naphthalene-methanol, 2-naphthalene-methanol, (2-methoxy-naphthalene-1-yl)-methanol, 4-hydroxy-methyl-naphthalene-1-ol, 4xe2x80x2-hydroxymethyl-biphenyl-4-ol, (4-hydroxymethylphenyl)-methanol, 4-(3-hydroxy-propenyl)-2-methoxy-phenol, 4-(3-hydroxy-propenyl)-2,6-dimethoxy-phenol, 3-(4-dimethylaminophenyl)-prop-2-ene-1-ol, 5-(4-(diethylamino-phenyl)-penta-2,4-diene-1-ol, thiophene-2-yl-methanol, (5-hydroxymethyl-thiophene-2-yl)-methanol, thiophene-3-yl-methanol, (1H-pyrrole-2-yl)-methanol, (1-methyl-1H-pyrrole-2-yl)-methanol, (5-methyl-furan-2-yl)-methanol, (1H-indole-3-yl)-methanol, and (6-methyl-1H-indole-3-yl)-methanol.
The use of the alcohol instead of the corresponding carbonyl compound of the present invention makes a rapid intensive dyeing of the fibers, especially of the keratin fibers possible in the presence of a compound with a nucleophilic center with the addition of an oxidizing enzyme. xe2x80x9cAn oxidizing enzymexe2x80x9d is understood here to be an enzyme, which is able to catalyze the oxidation of the alcohol to an aldehyde or ketone. The following are named as examples of such enzymes, which are, however, not limited to these: alcohol dehydrogenases (E.C. Classification 1.1.1.-), alcohol oxidases (E.C. Classification 1.1.2- and 1.1.3- and 1.1.99-), flavinoxidases (E.C. Classification 1.2.--), laccases (E.C. Classification 1.4.---), peroxidases (E.C. Classification 1.11.1.-), hydroxylases and monooxygenases (E.C. Classification 1.13.12- and 1.13.99-).
The enzyme is used preferably in an amount of 5 to 100 units per millimole of substrate (alcohol). A unit of enzyme activity refers here to the amount of enzyme, which is required to catalyze the oxidation of 1 micromole of alcohol per minute.
xe2x80x9cCompounds with a nucleophilic reaction centerxe2x80x9d are understood to be compounds, which are able to form unsaturated carbon-carbon or carbon-nitrogen bonds by reaction with the electrophilic carbonyl carbon of the aldehyde or ketone. Suitable compounds with a nucleophilic reaction center of the present invention are, for example, primary or secondary aliphatic or aromatic amines, nitrogen-containing heterocyclic compounds, amino acids, oligopeptides with 2 to 9 amino acid groups, aromatic hydroxy compounds and compounds with active CH group.
Suitable compounds with primary or secondary amino groups are, for example, primary aromatic amines such as N,N-dimethyl-p-phenylenediamine, N,N-diethyl-p-phenylenediamine, N-(2-hydroxyethyl)-N-ethyl-p-phenylenediamine, N,N-bis-(2-hydroxyethyl)-p-phenylenediamine, N-(2-methoxyethyl)-p-phenylenediamine, 2,3-dichloro-p-phenylenediamine, 2,4-dichloro-p-phenylenediamine, 2,5-dichloro-p-phenylenediamine, 2-chloro-p-phenylenediamine, 2,5-dihydroxy-4-morpholinoaniline dihydrobromide, 2-aminophenol, 3-aminophenol, 4-aminophenol, 2-aminomethyl-4-aminophenol, 2-hydroxymethyl-4-aminophenol, o-phenylenediamine, m-phenylenediamine, p-phenylenediamine, o-toluylenediamine, m-toluylenediamine, 2,5-diaminotoluene, 2,5-diaminophenol, 2,5-diaminophenethol, 4-amino-3-methylphenol, 2,4-diaminophenoxyethanol, 2-(2,5-diamino-phenyl)-ethanol, 2-(2,5-diaminophenoxy)-ethanol, 4-methylaminoaniline, 3-amino-(2-hydroxyethyloxy)aniline, 3,4-methylenediaminoaniline, 3,4-methylenedioxyaniline, 3-amino-2,4-dichloro-phenol, 4-methylamino-phenol, 2-methyl-5-amino-phenol, 3-methyl-4-amino-phenol, 2-methyl-5-(2-hydroxyethylamino)-phenol, 6-methyl-3-amino-2-chloro-phenol, 2-methyl-5-amino-4-chloro-phenol, 5-(2-hydroxyethylamino)-4-methoxy-2-methyl-phenol, 1,3-diamino-2,4-dimethoxybenzene, 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, 2,3-diaminobenzoic acid, 2,4-diaminobenzoic acid, 2,4-diaminobenzoic acid, 2,5-diaminobenzoic acid, 3,4-diaminobenzoic acid, 3,5-diaminobenzoic acid, 4-aminosalicylic acid, 5-amino-salicylic acid, 3-amino-4-hydroxy-benzoic acid, 4-amino-3-hydroxy-benzoic acid, 2-aminobenzenesulfonic acid, 3-aminobenzenesulfonic acid, 4-aminobenzenesulfonic acid, 3-amino-4-hydroxybenzenesulfonic acid, 4-amino-3-hydroxynaphthalene-1-sulfonic acid, 6-amino-7-hydroxy-naphthalene-2-sulfonic acid, 7-amino-4-hydroxynaphthalene-2-sulfonic acid, 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid, 3-amino-2-naphthoic acid, 3-aminophthalic acid, 5-aminoisophthalic acid, 1,3,5-triaminobenzene, 1,2,4-triaminobenzene, 1,2,4,5-tetraaminobenzene, 2,4,5-triaminophenol, pentaaminobenzene, hexaminobenzene, 2,4,6-triaminoresorcinol, 4,5-diaminopyrocatechol, 4,6-diaminopyrogallol, 3,5-diamino-4-hydroxypyrocatechol, aromatic anilines or phenols with a further aromatic group, such as 4,4xe2x80x2-diaminostilbene, 4,4xe2x80x2-diaminostilbene-2,2xe2x80x2-disulfonic acid monosodium salt or 4,4xe2x80x2-diaminostilbene-2,2xe2x80x2-disulfonic acid disodium salt, 4,4-diamino-diphenylmethane, 4,4-diaminodiphenylsulfide, 4,4-diaminodiphenylsulfoxide, 4,4-diaminodiphenylamine, 4,4-diaminodiphenylamine-2-sulfonic acid, 4,4xe2x80x2-diaminiobenzophenone, 4,4xe2x80x2-diamininobenzophenone diphenyl ether, 3,3xe2x80x2,4,4xe2x80x2-tetraaminodiphenyl), 3,3xe2x80x2,4,4xe2x80x2-tetraamino-benzophenone, 1,3-bis-(2,4-diaminophenoxy)-propane, 1,8-bis-(2,5-diaminophenoxy)-3,6-dioxaoctane, 1,3-bis-(4-aminophenylamino)-propane, 1,3-bis-(4-amino-phenylamino)-2-propanol, 1,3-bis-[N-(4-aminophenyl)-2-hydroxyethyl-amino]-2-propanol, N,N-bis-[2, (4-aminophenoxy)-ethyl]-methylamine and N-phenyl-1,4-phenylenediamine.
Suitable nitrogen-containing heterocyclic compounds are, for example, 2-aminopyridine, 3-aminopyridine, 4-aminopyridine, 2-amino-3-hydroxy-pyridine, 2,6-diaminopyridine, 2,5-diaminopyridine, 2,3-diaminopyridine, 2-dimethylamino-5-aminopyridine, 2-methylamino-3-amino-6-methoxy-pyridine, 2,3-diamino-6-methoxypyridine, 2,6-dimethoxy-3,5-diaminopyridine, 2,4,5-triaminopyridine, 2,6-dihydroxy-3,4-dimethylpyridine, 2,4-dihydroxy-5,6-diamino-pyrimidine, 4,5-triamino-pyrimidine, 4-hydroxy-2,5,6-triamino-pyrimidine, 2-hydroxy-4,5,6-triamino-pyrimidine, 2,4,5,6-tetraamino-pyrimidine, 2-methylamino-4,5,6-triamino-pyrimidine, 2,4-diamino-pyrimidine, 4,5-diamino-pyrimidine, 2-amino-4-methoxy-6-methyl-pyrimidine, 3,5-diaminopyrazole, 3,5-diamino-1,2,4-triazole, 3-aminopyrazole, 3-amino-5-hydroxypyrazole, 2-aminoquinoline, 3-aminoquinoline, 8-aminoquinoline, 4-aminoquinaldine, 2-aminonicotinic acid, 6-aminonicotinic acid, 5-amino-isoquinoline, 5-aminoindazole, 6-aminoindazole, 5-aminobenzimidazole, 7-aminobenzimidazole, 7-amino-benzothiazole, 5-amino-benzothiazole, 2,5-dihydroxy-4-morpholinoaniline as well as indole and indolin derivatives, such as 4-aminoindole, 5-aminoindole, 6-aminoindole, 7-aminoindole, 5,6-dihydroxyindole, 5,6-dihydroxyindoline and 4-hydroxyindoline.
The aforementioned amines and heterocyclic compounds can be used in free form as well as in the form of the physiologically tolerated salts, such as salts of inorganic acids like hydrochloric acid or sulfuric acid.
All naturally occurring and synthetic amino acids, such as arginine, histidine, tyrosine, phenylalalanine, dihydroxyphenylalanine, ornithine, lysine and tryptophane come into consideration as amino acids.
As oligopeptides, all naturally occurring or synthetic oligopeptides, as well as the oligopeptides contained in polypeptide or protein hydrolysates, can be used, provided that they are sufficiently soluble for use in the inventive dyeing agents. Glutathione or the oligopeptides, contained in the hydrolysates of collagen, keratin, casein, elastin, soybean protein, wheat gluten or almond protein, are named as examples. In this connection, the joint use of the oligopeptides with compounds with a primary or a secondary amino group or with aromatic hydroxy compounds is preferred.
Suitable aromatic hydroxy compounds are, for example, 2-methylresorcinol, 4-methylresorcinol, 5-methylresorcinol, 2,5-dimethylresorcinol, resorcinol, 3-methoxyphenol, pyrocatechol, hydroquinone, pyrogallol, phloroglucinol, hydroxyhydroquinone, 2-methoxyphenol, 3-methoxyphenol, 4-methoxyphenol, 3-dimethylaminophenol, 2-(2-hydroxyethyl)-phenol, 3,4-methylenedioxyphenol, 2,4-dihydroxybenzoic acid, 3,4-dihydroxy-benzoic acid, 2,4-dihydroxy-phenylacetic acid, 3,4-dihydroxyphenylacetic acid, gallic acid, 2,4,6-trihydroxybenzoic acid, 2,4,6-trihydroxy-acetophenone, 2-chlororesorcinol, 4-chlororesorcinol, 1-naphthol, 1,5-dihydroxynaphthalene, 2,3-dihydroxy-naphthalene, 2,7-dihydroxynaphthalene, 4-hydroxy-2-naphthalene sulfonic acid and 3,6-dihydroxy-2,7-naphthalene sulfonic acid.
As suitable compounds with an active CH group, 1,2,3,5-tetramethyl-3H-indolium iodide, 1,2,3,5-tetramethyl-3H-indolium methosulfate, 2,3-dimethyl-benzothiazolium iodide, 2,3-dimethyl-benzothiazolium-p-toluenesulfonate, rhodanine, rhodanine-3-acetic acid, 1-ethyl-2-quinaldinium iodide, 1-methyl-2-quinaldinium iodide, barbituric acid, thiobarbituric acid, 1,3-dimethyl-thiobarbituric acid, 1,3-diethyl-thiobarbituric acid, oxindole, 3-indoxyl acetate, cumaranone, 1-methyl-3-phenyl-2-pyrazolinone and enamines of Formula (II) or their salts of Formula (IIa),
in which R1 is a single ring or multi-ring aromatic group, especially a 5-membered or 6-membered aryl group (preferably a phenyl group), which optionally is substituted by a C1 to C4 alkyl group, a C1 to C4 hydroxyalkyl group, a hydroxy group, a methoxy group, a dialkylamino group or a halogen group (F, Ck, Br, I), a 5-membered or 6-membered hetero cyclic ring (preferably a pyridyl group or a naphthyl group), which optionally is substituted by a C1 to C4 alkyl group, a C1 to C4 hydroxyalkyl group, a hydroxy group, a methoxy group, a dialkylamino group or a halogen group (F, Cl, Br, I); R2 is a linear or branched C1 to C8 alkyl group, a linear or branched C1 to C8 hydroxyalkyl group or a C1 to C8 alkoyalkyl group, possibly with oxygen atoms between the carbon atoms of the alkyl chain; R3 is a linear or branched C1 to C8 alkyl group, a C1 to C8 alkoxyalkyl group, a linear or branched C1 to C8 alkylene group, a C1 to C8 alkoxy alkylene group, an oxygen atom, a sulfur atom, an xe2x80x94NH group or an xe2x80x94NR group, in which R is an alkyl group, an alkoxyalkyl group, a hydroxyalkyl group or hydrogen, a cyclic compound possibly being formed by the R1 and R3 groups together with the nitrogen atom and the carbon atom of the basic enamine structure, and R4 is hydrogen, a linear C1 to C4 alkyl group or a branched C1 to C4 alkyl group and Axe2x88x92 is the anion of an organic or inorganic acid.
Especially preferred compounds with a nucleophilic reaction center are the following enamines of Formulas (III) to (X), in which X is a carbon atom with two C1 to C4 alkyl groups, which may be the same or different (especially 2 methyl groups), a carbon atom, substituted by a C1 to C4 alkyl group, and a carbon atom, substituted by a hydroxy group, a sulfur atom, an alkylated nitrogen atom, a not alkylated nitrogen atom or an oxygen atom; and R2 is a linear or branched C1 to C8 alkyl group, a linear or branched C1 to C8 hydroxyalkyl group or a C1 to C8 alkoxyalkyl group, oxygen atoms possibly being present between the carbon atoms of the alkyl chain; R4 is hydrogen, a linear C1 to C4 alkyl group or a branched C1 to C4 group; R5, R6, R7 and R8 independently of one another are hydrogen, a linear or branched C1 to C4 alkyl group, a linear or branched C1 to C4 hydroxyalkyl group, a hydroxy group, a methoxy group, an amino group, a mono(C1 to C4) alkylamino group, a di(C1 to C4) alkylamino group, a benzyl group or a halogen atom (F, Cl, Br, I); and Axe2x88x92 is chloride, bromide, iodide, sulfate, hydrogen sulfate, toluenesulfonate, benzenesulfonate, monomethyl sulfate, hexafluorophosphate, hexafluoroantimonate, tetrafluoroborate, tetraphenylborate, formate, acetate or propionate, the chloride ion, tetrafluoroborate ion, the acetate ion and the hydrogen sulfate ion being particularly preferred.
The following compounds with nucleophilic reaction centers are preferred: 1,3,3-trimethyl-2-methylene-indoline as well as its salts,
1,3,3,4-tetramethyl-2-methylene-indoline as well as its salts,
1,3,3,5-tetramethyl-2-methylene-indoline as well as its salts,
1,3,3,6-tetramethyl-2-methylene-indoline as well as its salts,
1,3,3,7-tetramethyl-2-methylene-indoline as well as its salts,
1,3,3,6,7-pentamethyl-2-methylene-indoline as well as its salts,
1,3,3,5,7-pentamethyl-2-methylene-indoline as well as its salts,
1,3,3,4,7-pentamethyl-2-methylene-indoline as well as its salts,
5-chloro-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5-fluoro-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5-isopropyl-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5-hydroxy-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5-methoxy-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5-amino-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5-nitro-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5-N-acetylamino-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
6-hydroxy-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
6-methoxy-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5-methoxy-6-nitro-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5-methoxy-6-N-acetylamino-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5-methoxy-6-amino-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5,6-methylenedioxy-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5,6-dihydroxy-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5,6-dimethoxy-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
4,5-dihydroxy-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5,7-dihydroxy-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5-amino-6-methoxy-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5-amino-7-hydroxy-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5-hydroxy-7-amino-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
5-hydroxy-7-N-acetylamino-1,3,3-trimethyl-2-methylene-indoline as well as its salts,
1-methyl-3-spiro-cyclopropyl-2-methylene-indoline as well as its salts,
1-methyl-3-spiro-cyclohexyl-2-methylene-indoline as well as its salts,
1-methyl-3-spiro-cyclohexyl-5-hydroxy-2-methylene-indoline as well as its salts,
1-methyl-3-spirocyclohexyl-5-methoxy-2-methylene-indoline as well as its salts,
1-(2xe2x80x2-hydroxyethyl)-3,3-dimethyl-2-methylene-indoline as well as its salts,
1,3,3-trimethyl-2-methylene-3H-benz[e]indole as well as its salts and N-ethyl-2-methylene-benzthiazole as well as its salts; the 5-nitro-1,3,3-trimethyl-2-methylene-indoline, 5-methoxy-6-nitro-1,2,3,3-tetramethyl-3H-indolium-chloride, 5-N-acetylamino-1,2,3,3-tetramethyl-3H-indolium acetate, 1,3,3-trimethyl-2-methylene-indoline, 1,2,3,3-tetramethyl-3H-indolium chloride, 1,2,3,3-tetramethyl-3H-indolium bromide, 1,2,3,3-tetramethyl-3H-indolium iodide, 1,2,3,3-tetramethyl-3H-indolium sulfate, 1,2,3,3-tetramethyl-3H-indolium-hydrogen sulfate, 1,2,3,3-tetramethyl-3H-indolium methyl sulfate, 1,2,3,3-tetramethyl-3H-indolium hexafluorophosphate, 1,2,3,3-tetramethyl-3H-indolium hexafluoro antimonate, 1,2,3,3-tetramethyl-3H-indolium tetrafluoroborate, 1,2,3,3,5-pentamethyl-3H-indolium iodide, 1,2,3,3,7-pentamethyl-3H-indolium tetrafluoroborate, 1,2,3,3,6,7-hexamethyl-3H-indolium tetrafluoroborate, 1,2,3,3,5,7-hexamethyl-3H-indolium tetrafluoroborate, 1,2,3,3,4,7-hexamethyl-3H-indolium tetrafluoroborate, 5-chloro-1,2,3,3-tetramethyl-3H-indolium iodide, 5-fluoro-1,2,3,3-tetramethyl-3H-indolium iodide, 5-isopropyl-1,2,3,3-tetramethyl-3H-indolium iodide, 5-methoxy-1,2,3,3-tetramethyl-3H-indolium iodide, 5-hydroxy-1,2,3,3-tetramethyl-3H-indolium iodide, 1,2,3,3-tetramethyl-3H-benz[e]indolium chloride, 1,2,3,3-tetramethyl-3H-benz[e]indolium bromide, 1,2,3,3-tetramethyl-3H-benz[e]indolium iodide, 1,2,3,3-tetramethyl-3H-benz[e]indolium sulfate, 1,2,3,3-tetramethyl-3H-benz[e]indolium hexafluorophosphate, 1,2,3,3-tetramethyl-3H-benz[e]indolium methyl sulfate, 1,2,3,3-tetramethyl-3H-benz[e]indolium hexafluoro antimonate, 1,2,3,3-tetramethyl-3H-benz[e]indolium tetrafluoroborate, 1,2-dimethyl-benzthiazolium iodide, 5-methoxy-6-N-acetylamino-1,2,3,3-tetramethyl-3H-indolium acetate, 5-hydroxy-6-N-acetylamino-1,2,3,3-tetramethyl-3H-indolium acetate and N-ethyl-2-methylbenzthiazolium iodide being especially preferred.
The alcohol and the compound with a nucleophilic reaction center are used in each case in a total amount of about 0.05 to 25% by weight and preferably of 0.2 to 15% by weight, based on the ready-for-use agent.
As already mentioned above, alcohol dehydrogenases, alcohol oxidases, various flavin oxidases, laccases, peroxidases or similar enzymes can be used as oxidizing enzymes pursuant to the present invention. In a preferred embodiment of the invention, alcohol dehydrogenase as oxidizing enzyme, the oxidation being carried out in the presence of a suitable co-factor, such as the nicotinamide co-factor.
As xe2x80x9cnicotinamide co-factorxe2x80x9d NAD+, NADP+ and a variety of derivatives, which affect the enzymatic oxidation of alcohol to aldehyde advantageously, can be used, for example. Such co-factors and their derivatives are known to those skilled in the art. These co-factors can be used in an amount approximately equimolar to the amount of alcohol or, if desired, the co-factor can be recovered. A plurality of methods for recovering the co-factor has become known from the art and any of these known methods can be used in the present invention. Suitable methods for recovering the co-factor are described, for example, in G. L. Lemiere, J. A. Lepoivre, and F. C. Aldenweireldt, Tetrahedron Letters, 26, 4257 (1985); in xe2x80x9cEnzymes as Catalysts for Organic Synthesis,xe2x80x9d pp. 19-34, M. Schneider, Ed., Reidel Dordecht, 1986; Z. Shaked and G. M. Whitesides, J. Am. Chem. Soc. 102, 7104-5 (1980); J. B. Jones and T. Takamura, Can. J. Chem. 62, 77 (1984). In Preparative Biotransformations (S. M. Roberts, editor), Chapter 3, pages 3.1.1-3.1.6, John Wiley and Sons, Chichester, U.K. (1997), a recovery method is described, for which flavin mononeucleotides (FMN) are used, which transfer electrons to the oxygen, which functions as the ultimate oxidizing agent. For this method, 0.0005 to 0.05 moles of NAD+ or NADP+ are used per mole of diol, which is to be oxidized. This represents a recovery factor for a co-factor of about 20 to 2000.
In a further preferred embodiment of the present invention, flavin oxidase, which catalyses the oxidation of alcohol to aldehyde using molecular oxygen as oxidant, is used as oxidizing enzyme. The use of galactose oxidase as oxidizing enzyme is particularly preferred. Likewise, the use of vanillyl alcohol oxidase as oxidizing enzyme is particularly preferred. The use of derivative of the galactose oxidase or of the vanillyl alcohol oxidase is also particularly preferred. A xe2x80x9cderivativexe2x80x9d is understood here to be an enzyme variant, which is obtained by a mutagenesis of the original enzyme by means of known methods. Examples of such mutagenesis are selective evolution, DNA shuffling, molecular breeding, gene rearrangement and recombination, random mutation, point mutation, gene site saturation mutagenesis, etc. Such methods are also known from the art, for example, from U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,837,458, 5,965,408, 5,958,672 and 6,001,574. Gene encoding derivatives, if desired, can be planned and/or produced by inserting preferred codons.
Furthermore, compounds from the group of direct dyes, such as aromatic nitro dyes, azo dyes, anthraquinone dyes or triphenylmethane dyes, can be added alone or in admixture with one another to optimize the dyeing result and to produce special color effects.
Examples of nitro dyes are picramic acid, 4-(2xe2x80x2,3xe2x80x2-dihydroxypropyl)-amino-3-nitro-trifluoro-methylbenzene, 4,N-ethyl-N-(2xe2x80x2-hydroxyethyl)-amino-1-(2xe2x80x2-hydroxyethyl)-amino-2-nitrobenzene, 2-chloro-6-ethylamino-4-nitrophenol, 1-hydroxy-2-xcex2-hydroxy-ethylamino-4,6-dinitrobenzene, 4-(2xe2x80x2-hydroxyethyl)-amino-3-nitrochlorobenzene, 2-amino-6-chloro-4-nitrophenol and 4-(2xe2x80x2-hydroxyethyl)amino-3-nitro-methylbenzene.
Examples of azo dyes are 1-(2xe2x80x2-methoxyphenylazo)-2-hydroxy-7-trimethyl-ammonium-naphthalene (Basic Red 76), 4-(4xe2x80x2-sulfo-1-phenylazo)-1-(4xe2x80x3-sulfophenyl)-3-carboxy-5-hydroxypyrazolone (Acid Yellow 23), 4-amino-4xe2x80x2-bis[2xe2x80x3-hydroxyethyl]-amino-azobenzene (Disperse Black 9) and 1-(4xe2x80x2-aminophenylazo)-2-hydroxy-7-trimethyl-ammonium naphthalene (Basic Brown 16).
Examples of anthraquinone dyes are 1-methylamino-4-(2xe2x80x2-hydroxyethyl)amino-anthraquinone (Disperse Blue 3), 1-amino-4-hydroxy-anthraquinone (Disperse Red 15), 2-methoxy-1,4-diamino-anthraquinone (Disperse Red 11), 1,4-diamino-anthraquinone (Disperse Violet 1), 1-amino-4-methylamino-anthraquinone (Disperse Violet 4), 1,4-bis(2xe2x80x2,3xe2x80x2-dihydroxypropyl)amino-anthraquinone, 1-methylamino-4-(amino-n-propyltrimethyl-ammonium)-anthraquinone (Basic Blue 22), 1,4-bis-(2-hydroxyethyl)amino-5,8-dihydroxy-anthraquinone (Disperse Blue 7) and 1-methylamino-4-aminopropylamino-anthraquinone (HC Blue 8).
Examples of triphenylmethane dyes are [4-[[4-diethylamino]phenyl]-[4-(ethylamino)-1-naphthalinyl]methylene]-2,5-cyclohexadiene-1-ylidene]-N-ethyl-ethanamine (Basic Blue 7) and 4xe2x80x2,4xe2x80x2,4xe2x80x3-triamino-3-methyltriphenyl-carbenium chloride (Basic Violet 14, Fuchsin AN).
The amount of direct dyes added preferably is 0.01% to 5% by weight and especially 0.1% to 4% by weight.
The inventive agent represents a mixture of the components with the additives, which are customary for such preparation.
Conventional, cosmetic additives, are, for example, solvents such as water, low molecular weight, aliphatic, monohydric or multihydric alcohols, their esters and ethers, such as alkanols, especially with 1 to 4 carbon atoms in the alkyl chain, such as ethanol, n-propanol or i-propanol, butanol, i-butanol, dihydric or trihydric alcohols, especially those with 2 to 6 carbon atoms, such as ethylene glycol, propylene glycol, 1,3-dihydroxypropane, 1,4-dihydroxybutane 1,5-dihydroxypentane, 1,6-dihydroxyhexane, 1,2,6-trihydroxyhexane, glycerin, diethylene glycol, dipropylene glycol, polyalkylene glycols, such as triethylene glycol, polyethylene glycol, tripropylene glycol and polypropylene glycol, lower molecular weight alkyl ethers of multihydric-alcohols, such as ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monopropyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether or diethylene glycol monoethyl ether, triethylene glycol monomethyl ether, triethylene glycol monoethyl ether, ketones and ketoalcohols, especially those with 3 to 7 carbon atoms in the molecule, such as acetone, methyl ethyl ketone, diethyl ketone, methyl isobutyl ketone, methyl phenyl ketone, cyclopentanone, cyclohexanone and diacetone alcohol, ethers, such as dibutyl ether, tetrahydrofuran, dioxane or diisopropyl ether esters, such as ethyl formate, methyl formate, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, phenyl acetate, ethylene glycol monoethyl ether acetate or hydroxyethyl acetate, amides, such as dimethylformamide, dimethylacetamide or N-methyl-pyrrolidone; as well as urea, tetramethylurea and thiodiglycol, furthermore wetting agents or emulsifiers from the classes of anionic, cationic, amphoteric, nonionic or zwitterionic, surface active substances, such as fatty alcohol sulfates, alkylsulfonates, alkylbenzenesulfonates, alkyltrimethylammonium salts, alkylbetaines, xcex1-olefinsulfonates, ethoxylated fatty alcohols, ethoxylated nonylphenols, fatty acid alkanolamines, ethoxylated fatty acid ester, fatty alcohol polyglycol ether sulfates, alkyl polyglucosides, thickening agents, such as higher molecular weight fatty alcohols, starch, cellulose derivatives, Vaseline, paraffin oil, fatty acids and other fat components in emulsified form, water-soluble, polymeric thickening agents, such as natural gums, guar gum, xanthan gum, carob seed flour, pectin, dextran, agar-agar, amyloses, amylopectin, dextrins, clays or fully synthetic hydrocolloids, such as polyvinyl alcohol, moreover, cosmetics such as lanolin derivatives, cholesterol, pantothenic acid, water soluble cationic polymers, protein derivatives, pro-vitamins, vitamins, plant extracts, sugar and betaine, auxiliary materials, such as electrolytes, antioxidants, fatty amides, sequestering agents, film-forming agents, and preservatives.
The constituents mentioned are used in amounts customary for such purposes. For example, the wetting agents and emulsifiers are used in concentrations of about 0.5 to 30% by weight, the thickeners in an amount of 0.1 to 25% by weight and the cosmetics in a concentration of about 0.1 to 5.0% by weight.
Normally, the individual constituents of the inventive dyeing agents must be stored separately from one another and are mixed together immediately before use. The inventive agents can be produced in various ways. For example, the agents may be in the form of a 2-component kit, which consists of a component (A) containing the compound with a nucleophilic reaction center, the alcohol, optionally the nicotinamide co-factor and/or the buffer, and a component (B), containing the oxidizing enzyme as well as optionally the nicotinamide co-factor and/or the buffer. In a further, preferred, embodiment, the 2-component kit consists of a component (A), containing the compound with a nucleophilic reaction center, the alcohol and the oxidizing enzyme, as well as, optionally, the nicotinamide co-factor and/or the buffer, and a component (B), containing the alcohol, as well as, optionally, the nicotinamide co-factor and/or the buffer. Preferably, components (A) and/or (B) are anhydrous, that is, they contain not more than 1% by weight of water, and are mixed with water, which may contain additional, conventional cosmetic additives, only before use.
The constituents of the inventive dyeing agent, that is, the alcohol, the compound with a nucleophilic carbon and the oxidizing enzyme, as well as, optionally, the nicotinamide co-factor and/or the buffer, can also be packed together, provided that they are anhydrous, that is, provided that they do not contain more than 1% by weight of water, and are mixed with water, which may contain additional conventional cosmetic additives, only before use.
It is also possible to package the co-factor and the buffer separately and to mix them before use with the remaining components of the agent (3- or 4-component kit).
The dyeing of keratin fibers is usually carried out in an aqueous medium. Mixtures, which contain at least 60% by weight of water and more preferably at least about 70% by weight of water are regarded as xe2x80x9caqueous mediaxe2x80x9d.
The pH of the ready-for-use dyeing agent is about 2 to 12, preferably about 4 to 10 and particularly about 6 to 9.
The individual components are mixed together before use and the ready-for-use agent, so obtained, is then applied on the keratin fibers, which are to be dyed, water or an aqueous preparation, containing the usual cosmetic additives, optionally being added. The mixture is left on the fibers, for example, on hair, for about 10 to 45 minutes and preferably for about 30 to 40 minutes at about 10xc2x0 to 70xc2x0 C. and preferably at about 15xc2x0 to 50xc2x0 C., after which the fibers are rinsed with water and dried.
Dyeings with outstanding fastness properties, especially with respect to light fastness, wash fasteners and rubbing fastness, especially on hair, are made possible by the inventive dyeing agents and dyeing method.
The object of the invention is described in greater detail by the following examples, without being limited to these

Agent and method for dyeing keratin fibers


A device for a continuous indication of illicit substances in the exhalation air of face of the machine operator comprises an IR source and an IR detector being sensitive to wave length having an absorbance of the illicit substance, whereby the beam path of the IR source to the detector runs in front of the face of the operator.

Operators/drivers of vehicles, boats, aircraft, trains, traverses and other machines are in a position demanding tense watchfulness and high reaction ability so as not to cause accidents. The influence of different substances, such as alcohol may seriously deteriorate judgment, time of reaction and also other abilities giving rise to an increased risk for accidents.
So called alcohol interlocks were initially used in Canada and the US and are now also in use e. g. in Sweden. Initially the systems were only used in vehicles used by persons convicted in court to install them in order to keep their drivers' licenses. The monitoring system implied that they should, within short intervals, visit a service firm which scrutinized the information stored in the system concerning the defects and violations.
In Sweden this type of alcohol interlocks has also achieved great use for voluntary mounting in primarily commercial vehicles. The operation normally implies that the vehicle cannot be started if the concentration of alcohol of the exhalation air exceeds a predetermined value.
The interest in monitoring the soberness of drivers in various types of vehicles has considerably increased lately. Several manufacturers market alcohol interlocks for vehicles. All these products are based on active systems which require the driver to make a heavy blow through a mouth piece. Detection is made by means of a fuel cell or a semiconductor.
Although there has been a development aiming at preventing the manipulation of the alcohol test result, several essential drawbacks remain in existing systems. Thus none of the presently used systems has any function for checking if the driver is the person providing the sample. A certain check-up of his identity may however be performed by demanding from the driver after some driving a further exhalation air sample. The simplest systems do not even imply a feature checking that it is a human being who provides the exhalation air sample. Systems approved by the authorities do however demand, e.g. that the blowing person simultaneously must perform a "hum tone", the purpose thereof being to prevent that a sample delivered by an air pump or a balloon may be approved. Furthermore the exhalation air sample should derive from the deep lung air in order to give a fully correct result. That this is the fact is usually checked by requiring a certain minimum volume passing through the instrument before approval of the sample, which implies that persons with a decreased lung capacity may experience a difficulty in giving an approved sample. Thus, a driver may in chilly weather by taking a few deep breaths chill the mouth cavity and thereupon deliver a sample giving rise to a lower measuring result.
It can furthermore be noted that existing systems are not particularly user-friendly. It may e.g. at a low temperature require several minutes before the instrument by means of heating reaches a temperature enabling sampling. Also the detector of the instrument has to be initially heated to reach the temperature needed to give correct results and condensation in hoses and tubes may disturb the measuring and have to be avoided. Furthermore existing systems comprise a measuring unit formed by a separate hand-hold device which is difficult to locate in present-day vehicles without the unit itself or its connection to the electrical system of the vehicle being in the way. The cable connecting the measuring unit is usually the part of the system which is most frequently damaged. Furthermore many drivers experience the personal integrity to be affected. There exists a general disinclination for using an equipment requiring a fully visible blowing through a measuring instrument before a vehicle may be used.
Thus there is a need for a device for a non-active system, not requiring the blowing through a mouth-piece by the driver.
In the European Patent Application 1 688 741 there is described a chemical vapor sensor which, in addition to the conventional active (mouthpiece) measuring mode which in a conventional way is used for enabling the starting up of the vehicle, also provides for a passive measurement mode also using a measuring chamber used e.g. for detecting the ethanol vapor in a vehicle cabin. However, since this measurement is made without any reference value it requires an absorber and a heater in order to make an accumulated measurement over an extended period of time and it is even so only an estimate of the alcohol concentration in the cabin not necessarily related to the exhalation air of the driver is made.
It is therefore an object of the present invention to provide a device which eliminates the disadvantages of the active systems described above, i.e. essentially makes it impossible to manipulate and is furthermore extremely user-friendly. The characteristics of the invention will appear from the claims following the specification.
In FIG. 1 reference 1 denotes an IR source transmitting a light beam 6 through the air to be analyzed. The beam path 6 is arranged closed to the face of an operator (not shown), e.g. a vehicle driver, and reaches one or several optical analysers 2 located in front of an IR detector 3 measuring the intensity of the IR light. According to the example the optical filters 2 are adapted to wavelengths having a high absorption for ethanol and a reference wavelength not being affected by ethanol and the IR source and the optical filters are stationary mounted in front of the driver in the cabin of a vehicle, i.e. where possibly exhalated alcohol has its highest concentration. The signal is transmitted from a detector 3 for electronic signal processing to a processor 4 for a digital signal conversion and further to an interface 5 into the machine which the signal generated possibly shall affect. Reference 7 denotes the filtered and deflected IR light having specific wavelengths, reference 8 a filtered and adjusted analogue signal and reference 9 denotes computerized digital information. Reference 10 denotes a machine specific signal which in some way or other affects the machine handled by the operator. The effect might e.g. be that the vehicle will not be able to start if the concentration of ethanol in the IR light 6 exceeds a certain level.
In the device schematically shown in FIG. 1 where the infra-red beam passes through the air in front of the driver there is a full certainty that the sample is delivered by the operator. The inhalation and exhalation air of the driver is continuously analyzed and compared and e.g. an increase of ethanol in the exhalation air over the inhalation air will be immediately detected and can be measured with acceptable accuracy. Furthermore any attempt of manipulating the sample will be detected immediately by the instrument as the signal of the detector will deviate from the curve shape normally delivered and furthermore there will be carried out a measuring of normal breathing and not only the deep lung air. Also an abnormal breathing aiming at chilling the respiratory passages will be detected after a few breaths.
The device according to FIG. 1 will furthermore be extremely user-friendly since the sample is made at normal breathing and it is easy for anyone to give samples which can be analyzed. Furthermore the measurement is made in open air by using IR technique which means that the measuring can be started immediately when the operator takes the seat monitored. The system for measuring in open air has no movable parts. All parts are solidly mounted and integrated which protects the personal integrity as the operator can act as usual and no-one will note that his soberness is checked.
If convenient the device according to FIG. 1 may be modified in that the light-source 1 is located in some more suitable place and the IR light is transmitted to a suitable position via a system of one or several mirrors or light conductors.
The processor 4 suitably comprises software treating the measuring signals and compares the result with parameters stored. A result indicating presence of alcohol will be transmitted to the electronics of the driver and will activate a warning system and/or functions which prevent a driver affected by alcohol from using the vehicle. If the exhalation air contains alcohol the system may thereby demand the performance of an active exhalation sample before further driving of the vehicle is possible.
FIG. 2 schematically shows a sampling chamber which can be used for the performance of such an active exhalation air sample and thereby using the light source and detection equipment of the device according to the invention. This sampling chamber can also be used for the calibration and verification of the analysis system and for the analysis of other gases. In FIG. 2 reference 11 denotes a measuring chamber suitably introduced in the device according to FIG. 1 and where the gas to be analysed will be introduced via a tube 12 provided with a non-return valve 14. The air will then pass through and out from the chamber via an exit tube 13 likewise provided with a non-return valve 15. The spring loaded non-return valves make the final air stay in the chamber until the analysis is terminated. The chamber is further provided with lenses 16 to enable the IR beam according to FIG. 1 to penetrate the sample chambers towards the detector. In case of a reflected beam the chamber may alternatively be provided with a lens at one end and a reflector at the other end.
In the analysis the measuring chamber is located so as to make the IR beam penetrate into the chamber and either being reflected against the IR detector or being directed directly towards the detector. The concentration of ethanol in the expiration air in the measuring chamber will then be measured at high precision. The IR measuring should be made continuously during the blowing moment. This will only be possible if the chamber is located within the IR beam, i.e. it is located in front of the face of the driver aligned to the beam. The correct location can be ensured by having a tone signal indicating this location.
Thus the present invention provides for a system which completely eliminates the disadvantages of the active systems presently existing by providing a passive system having an accuracy and a sensitivity which it has hitherto not been able to achieve. The device according to the invention can not be manipulated and is extremely user friendly and furthermore protects the personal integrity as the operator can behave in a normal way without the environment being able to detect that the soberness is being checked.

Device for indicating illicit substances in the exhalation air of a machine operator


A glass pane including at least a glass sheet provided with heating strips powered by collectors, the heating strips being divided in groups. The power supply of the different groups is provided sequentially, each group being powered once per sequence. The supply is provided by a device equipped to measure the outside temperature and the temperature of the outer surface of the glass, to determine a minimum heating time for each group according to the outside temperature, and to maintain the heating in the group as long as the outer surface temperature of the glass opposite the group is not higher than the melting point of ice.

1. Field of the Invention
The present invention concerns an electrically heated glass pane, in particular for a motor vehicle.
2. Discussion of Background
The heated glass panes used in motor vehicles are generally composed of at least a glass sheet provided on one of its faces with a heating network consisting of strips of resistance connected in parallel. This network generally enables the deicing or demisting of the glass pane. The strips of resistance may have a constant cross section, but it is also known to have their width vary from one end to the other of the glass pane in order to have intensified heating areas and preferential fields of view. In all these glass panes, the same nominal power is applied to all the resistance strips for the duration necessary for deicing or demisting the glass pane.
The heating network of such glass panes may also include several heating areas operating sequentially and in cycles (see U.S. Pat. No. 3,982,092). The heating of each area is controlled by means of a differential thermostat functioning over a narrow temperature range, for example 10 to 30xc2x0 C. With this type of glass pane, we cannot, however, adapt the duration or power of heating to outside conditions such as temperature, the thickness of the ice or the amount of mist. What happens is that the first area is heated as soon as the outside temperature is less than the lower temperature (10xc2x0 C.) of the thermostat, and the heating of this area stops, to pass on to the next area, only when the temperature reaches the upper temperature (30xc2x0 C.) of the thermostat.
In the motor vehicle sector, the tendency is towards the multiplication of powered devices of all kinds (seat adjustment, door locks, electric windows, wing mirrors, etc.). For this purpose, we seek to reduce the energy consumption of electrically heated glass panes by reducing the power consumed or the duration of heating without affecting the efficiency of the deicing or demisting.
In order to achieve this one can, as it is disclosed in U.S. Pat. No. 5,496,986, provide the glass pane with an electronic device which permits to control the external temperature and to apply the minimal power necessary to heat the glass surface.
The purpose of the present invention is therefore to propose an economic heated glass pane, especially a heated glass pane that can be used advantageously as a rear window in a motor vehicle, said glass pane being used for demisting or efficient deicing with a reduced duration of heating and/or a gain in the power consumed.
This purpose is achieved by the glass pane according to the invention comprising at least a glass sheet provided with heating strips powered by collectors, the heating strips being divided in groups, the power supply to the different groups being provided sequentially, each group being powered once per sequence, the supply being provided by a device equipped with means enabling it to measure the outside temperature and the temperature of the outer surface of the glass, to determine a minimum heating time for each group according to said outside temperature, and to maintain the heating in said group as long as the outer surface temperature of the glass opposite said group is not higher than the melting point of ice.
The glass pane according to the invention is preferentially a motor vehicle rear window, but it can also be a windscreen or any other glass pane for the motor vehicle or a glass pane used in applications other than a motor vehicle. This glass pane may comprise one or more sheets of glass and possibly one or more plastic sheets. In most cases, it is a monolithic glass pane comprising a tempered sheet of glass or, possibly, it is a laminated glass pane comprising at least two sheets of glass generated by a plastic insert or an armored glass pane further comprising at least a sheet having the required armoring properties. The glass pane may also be convex. The heating strips are located on at least one face (and generally only on one face) of a sheet of glass the glass pane and/or, where appropriate, are located on or are embedded in a plastic insert of the glass pane.
The heating strips are generally electrically conductive transparent layers with a suitable resistance (generally in the region of 0.1 xcexa9 and capable of going up to 200 xcexa9), for example a layer containing a metal oxide such as tin oxide, or are metal conductor wires of suitable resistivity, for example thin tungsten wires, or are wires with an electrically conductive composition (generally enamel) also with a suitable resistivity (i.e. generally in the region of a few xcexcxcexa9. cm, able to go up to 50 xcexcxcexa9.cm in the present invention). The conductive compositions used are generally in the form of a suspension of metallic silver and glass frit in an organic binder, and are generally deposited by screen printing or any other equivalent technique before being dried and baked at high temperature (for example during glass bending or tempering treatment) on the glass pane. Such wires may also be reinforced subsequently, by an electrolytic process or by metal deposition not using electrical current to achieve the resistance value wanted. Preferably screen-printed wires are used for glass panes formed of a tempered sheet of glass and for laminated glass panes, and tungsten wires are used for laminated glass panes.
The heating strips may be wavy or straight and are preferably narrow strips, for example approximately 10 mm in the motor vehicle sector and up to 100 mm for glass panes used in building for the conductive layers, in the region of 0.2 to 0.8 mm for screen-printed wires and in the region of 25 to 50 xcexcm for tungsten wires. Also preferably, the strips are arranged with a gap between neighboring strips that may attain several centimeters. Advantageously, these strips are also arranged horizontally (generally along the larger dimension of the glass pane) in the position of use of the glass pane, especially for a rear window, and are isoresistant, i.e. they have constant resistivity over their whole length. For example, the strips are transparent layers or screen-printed wires uniform both in terms of conductive composition and thickness, or are metal wires of constant cross section.
The heating strips, at the two lateral edges of the glass pane, are connected by electrical connection elements to the current supply cables, these elements being called connectors or current supply terminals or collector strips or xe2x80x9cbusbarsxe2x80x9d. In the present invention, these elements are referred to hereafter more simply as xe2x80x9ccollectorsxe2x80x9d. These collectors may come, for example, in the form of metal strips or sections (for example in the form of tinned copper tabs) attached for example by welding onto the glass pane. Each separately powered group has its own collectors. These may be obtained by splitting the normal collecting strips into several parts separated by insulators or several collectors may be used, the number of collectors or separate parts in the collectors depending on the number of groups to be powered.
According to the definition of the invention, power is supplied sequentially, each group being powered by its own collectors and each group being powered only once per sequence. xe2x80x9cSequencexe2x80x9d here is understood to mean the operation consisting in heating each group of heating strips just once in a specified order with a view to achieving the deicing and/or demisting of the glass pane. The sequential power supply of the groups is controlled by a device equipped with means for establishing the power supply sequence, i.e. the order in which the different groups are powered one after the other, and for determining the duration of heating of each of these groups. For this purpose, an electronic control box or a control device may be used connected to the collectors comprising means of measuring the outside temperature and the surface temperature of the glass, the control box or device being programmed to associate a heating time or minimum power supply time with each group according to the temperature measured, and which maintains the heating for as long as the ice remains (i.e. as long as the temperature of the outer surface of the glass with respect to the heating group is less than the melting temperature of ice). In general, the melting temperature is equal to the melting point of ice measured under normal conditions (0xc2x0 C.) but it may vary considerably according to altitude and the purity of the water. The minimum heating time depends on several parameters such as the nature of the glass pane (dimensions, number of heating groups, number and nature of heating strips, etc.) and the nature of the deposit (ice, mist). In the case of deicing and for a given glass pane, the minimum heating time for a given outside temperature (in general negative) is determined experimentally by measuring the time during which it is necessary to heat each group to completely remove a layer of ice of given characteristics. Once programmed, the electronic box or control device associates a minimum heating time with the measured temperature for each group and maintains the heating for as long as the surface temperature of the glass is less than the reference value. To determine the various temperatures, any kind of suitable instrument may be used, for example a temperature sensor. The outside temperature sensor may be placed anywhere on the vehicle, so long as it is away from a source of heat or exposure to the sun""s rays, for example under the hood of a motor vehicle. The glass surface temperature sensor may be placed on the outer face or the inner face of the glass pane, it is generally placed close to the power supply collector, basically for reasons of ease of use and cost. In preference, each heating group is provided with a glass surface temperature sensor. Generally and in preference, the glass surface temperature sensor is placed on the inner face of the glass pane (in order to avoid any protrusion on the outer surface), which requires adjusting the reference value chosen or the value measured by the sensor by applying a suitable adjustment factor to the glass pane in question.
In an especially preferred way, the glass pane is equipped with an electronic control box or a collector control device, this box or device being connected to a sensor for measuring the outside temperature and with as many glass surface temperature measuring sensors as heating groups, each heating group only being connected to a single glass surface temperature sensor.
Deicing or demisting may be activated manually, the outside temperature sensor measuring the temperature at the moment when the deicing is activated. According to the temperature measured, the first heating group is powered for the duration corresponding to the time of minimum heating, or according to the measured temperature of the glass surface, then the next group is powered. When the last group has been heated, the power supply is switched off or, where necessary, a new sequence is initiated. In general, a single sequence is sufficient to reach a satisfactory level of deicing or demisting.
Deicing or demisting may also be automatic and free of any manual starting, the control box or device being for example programmed to maintain visibility in case of ice or mist, this also having an advantage in terms of convenience and saving current. The glass pane may include, for example in series, on its power supply circuit, a device conducting current when it is covered with ice or mist and switching the power supply current off when the ice or mist has disappeared, for example an electronic device including at least a power transistor. It may further include a special antimist device and other detector(s) sensitive to atmospheric conditions (mist, moisture detector, systems of electrodes, heat detector or sensor, etc.) placed for example on the inner face of the glass pane, this (these) detector(s) controlling a heating band power supply sequence. A threshold (3xc2x0 C. for example) may also be set, above which deicing may not be started for safety reasons.
In preference, with the strips being placed horizontally, heating takes place starting with the group located in the upper part of the glass pane in the position of use so that the water flow occurring as a result of this heating contributes to the deicing of the lower parts. This does not, however, exclude starting with another group, for example with a median group (where appropriate) or with a lower group. The groups are powered one after the other according to the sequence chosen. Advantageously, only one group is powered at a time, each group only being powered once per sequence. The powering of a group or of several groups successively may possibly be restarted in the event of ice or mist reappearing, at the end of the sequence.
The particular structure and implementation of the glass pane according to the invention enable rapid deicing helping to improve driving safety, and are especially advantageous when the available electrical power is limited. In general, at the end of 2 to 6 minutes, the first group of strips, especially when it involves wires, is completely deiced whereas on existing glass panes, it requires something in the region of 12 minutes to have a preferential area deiced. Advantageously, for the same dissipated power, 30% deicing or demisting time is gained with glass panes according to the invention by comparison with existing glass panes working in a nonsequential way, or if the same deicing or demisting time is used, the average dissipated power is 30% less than that of these same glass panes. It should also be noted that the dissipated power is more constant whereas in existing glass panes it can vary by about 10% during operation. Another advantage is that it is not necessary to provide resistances with intensified heating areas (especially in the horizontal direction), for example by varying the composition or thickness of the layers, the width or the thickness of the screen-printed wires, or the cross section of the metal wires in the lesser heating areas, the production of such glass panes being clearly more complicated. Advantageously, the glass pane according to the invention may be provided with conductors with a constant cross section and thickness (even if using conductors with a variable cross section and/or thickness and/or composition is not ruled out) and may therefore be produced easily and economically.
Each group comprises one or more strips, preferably comprises from two to 10 heating strips, and in an especially preferred way comprises three to 7 heating strips, the heating strips being connected in parallel within each group. Each group has its own collectors and each collector powers a single group. The glass pane presents several heating areas (groups) in a vertical direction separate from one another and several separate power supply areas (one separate power supply per group), power being supplied area by area.
When the glass pane according to the invention is intended for motor vehicle use, the number of heating groups is preferably 3. Beyond 3 groups, the gain in power or deicing or demisting time is not significant.
For motor vehicle applications in particular, the electrical system powering the heating strips may operate at 12 Volts (as normally used in the motor vehicle sector) but in preference it operates at a higher voltage, for example at 24, 30 or 42 Volts or more (nominal voltage of the battery used), the use of a higher voltage having the advantage of enabling less energy consumption, improving the efficiency of the electrical system conducting the current to the heating strips (reduced line losses for the same powerxe2x80x94in the region of 180 to 250 Watts for deicing or demisting a glass panexe2x80x94these line losses reducing the efficiency of the electrical system) and preventing heating effects in this system, this increase also enabling the space requirement of said system to be reduced (thinner power supply wires, smaller power transistors for power supply and cutouts, more economic equipment) and facilitating its production.
In the case of using this higher voltage and in order to have the necessary power for deicing or demisting the glass pane without excessive heating, the resistance of the heating strips may advantageously be increased, for example by lengthening the heating strips by deviations (even if it means reducing the number of wires per group where necessary) and/or by reducing the cross section (thickness and/or width) of the strips and/or where necessary by reducing the tin oxide content in the composition of the layers or the metallic silver content in the composition of the screen-printed wires.

Heated glass panes, in particular for vehicles


A high resolution objective lens assembly (20, 120, 220, 320) is implemented with refractive lens elements made of modified fused silica for operation at deep ultraviolet (DUV) wavelengths to reduce birefringence-caused image aberrations. The high resolution is achieved by an optical design that is characterized by a very high numerical aperture (NA) and short wavelength operation.

A microlithographic lens is a high resolution lens used in the manufacture of semiconductor devices. The microlithographic objective lens forms images carried by light propagating from a reticle illuminated by a laser and incident on a silicon wafer coated with a photoresist material. State of the art lithographic optical systems use a 193 nm ArF excimer laser to illuminate the reticle. A high resolution microlithographic objective lens ideally causes fractional magnification of the image carried by the light in the absence of significant image-distorting aberrations to form an image in fine detail. The formation of images in fine detail enables the manufacture of high density semiconductor devices. High resolution lens performance can be achieved by one or both of decreasing the wavelength of image-carrying light and increasing the numerical operature (NA) of the objective lens.
Calcium fluoride is a crystalline material typically used in the manufacture of lens components of 193 nm lithography tools. Intrinsic birefringence of calcium fluoride crystal material presents a serious threat to the optical performance of very high NA 157 nm lithography tools. What is needed, therefore, is a high resolution objective lens that is formed with optical components made from alternative refractive lens materials that degrade to a lesser degree the optical performance of the tool in which the lens is incorporated.


The invention relates to a process for the preparation of a purified version of a solvent-soluble dyestuff, comprising the steps of

This invention relates to organic dyestuffs and more particularly to improved dyestuffs suitable for use in printing and imaging technologies, especially those suitable for coloration of substrates such as paper, plastics, textiles, metals and glass by printing processes such as Ink jet.
Ink jet printing is a non-impact printing technique which involves ejecting, thermally or by action of an oscillating piezo crystal, droplets of ink continuously or on demand from a fine nozzle directly onto a substrate such as paper, plastics, textile, metal or glass. The ink may be aqueous, solvent or hot melt based and must provide sharp, non-feathered images which have good water fastness, light fastness and optical density, have fast fixation to the substrate and cause no clogging of the nozzle.
Commercially available Solvent dyes usually exhibit a sulfate content of about 500 ppm and a chloride content of about 1000 ppm which salt content leads to the above mentioned clogging problems.
Therefore, a special quality of solvent dyes is needed to make sure that it contains no insoluble residues that may cause clogging of the nozzle.
It has now been found that an unusually high quality, especially a very low salt content, of solvent-soluble dyestuffs is attained by a process as hereinafter described.
There is provided, according to the present invention, a process for the preparation of a purified version of a solvent-soluble dyestuff, comprising the steps of
(a) mixing an aqueous presscake of the dyestuff into a water-miscible liquid;
(b) distilling off the water until a water content of not more than 5% by weight is attained;
(c) filtering the dyestuff composition comprising solvent-soluble dyestuff and water-miscible liquid, thereby removing all insoluble contaminants and
(d) diluting the filtered dyestuff composition in de-ionized water, thereby performing the precipitation of the solvent-soluble dyestuff which is then isolated after filtration and washing with de-ionized water.
This process yields an unusually low salt content quality of solvent dyes which shows no residues when dissolved in organic solvents which are commonly used for printing inks.
For example, an improved Solvent Black 27 has a sulfate level below 50 ppm and a chloride content of 150 ppm (measured by ion chromatography).
In GB 1,062,256 a similar process is described for purifying azo pigments which process is, however, not comparable as it concernes compounds which are insoluble in all solvents, i.e. it solves a different problem from the one described above. More specifically, step c) of the actual process of the invention is the reverse of the disclosed process (in which the pigments are filtered, decantated or centrifuged) and step d) of the actual process of the invention is not comparable with the optional washing step of the already isolated pigments in the disclosed process.
In GB 1,444,108 a process for preparing chromium complexes of metallisable dyestuffs is described which could be interpreted to be similar to the process of the invention when these chromium complexes are solvent-soluble. The described process, however, relates to the preparation of the complexes, i.e. the reaction with chromium compounds, and the obtained solution is evaporated as a final step. In other words, the critical step d) of the process of the invention is missing.
Any solvent-soluble dyestuffs known in the art may be treated according to the process of the present invention, particularly dyestuffs of the 1:2-metal complex type, more particularly when the metal is chromium, cobalt, iron or aluminum. Examples of preferred dyestuffs are:
C.I. Solvent Black 27, C.I. Solvent Black 45, C.I. Solvent Black 29, C.I. Solvent Blue 44, C.I. Solvent Brown 28, C.I. Acid Violet 66, C.I. Solvent Orange 41, C.I. Solvent Orange 62, C.I. Solvent Red 8, C.I. Solvent Red 89, C.I. Solvent Red 91, C.I. Solvent Red 92, C.I. Solvent Red 122, C.I. Solvent Red 124, C.I. Solvent Red 127, C.I. Solvent Yellow 83, C.I. Solvent Yellow 83:1, C.I. Solvent Yellow 62, C.I. Solvent Yellow 79.
The water-miscible liquid which is used in step (a) may be any high boiling water-miscible organic solvent known to the art having boiling points higher than 110xc2x0 C., preferably higher than 115xc2x0 C., under atmospheric pressure. Blends of solvents may also be used. Typical classes of solvents are, for example, alkanols, ethers and ketones, preferably propylene glycols and tetrahydrofurfuryl alcohol. An especially preferred class are glycol ethers, from the point of view of both performance and environmentally innocuousness. Particularly preferred are the dialkylene glycol monoalkyl ethers, most particularly dipropylene glycol monomethyl ether.
The aqueous presscake which is used in step (a) may have a water content of 20 to 80% by weight, preferably of 40 to 60% by weight.
The process of the invention comprises, first of all, mixing the aqueous presscake into the water-miscible liquid. The amount of said liquid may be the 0.5 to 10 fold amount (by weight) relative to the aqueous presscake.
The mixing may be done by breaking up the presscake into suitably-sized fragments and stirring them into the solvent, with heating if necessary. When this has been done, the temperature of the mixture is raised to a temperature at which water may be readily distilled off. The optimum temperature will vary, depending on the solvent and the quantity of water, but is expediently from 110 to 180xc2x0 C., preferably from 130 to 160xc2x0 C. The distillation is continued until the water content of the mixture drops below 5%.
This process allows the colorant to be entirely dissolved in the solvent, whereas the insoluble contaminants remain undissolved and may even settle on the bottom of the reactor depending on their quantity. These insolubles are then removed by a filtration process where a filter aiding agent, e.g. filter earth, can be used if necessary. The clarified solution of the colorant is then poured into a large excess, e.g. a 5 to 20 fold excess (by weight), of de-ionized water under vigorous stirring to make sure that the colorant precipitates as a solid which can be easily recovered by filtration from the reaction mixture. The resulting cake is finally washed until solvent free with de-ionized water before being dried and ground.
An improved quality, especially a very low salt content (below 500 ppm), of solvent dyes is thereby obtained which shows no residues when dissolved in organic solvent which are commonly used for printing inks. A further advantage of the obtained solvent dyes is the absence of any solvent which could interfere in the preparation of ink compositions.
Solvent based ink compositions are used where fast drying times are required and particularly when printing onto hydrophobic substrates such as plastics, metal or glass. For this application, the solvent is preferably selected from ketones, alkanols, aliphatic hydrocarbons, esters, amides or mixtures thereof. Where an aliphatic hydrocarbon is used as the solvent, a polar solvent such as alcohol, ester, ether or amide is preferably added. Preferred solvents include ketones, especially methyl ethyl ketone and alkanols, especially ethanol and n-propanol.
Where the medium for an ink composition is a low melting point solid the melting point of the solid is preferably in the range from 60xc2x0 C. to 125xc2x0 C. Suitable low melting point solids include long chain fatty acids and alcohols, preferably those with C18-20 chains or sulphonamides. The dyes may be dissolved in the low melting point solid or may be finely dispersed in it.
Therefore, these dyestuffs are also suited for use in color filters and in toner resins. A preferred toner resin is a styrene or substituted styrene polymer or copolymer such as polystyrene or styrene-butadiene copolymer, especially a styrene-acrylic copolymer such as a styrene-butyl methacrylate copolymer. Other suitable toner resins include polyester, polyvinylacetate, polyalkenes, polyvinylchloride, polyurethanes, polyamides, silicones, epoxyresins and phenolic resins.
These improved dyestuffs are also suited for inks based on micro-emulsion systems such as described in EP 0 839 883 A2.
The invention is further illustrated by means of the following examples in which all parts are by weight.

Process for preparing purified dyestuffs

Abstract: (a) mixing an aqueous presscake of the dyestuff into a water-miscible liquid; and

Abstract: (b) distilling off the water until a water content of 5% by weight maximum is attained; and

Abstract: (d) diluting the dyestuff composition comprising solvent-soluble dyestuff and water-miscible organic solvent in de-ionized water, thereby performing the precipitation of the solvent-soluble dyestuff which is then isolated after filtration and washing with de-ionized water

Abstract: This ink jet grade version has the important advantage of being virtually salt-free and solvent-free.


A scuff mark removal tool used to remove tar, dirt, and rubber scuff marks from an uncarpeted floor surface. The tool has a handle portion and a felt-covered ball attached to the handle portion. The felt-covering on the ball effectively removes the scuff marks when rubbed against the marks without scratching the floor surface.

The invention relates to a scuff mark removal tool. In particular, the invention is a tool that aids a person in removing scuff marks from uncarpeted floor surfaces while remaining in an upright standing position.
People often try to keep a clean environment in their homes and workplaces. This is accomplished by routine cleaning of the area by the home owner or by a custodian of an office, store, or school. One important chore involved in the routine cleaning is removing scuff marks from uncarpeted areas of the floor. Scuff marks result when shoe soles, chairs, furniture, or similar items rub across the uncarpeted floor surface and leave residue, thereby causing black marks on the floor surface. Scuff marks are particularly prevalent on linoleum and wood floors. These black marks are often composed of dirt, tar, or rubber and are difficult to remove with ordinary cleaning products. Further, because of the stubborn nature of the marks, cleaning the floor with a mop normally does not rid the surface of the marks.
In order to rid the floor surface of these unsightly scuff marks, it is often necessary for a person to scrub the area on his or her hands and knees. Direct and concentrated contact with the marks is sometimes the only means of removing said marks. This can cause a great strain on a person""s body, particularly his or her knees and shoulders. Moreover, kneeling down on the floor typically causes the person to become dirty.
Thus, there exists a need for a tool that enables a person to thoroughly clean scuff marks from an uncarpeted floor surface without having to kneel down to the floor. Such a tool would be held by the user while the user is standing. The distal end of the tool is rubbed over the marked area, thereby removing any tar, dirt, or rubber marks therefrom.
While the units available may be suitable for the particular purpose employed, or for general use, they would not be as suitable for the purposes of the present invention as disclosed hereafter.
In view of the foregoing disadvantages inherent in the prior art, the present invention provides an improved scuff mark removal tool. As such, the general purpose of the present invention, which will be described subsequently in greater detail, is to provide a new and improved scuff mark removal tool which has all the advantages of the prior art and none of the disadvantages.
To attain this, the present invention essentially comprises a scuff mark removal tool used to remove tar, dirt, and rubber scuff marks from an uncarpeted floor surface. The tool has a handle portion and a felt-covered ball attached to the handle portion. The felt-covering on the ball effectively removes the scuff marks when rubbed against the marks without scratching the floor surface. The ball allows the tool to be effectively used while the handle is held at almost any angle with respect to the floor.
It is an object of the invention to produce a scuff mark removal tool that allows a user to effectively removes marks from an uncarpeted floor surface while remaining in a standing position. Accordingly, the user holds the proximal end of the tool and positions the distal end of the tool over the marked area of the floor. Downward pressure is then applied to the tool as the user maneuvers the tool distal end over the area until the mark is removed.
To the accomplishment of the above and related objects the invention may be embodied in the form illustrated in the accompanying drawings. Attention is called to the fact, however, that the drawings are illustrative only. Variations are contemplated as being part of the invention, limited only by the scope of the claims.


A method is described for the detection of a rare deletion mutant allele in a population of wild-type individuals. DNA samples from the individuals are subjected to a modified form of polymerase chain reaction (PCR) which favors the replication of truncated DNA strands over the synthesis of wild-type full length strands. The preferred way to bias the process toward the synthesis of truncated DNA strands is by limiting the extension step in the PCR reaction protocol. This process permits the convenient detection of a deletion mutant allele from a population of 1000 wild type individuals with the full length allele.

Not applicable.
To be determined.
One of the objectives of much genetic research is to identify the genes responsible for selected phenotypic traits in individuals. While much effort is being undertaken to develop genomic information from a large number of organisms, often the information about the function of a gene is more important than information as to the sequence of the gene itself. One way in which the function of individual genes is studied is to look for mutated versions of the gene of interest. Sometimes the search for mutated versions of a gene and the study of the mutated genes is referred to as xe2x80x9creverse genetics.xe2x80x9d If one finds a gene which is mutated so as to render the mutated gene inoperative, one can discern what phenotypic change has been made to the organism that renders it different from organisms not carrying the mutated version of the gene. Such studies can often lead to insightful information about the function of the gene which has the mutation.
Various strategies have been developed for using reverse genetics to study the functioning of genes in the model plant species Arabidopsis thaliana. For example, one laboratory at the University of Wisconsin has created a large population of Arabidopsis plant lines each of which has been transformed using the transferred-DNA (T-DNA) from the bacterium Agrobacterium tumefaciens, which has the native ability to transfer its T-DNA into the genome of a plant cell. Since the mechanism for DNA transfer operated by Agrobacterium results in a random insertion of the transferred DNA into the plant genome, the use of the Agrobacterium transformation technique results in large populations of plants each of which has a potentially disruptive insert at a different place within the plant genome. The problem then becomes to identify the particular gene which has been disrupted within each of the plants and to determine what the function of the gene might have been prior to disruption.
Another technique for introducing mutations into genes in an individual species under study, as such as a plant, is to introduce transposable elements into the genome of the organism. Various transposable elements are known in plants and many are known to be immobilized in Arabidopsis plants. A transposable element transports itself within the genome to insert itself randomly within the genome of the plant. Such random insertion mutations also could help identify the function of genes by disrupting the function of those genes and permitting the disruption to be observed at a phenotypic level.
Another method used to produce random mutations in the genome of various species, such as plants, is to expose the tissues of the plant to ionizing radiation. Ionizing radiation is known to produce deletion mutations in the genome of many plant species. The deletions created range in size from less to 1 kilobase to large chromosomal scale deletions that remove entire arms of chromosomes. Inducing such deletions in the genome of a plant is as simple as exposing a bag of seeds to the ionizing radiation. The radiated seeds can then be planted and each resulting plant carries a different deletion mutation. By collecting the seeds of the resultant plants one can easily generate a large population of plants that carry deletion mutations throughout the entire genome. No special transformation or transposon protocols are needed, and one is limited only by the amount of space available to grow the plants.
Whichever method of mutation creation is used, the problem still becomes to rapidly localize the rare organisms that carry a mutant version of the particular gene of interest. Thus what is needed is a method for rapidly identifying relatively rare deletion mutations within a population of organisms, such as a plant.
The present invention is summarized in that a technique based on a polymerase chain reaction (PCR) has been developed that permits the rapid and efficient characterization of deletion mutants in individual organisms.
The present invention is also summarized in a method that allows one to identify individual Arabidopsis thaliana plants which contain a single deletion mutation in their genome, the deletion resulting in a functional mutation of a gene of interest.
Other objects, advantages and features of the present invention will become apparent from the following specification.
Not applicable.
The method described here was developed for a situation in which one individual in a population of individuals of that species carries a deletion mutation in a particular gene, the function of which is sought to be studied. The sequence of the gene is known, and enough random deletion mutants have been made that the statistical likelihood is high that an individual exists in the population with a deletion mutation in the gene of interest, but the identity of that particular individual is not known. This method provides a very practical and efficient method that may be used to identify the individual with a deletion mutation in the desired locus.
The method is based on the polymerase chain reaction, or PCR, which is now a standard protocol of molecular biology and genetic laboratories. In a standard PCR reaction, the object is to amplify, or replicate exponentially copies of a particular sequence of a target DNA sequence possibly present in a sample. The PCR reaction in performed in a series of rounds, and in each round there are steps of denaturing, annealing and extending. In the denaturing step, the double stranded DNA is heated so as to denature the double stranded DNA so that it becomes single stranded. In the annealing step, the temperature is lowered so that conditions permit the annealing of specially made primers, which have sequences selected to bind to the target sequence, to the target DNA forming partially double stranded complexes. Then in the extension step, a DNA polymerase replicates the complement of the target strand by beginning at the double stranded complex of the primer and the target and extending the sequence of the primer using target sequence as a template. The product of each round of replication becomes a template for the next round. In the presence of excess primer, the target sequence is exponentially amplified. These three steps of denaturing, annealing, and extension are conducted repeatedly, each amplifying the complement of the target sequence, if it is present at all, until sufficient amplified DNA is made to be detectable by some detection procedure. In normal PCR, the object is to amplify the full length target sequence and thus the parameters of the process are normally arranged so as to maximize the yield of full length product made.
In the method of the present invention, the object is not to make full length target strands but to find, in essence, shorter sequences. The PCR process is adjusted by manipulating the three basic steps so that the shorter target sequences, representing the deletion mutations, are selectively amplified in preference over the full length sequences. This can be done most conveniently by limiting the time of the extension step in the process. Making this modification to standard PCR results in a process which selectively favors the amplification of shorter DNA species that still bind to the primers. In other words, this modified form of PCR reaction results in selective amplification of deletion mutant forms of native genes.
It is envisioned that this process will be used with pooled DNA from many individuals, for reasons of practical convenience. To screen large populations of individuals for presence of a desired deletion mutation, DNA from the individuals is obtained and processed in large pools, each pool containing the DNA from thousands of individuals. Then the pools can be tested for the presence of the deletion mutation in that pool. For any pool in which a deletion mutation is found, the pool can be divided into sub-pools which can then be retested. This process can be repeated until the individual organism with the deletion mutant allele is identified. Thus, for reasons of practicality, it is desired that the process can detect one mutant shortened allele in a pool of 1000 copies of the wild-type full length alleles.
Tables 1 and 2 illustrate a comparison of a standard PCR reaction with the modified reaction for deletion mutant amplification. Both reaction use primers selected to bind to the target sequence and use the steps of denaturing, annealing and extension repeated cyclically. In the standard PCR reaction of Table 1 the denaturing step is conducted at 94xc2x0 C. for a time of 10 seconds. Then the temperature of the reaction mixture is lowered to 65xc2x0 C. for the annealing step which is conducted for 30 seconds. Lastly, the extension step is conducted at 72xc2x0 C. for a time period of two minutes, normally the longest step in the reaction by far. The temperatures of the three steps are normally selected with the optimum for each object in mind. The denaturing step and the annealing step requires the solution to be adjusted to temperatures consisted with only single or double stranded DNA species being present in the mix. The temperature of the extension step is chosen for the optimum performance of the DNA polymerase. The time periods of the reaction steps are chosen so that the reaction taking place in each step can be completed. The long time period of the extension step is necessary to ensure that full length replications of the entire target sequence can be constructed by the DNA polymerase.
In the amplification process for deletion mutant genes, the process is altered to favor the production of the shortened amplified species. This is illustrated in Table 2. The denaturing time is left at 10 seconds at 94xc2x0 C. The time for the annealing step is compressed to 1 second at 65xc2x0 C. Most critically, the time for the extension step is reduced to 10 seconds, still at 72xc2x0 C. In this way, there is sufficient time period for shorter target strands to be replicated by the DNA polymerase, but there will be insufficient time period for a population of full length target strand to be made.
In a modem molecular biology laboratory, PCR reactions are performed in devices known as thermocyclers. These instruments are programmed by the user to take a reaction mixture and subject that mixture to a series of different temperatures for preselected times so that defined reactions occur. All of the PCR reactions described here, both the conventional reactions and the deletion mutation enrichment reactions, can conveniently be performed in such thermocyclers programmed to provide the temperatures and times as suggested in this document. Also, unless stated otherwise, standard commercially available reagents for PCR reactions, including DNA polymerases and buffer, can be used in these reactions, and such reagents are commercially available from a number of manufacturers.
The altered PCR deletion enrichment procedure of Table 2, if applied to a mixture of DNA strands, some of which might contain a deletion mutation. The deletion mutant can be rare, perhaps 1 copy of a deletion mutation in amongst 1000 copies of a wild-type full length gene. Yet this process will selectively amplify or multiply only the deletion mutation version of the gene, not the native full length version. Thus by conducting pooled experiments, it becomes convenient to determine if a desired mutation has occurred in any of the individuals represented in large pools of DNA from many individuals. Once the existence of the deletion mutation in a pool is demonstrated, subsequent division of the pool and re-testing can lead to successful identification of the individual carrying the deletion mutation.
The time period for the extension step may need to be varied for different length deletions, and for different target DNA sequences, but the principle here is clear. It is possible to perform a PCR reaction process and manipulate the conditions to favor the multiplication of copies of gene which have deletion mutations in them in preference to full length genes without the deletion mutations. The extent to which the amplification of the deletion mutants is favored over the full length copies may vary somewhat in its magnitude depending on the parameters of the PCR process. At least the amplification of the deletion copy will be greater than the amplification of the non-mutant versions but often, as in the examples below, only the mutant version of the gene will amplified by the process.
Sometimes the amplification of the deletion mutant will not be sufficient in one PCR process to result in enough amplified DNA to be visible by a technique such as agarose gel electrophoresis. In such cases, it is possible and recommended to conduct a second round of PCR using primers nested inside the primers for the first reaction, but primers which will still bind to a mutant version of the target gene. It has been found, as again shown in the examples below, that such a second round of PCR is useful to increase the number of copies of the mutant version of the target DNA sequence in order to make possible a visual indication that the mutant gene has been found.
There are also other ways to vary the PCR process to selectively favor the creation of copies of truncated genes in preference to full length genes. Temperature is another parameter which can be altered. If the time of the elongation step were not changed, but the temperature were reduced, the speed of the reaction could be slowed in a manner that is the equivalent of shortening the reaction time for the elongation reaction. The lowering of the temperature might allow for a broader range of times to be used in the extension reaction while still favoring the complete synthesis of deletion mutant over the longer wild type versions.
Another approach is to use dideoxynucleotides in the extension reactions. The chemical nature of dideoxynucleotides is such that they are recognized by DNA polymerases and are incorporated into growing DNA strands. However, once a dideoxynucleotide is incorporated into a growing DNA strand, the strand can grow no further since a new nucleotide cannot be added to the dideoxynucleotide. One could conduct the extension reaction with a mixture of normal nucleotides and dideoxynucleotides which would limit the length to which a daughter strand could grow, at least as a statistical matter. The more dilute the dideoxynucleotides were, the longer the strands could grow before they were terminated by the incorporation of dideoxynucleotide. By manipulating the ratio of normal to dideoxynucleotides, one could vary the length to which the growing DNA strands grow and thus favor the truncated strands over the longer ones. A DNA strand which is shorter than the average length of the terminated DNA strands would be favored using this strategy.
Yet another way to favor the synthesis of deletion mutations over full length genes would be to use a less efficient DNA polymerase. A DNA polymerase that was slower, had lower processivity, or which had less affinity for the template strand, would also favor the synthesis of shorter strands over longer ones. It might be possible to identify a DNA polymerase or a combination of polymerases or additives which results in a controllable bias toward shorter strands over longer ones.
But the preferred method of biasing the reaction toward shorter strands is to limit the time of the extension portion of the reaction. The preferred reaction is to use a temperature of about 72xc2x0 C. and a time period of about 10 seconds. For the HY-4 example given below, good results were obtained using reaction times of between 7 and 14 seconds. If an extension time of longer than 14 seconds is used, the danger becomes that the deletion template may lose its competitive advantage over the wild-type templates, and the deletion product might not be detectable. Again, however, this parameter may need to be varied somewhat depending on the difference in length between the truncated template and the full-length template. The larger the gene of interest, the longer the extension time that should be used. It is possible to test empirically how long the time can be extended and still not result in large amounts of the wild-type DNA being replicated in the reaction.
All of these strategies have one common feature. They favor the synthesis in the extension step of a PCR reaction, of shorter daughter DNA strands over the synthesis of longer strands. Such favoring does not have to be absolute, it just must be sufficient so that detectable quantities of a rare deletion mutant can be favored so that its DNA becomes detectable in some fashion.

Method for rapidly identifying deletion mutations


A system highlights search terms in documents distributed over a network. The system generates a search query that includes a search term and, in response to the search query, receives a list of one or more references to documents in the network. The system receives selection of one of the references and retrieves a document that corresponds to the selected reference. The system then highlights the search term in the retrieved document.

A. Field of the Invention
The present invention relates generally to information retrieval systems and, more particularly, to systems and methods for highlighting query terms in search results.
B. Description of Related Art
Conventional techniques for searching a hypertext medium, such as the World Wide Web ("web"), are replete with deficiencies. To perform a search on the web, a user typically uses a web browser, such as the Netscape Navigator and Microsoft Internet Explorer browsers. The user enters one or more search terms into the browser. In response, the browser generates a query request to a search engine. The search engine typically returns a list of result links to the browser, which, in turn, displays the list to the user.
To view a document associated with one of the links, the user typically selects the link by, for example, clicking on it using a mouse. Sometimes, the user desires to determine where the search terms are located in the document to determine the document's relevance. Conventional information retrieval systems require that the user perform a word search on the document to locate each occurrence of the search terms.
As a result, there exists a need for mechanisms for highlighting search terms in a retrieved document to aid a user in determining the document's relevance.

Highlighting occurrences of search terms