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The invention relates to a method for producing polyvinyl alcohol shaped bodies involving thermoplastic processing of at least one polymer (A), of at least one softener and, optionally, of water and/or other additives, whereby polymer (A) contains: a.) 15.0 to 99.9 wt. % structural units of formula (1); b.) 0.0 to 50.0 wt. % structural units of formula (2), and; c.) 0.0 to 50.0 wt. % structural units of formula (3), each with regard to the total weight of polymer (A), whereby radicals R1, R2, R3, R4, R5, and R6 are defined according to the description. The method is characterized in that polymer (A) and the softener are placed into the extruder without being mixed beforehand and that the quantity of water is less than 2 wt. % with regard to polymer (A). The invention also relates to the polyvinyl alcohol shaped bodies, which can be obtained by using the method, and to their use. |
1-8. (canceled) 9. A process for producing plastics moldings via thermoplastic processing of at least one polymer (a), of at least one plasticizer, and optionally of water, wherein polymer (a) comprises: a.) from 15.0 to 99.9% by weight of structural units of the formula (1) wherein R1 is hydrogen or methyl; b.) from 0.0 to 50.0% by weight of structural units of the formula (2): wherein R2 is an alkyl radical having from 1 to 6 carbon atoms, c.) from 0.0 to 50.0% by weight of structural units of the formula (3) wherein each of R3, R4, R5 and R6, independently of the others, is a radical having a molar mass in the range from 1 to 500 g/mol, based in each case on the total weight of the polymer (A), characterized in that the polymer (A) and the plasticizer are introduced with no prior mixing into the extruder, and that the proportion of water is less than 2% by weight, based on the polyvinyl alcohol. 10. The process of claim 9 wherein R3, R4, R5 and R6 are independently aliphatic or cycloaliphatic and which are optionally substituted with one or more carboxylic acid, carboxylic anhydride, carboxylic ester, carboxamide and/or sulfonic acid groups. 11. The process of claim 9, wherein Polymer A has a degree of hydrolysis in the range from 70 to 100 mol % and a viscosity, measured on a 4% by weight aqueous solution, in the range from 2 to 70 mPas. 12. The process of claim 9, wherein the plasticizer comprises polyhydric alcohols, derivatives of polyhydric alochols, polyethylene glycols, glycerol, diols, or triols, or mixtures thereof. 13. The process of claim 9, wherein the polymer(s) and the plasticizer(s) and, when present, the water is processed with a lubricant, an antiblocking agent, an anti-oxidant, a pigment, a dye, a solid plasticizer, and/or a filler. 14. The process of claim 9, wherein the thermoplastic processing takes place by means of melt extrusion. 15. The process of claim 9, wherein polymer (A) has been internally plasticized. 16. A polyvinyl alcohol molding prepared by the process of claim 9. 17. A polyvinyl alcohol molding prepared by the process of claim 10 18. A polyvinyl alcohol molding prepared by the process of claim 11. 19. A polyvinyl alcohol molding prepared by the process of claim 12. 20. A polyvinyl alcohol molding prepared by the process of claim 13. 21. A polyvinyl alcohol molding prepared by the process of claim 14. 22. A polyvinyl alcohol molding prepared by the process of claim 15. 23. A packing material comprising the molding of claim 16. 24. A packing material comprising the molding of claim 17. 25. A packing material comprising the molding of claim 18. 26. A packing material comprising the molding of claim 19. 27. A packing material comprising the molding of claim 20. 28. A packing material comprising the molding of claim 21. 29. A packing material comprising the molding of claim 22. |
Materials sustainedly releasing drug in vivo |
Disclosed is a material for sustained chemical-release in vivo, which comprises a hydroxyapatite/collagen (HAp/Col) nanocomposite formed as a coprecipitate, and a chemical consisting at least one of a drug and a bioactive substance including a growth factor and a DNA enzyme. The hydroxyapatite (HAp) has a specific surface area in the range of 50 to 300 m2/g. The hydroxyapatite (HAp) and the collagen (Col) are combined in such a manner that the crystals of the hydroxyapatite (HAp) are oriented in the C-axis direction around the fibers of the collagen (Col). The chemical is carried on the surface of the hydroxyapatite (HAp), the surface of the collagen (Col) and in a hydrated water of the hydroxyapatite (HAp) and the collagen (Col). The sustained chemical-release material can be prepared in the form of a bioabsorbable capsule, or in the form of allowing in vivo administration by injection and in-vivo gelation after said administration. |
1. A material for sustained chemical-release in vivo, comprising: a hydroxyapatite/collagen (HAp/Col) nanocomposite formed as a coprecipitate; and a chemical consisting at least one of a drug and a bioactive substance including a growth factor and a DNA enzyme, wherein said hydroxyapatite (HAp) has a specific surface area in the range of 50 to 300 m2/g, said hydroxyapatite (HAp) and said collagen (Col) are combined in such a manner that the nanocrystals of said hydroxyapatite (HAp) are oriented in the C-axis direction around the fibers of said collagen (Col), and said chemical is adsorbed and carried on the surface of said hydroxyapatite (HAp), the surface of said collagen (Col) and in a hydrated water of said hydroxyapatite (HAp) and said collagen (Col). 2. The material as defined in claim 1, which further includes glycosaminoglycan or hydrogel which has high hydration ability, wherein said glycosaminoglycan or hydrogel is combined with said HAp/Col nanocomposite so that said HAp/Col nanocomposite serves as a bioabsorbable capsule capable of enclosing said growth factor therein. 3. The material as defined in claim 1, which is prepared in the form of allowing in-vivo administration by injection and in-vivo gelation after said administration. |
<SOH> BACKGROUND ART <EOH>In conventional sustained drug-release systems, the following drug carriers have been typically used. (1) A material having a high water-storage capacity, such as collagen, is used to carry a drug thereon. When the material with the drug is introduced in vivo, the drug is diffusedly released. (2) A biodegradable biomaterial, such as polylactic acid, is used, and mixed with a drug in the production process thereof. The drug is released in conjunction with in-vivo degradation of the biomaterial. In relation to the above (1), there has been known a collagen-based product designed for administration by injection and long-term sustained release (Japanese Patent Publication Nos. 05-12328, 05-71566 and 06-94418, and Japanese Patent Laid-Open Publication No. 08-34747). There has also been known a collagen/calcium phosphate composite material for use as a film for periodontal guided tissue regeneration (GTR), a hemostatic agent, a bone filler material, a cartilage filler material and a substrate for 3-dimensional culture of hard tissue cells (Japanese Patent Laid-Open Publication No. 06-304242). This composite material is obtained by causing a reaction between calcium phosphate compound and collagen in a calcium phosphate solution to form a complex thereof, and drying the complex. Further, a method of preparing a composite material with a high degree of orientation and for use as artificial bones etc. has been known (Japanese Patent Laid-Open Publication No. 11-199209). The method comprises coprecipitating calcium phosphate and collagen, and then pressing the obtained coprecipitate to form the composite material. On an experimental basis, collagen has been used as a carrier for various cytokines, growth factors and others in view of its high water-storage capacity or capability of being introduced in vivo while carrying a volume of drug solution. However, a growth-factor carrier using collagen or a hydrous gel substance, such as gelatin, releases a drug therefrom at a high rate after introduction, and thereby can sustain the effect of the drug only in a relatively short period of time. Thus, the carrier consisting of such a martial without modification involves a requirement of using a large volume of growth factor. Hydroxyapatite is widely used as a biomaterial having high bone-inducing ability, and a number of attempts are being made to use the bone filler material as a carrier for growth factors. It is also known that hydroxyapatite can be attached onto the crystal surface of protein or DNA because of its crystal structure having a significantly strong polarity and a high electrical affinity to protein and DNA. In the field of biochemistry, a nanocrystalline hydroxyapatite has been used as a material for chromatography. Nanocrystal as used in chromatography is in the form of power practically unavailable for biomaterials, and thus a processed powder material, such as a sintered material, is used as biomaterials. However, such a material inevitably has a significantly reduced surface area for adsorbing a drug, and practically contributes little to a carrier for drugs. |
Pharmaceutical compositions, and a method of preparing and isolating said pharmaceutical compositions, and use of said pharmaceutical compositions for prophylactic treatment of lesions and carcinomas |
This invention provides isolated nucleotide sequences disclosed in the specification, amino acid sequences deduced from the nucleotide sequences, constructs and/or vectors comprising and or expressing the nucleotide sequences, and there use in transforming plant cells to produce proteins or fragments of proteins. These proteins or fragments can be used in the manufacture of pharmaceutical components that can be used in a prophylactic treatment against causative agents of lesions and carcinomas, in particular cervical lesions and carcinomas. |
1. An isolated nucleotide sequence set out in any one of FIGS. 4 to 7, or part thereof. 2. A nucleotide sequence which is: (i) a homologue, variant, and/or derivative of the nucleotide sequence of claim 1; (ii) a complement of the nucleotide sequence of claim 1; or (iii) a complement of the homologue, variant or derivative of (i). 3. A gene construct comprising a nucleotide sequence according to claim 1, and a promoter. 4. A vector comprising and/or expressing: (a) an isolated nucleotide sequence set out in any one of FIGS. 4 to 7, or part thereof; (b) a homologue, variant or derivative sequence set out in any one of FIGS. 4 to 7, or part thereof; (c) a complement of the isolated nucleotide sequence set out in any one of FIGS. 4 to 7, or part thereof; (d) a complement of the homologue, variant or derivative of the isolated nucleotide sequence set out in any one of FIGS. 4 to 7, or part thereof; or (e) a nucleotide sequence that is capable of hybridizing to: (1) the isolated nucleotide sequences, or parts thereof; or (ii) the homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or (iii) the complements of the isolated nucleotide sequences, or parts thereof; or (iv) the complements of the homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or (v) any combination of (e)(i)-(e)(iv); or (f) any combination of (a)-(e), and a promoter, wherein the promoter is operably linked to the sequence of any one of (a)-(f) or any combination of (a)-(f). 5. A method of producing a papillomavirus protein, or parts thereof, comprising the steps of: 1) contacting a plant cell with a gene construct of claim 3; 2) cultivating the plant cell under conditions suitable for the production of a protein or fragment of a protein of: (a) the isolated nucleotide sequences of any one of FIGS. 4 to 7, or parts thereof; or (b) homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or (c) complements of the isolated nucleotide sequences, or parts thereof; or (d) complements of the homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or (e) a nucleotide sequence that is capable of hybridizing to: i) the isolated nucleotide sequences, or parts thereof; or ii) the homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or iii) the complements of the isolated nucleotide sequences, or parts thereof; or iv) the complements of the homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or v) any combination of (e)(i)-(e)(iv); (f) any combination of (a)-(e). 6. A method according to claim 5, wherein the plant cell is a Nicotiana benthamiana plant cell. 7. A method according to claim 5, wherein the conditions in step 2) permit the spontaneous assembly of the proteins or fragments of the proteins into immunogenic-response eliciting virus-like particles. 8. The use of a papillomavirus protein, or fragment of a papillomavirus protein, which comprises: a) the amino acid sequence of the nucleotide sequence set out in any one of FIGS. 4 to 7; or b) the amino acid sequence of a nucleotide sequence having at least 80% homology, after optimal alignment, with the nucleotide sequence set out in any one of FIGS. 4 to 7; or c) an amino acid sequence of a fragment of a papillomavirus protein as defined in a), in the manufacture of a pharmaceutical composition for use in a prophylactic treatment against causative agents of lesions and carcinomas. 9. The use of claim 8, wherein the pharmaceutical composition is used in a prophylactic treatment of cervical lesions and carcinomas. 10. A method of producing a papillomavirus protein, or parts thereof, comprising the steps of: 1) contacting a plant cell with a vector of claim 4; 2) cultivating the plant cell under conditions suitable for the production of a protein or fragment of a protein of: (a) the isolated nucleotide sequences of any one of FIGS. 4 to 7, or parts thereof; or (b) homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or (c) complements of the isolated nucleotide sequences, or parts thereof; or (d) complements of the homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or (e) a nucleotide sequence that is capable of hybridizing to: i) the isolated nucleotide sequences, or parts thereof; or ii) the homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or iii) the complements of the isolated nucleotide sequences, or parts thereof; or iv) the complements of the homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or v) any combination of (e)(i)-(e)(iv); (f) any combination of (a)-(e). |
<SOH> BACKGROUND OF THE INVENTION <EOH>The invention relates to the use of papillomavirus proteins for preparing pharmaceutical compositions intended to generate an immunogenic response against papillomaviruses. The invention further comprises the use of the compositions for prophylactic treatment of lesions and carcinomas associated with papillomavirus infection. The invention further comprises diagnostic kits for use in the diagnosis of papillomavirus infection. In this specification the following terms, phrases and/or clauses are to be understood to mean: Construct—as used herein is synonymous with terms such as “conjugate”, “cassette”, and “hybrid” and includes the nucleotide sequence according to the invention, or parts thereof, which may be directly or indirectly linked to a promoter. The construct may contain or express a marker, which allows for the selection for the construct in a host cell. “Expression vector”—as used herein means a construct capable of in vivo or in vitro expression. “Expression” is understood to mean the production of a protein from a DNA template via transcription and translation. “Fragment of the papillomavirus protein”—as used herein is intended to denote, in particular, any fragment of an amino acid sequence included in the amino acid of a papillomavirus protein which is capable of generating an immunogenic response directed against a papillomavirus, said fragment comprising at least 5 amino acids, preferably at least 10 amino acids and, more preferably at least 15 amino acids. “Homologue”—as used herein means an entity having a certain homology with amino acid sequences or nucleotide sequences. “Hybridisation”—as used herein encompasses the use of nucleotide sequences that are capable of hybridising to a nucleotide sequence of the present invention, or any fragment, variant, derivative, or homologue thereof. “In silico”—parts of assays and/or processes described herein may be performed by use of suitable computational software. Such assays and/or processes so performed are to be understood to be encompassed herein. “Nucleotide sequence”—as used herein is synonymous with the term “nucleotide acid sequence” and/or the term “polynucleic acid” and/or the term “polynucleotide” and includes genomic DNA, cDNA, recombinant DNA, synthetic DNA, and RNA, and any combinations of the aforementioned, also the nucleotide sequence may be double-stranded or single-stranded whether representing the sense or the antisense strand. Preferably, the term “nucleotide sequence” means DNA. “Operably linked”—as used herein refers to a juxtaposition wherein the promoter which is “operably linked” to a coding sequence is ligated in such manner that expression of the coding sequence is achieved under conditions compatible with the control sequences (i.e. inter alia promoters, enhancers and other expression regulation signals). “Promoter”—as used herein is a nucleotide sequence that directs the transcription of a nucleotide sequence. “Protein”—as used herein is intended to denote both peptides and polypeptides. “TMV”—as used herein indicates Tobacco Mosaic Virus. “Vector”—as used herein includes expression vectors, replicable vectors, transformation vectors, shuttle vectors, cosmids, plasmids, phages, viruses and yeast artificial chromosomes or any combination thereof. “VLP”—as used herein indicates virus-like particles. Papillomaviruses (PVs) belong to the taxonomic family Papillomaviridae, which are small, non-enveloped, double-stranded (ds) DNA viruses. These viruses infect a wide range of higher vertebrates and are highly species-specific. Upon infection, human papillomaviruses (HPVs) have a particular tropism for undifferentiated squamous epithelial cells and apart from being associated with plantar warts, flat warts, and genital warts (condyloma acuminata), HPV infections are also linked to a disease called epidermodysplasia verruciformis, a rare lifelong disease characterized by disseminating papillomas. There has, furthermore, been an epidemiological and biochemical link of certain papillomavirus isolates with urogenital cancers, including cervical, vulvar, penile cancer, and malignant transformation of epidermodysplasia verruciformis lesions. Of the characterized HPV types, about 27 have been identified as the causal agents of anogenital infections. HPV types 6, 11, 16, 18, 31, 33, 35 and 42 have been identified as being the most prevalent, whereas types 16, 18, 31, 33, 51 and 54 have been associated with anogenital carcinomas, believed to be of high risk, more particularly HPV-16 has been found in 95% of cervical cancers. Until recently it was believed that high-risk genital HPVs were only transmitted during sexual intercourse, however studies indicate that infants acquire high-risk HPV infections from their mothers at birth. In addition to non-sexual parent-to-child transmission, investigators detected HPV DNA in vaginal swabs from women, in cervical-vaginal specimens from young girls, and in vulval swabs from 9 out of 61 women who claimed no history of sexual contact. Initial HPV infection causes cervical intraepithelial neoplasia (CIN), more commonly known as pre-cancerous lesions. It has been found that in some women there is a spontaneous regression of CIN, but this is not always the case and lesions can persist for years, increasing the likelihood of progression towards cervical cancer. Treatment methods ranging from cryotherapy, surgical excision and topical treatments do not often alleviate problems and for patients who do respond to the treatment, recurrence is often a problem. Ultimate destruction of all infected tissue does not appear to be feasible, since multifocal disease and latency are common features of the infections. Antiviral drugs designed to target and arrest viral replication seem to be ineffective in the case of HPV infections, firstly because the virus does not replicate in the cells that maintain the infection and secondly due to the fact that viral replication genes seem to get lost through integration, rearrangement and deletions. Traditionally most prophylactic vaccines have consisted of live, attenuated virus or formalin-inactivated virus. Due to the difficulties and risks involved in generating large quantities of these traditional vaccines there has been great emphasis on developing a viral protein subunit vaccine. |
<SOH> SUMMARY OF THE INVENTION <EOH>In accordance with the invention there are provided isolated nucleotide sequences set out in any one of FIGS. 4 to 7 (SEQ ID NO 1-4), or parts thereof, more particularly an isolated recombinant nucleotide sequence containing a papillomavirus coding sequence, still more preferably the isolated recombinant nucleotide sequence expresses a papillomavirus capsid protein, even more preferably the isolated nucleotide sequence is a plant/viral chimeric nucleotide sequence, most preferably the plant/viral chimeric nucleotide sequence is a human papillomavirus/plant chimeric sequence encoding an isolated nucleotide sequence selected from the group consisting of: (i) papillomavirus L1 capsid protein; (ii) a truncated papillomapapillomavirus L1 protein coding sequence which has been altered to include an in-frame C-terminal fusion peptide, preferably of a length determined not to interfere with self-assembly of the papillomapapillomavirus L1 protein into pentamers or larger aggregates; most preferably wherein the in-frame C-terminal fusion peptide sequence is derived from the L2 capsid protein, still more preferably wherein the in-frame C-terminal fusion peptide sequence is derived from the group consisting of: a. papillomavirus E2 protein; b. papillomavirus E7 protein; c. papillomavirus E6 protein; (iii) papillomavirus L2 capsid protein; preferably wherein the L2 protein coding sequence has been altered to include an in-frame C-terminal fusion peptide selected from the group consisting of: a. papillomavirus E6 protein; b. a derivative of papillomavirus E6 protein; c. papillomavirus E7 protein; d. a derivative of papillomavirus E7 protein; e. papillomavirus E2 protein; f. a derivative of papillomavirus E2 protein (iv) a papillomavirus non-structural protein selected from the group consisting of: a. E2 non-structural protein or part thereof; b. E6 non-structural protein or part thereof; c. E7 non-structural protein or part thereof, (v) a papillomavirus L1 capsid protein modified as follows: a. HPV 11 OR HPV 16 L1 gene lacking nuclear localisation signal (NLS-, Δ483) b. HPV 11 OR HPV 16 L1 gene lacking 10 N-terminal codons (ΔN10) c. HPV 11 OR HPV 16 L1 gene lacking 10 N-terminal codons and NLS (ΔN10Δ483) d. HPV 11 OR HPV 16 L1 gene with a C to G mutation (pen) at aa428 (pen504) e. HPV 11 or HPV 16 L1 genes with pen and ΔN10 modifications f. HPV 11 or HPV 16 L1 genes with pen and Δ483 modifications g. HPV 11 or HPV 16 L1 genes with pen and ΔN10Δ483 modifications (vi) any combination of (i) to (v). Furthermore, there are provided deduced amino acid sequences of said isolated nucleotide sequences or parts thereof, more preferably the deduced amino acid sequence reflects preferred plant codon usage. Furthermore, in accordance with the invention there are provided homologues, variants, and/or derivatives of the isolated nucleotide sequences, or parts thereof. Still furthermore, in accordance with the invention there are provided complements of the isolated nucleotide sequences, or parts thereof. Still furthermore, in accordance with the invention there are provided complements of the homologues, variants and derivatives of the isolated nucleotide sequences, or parts thereof. Still furthermore, in accordance with the invention there are provided construct(s) and/or vector(s) comprising and/or expressing: a. the isolated nucleotide sequences set out in any one of FIGS. 4 to 7 , or parts thereof; or b. homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or c. complements of the isolated nucleotide sequences, or parts thereof; or d. complements of the homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or e. a nucleotide sequence that is capable of hybridising to: (i) the isolated nucleotide sequences, or parts thereof; or (ii) the homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or (iii) the complements of the isolated nucleotide sequences, or parts thereof; or (iv) the complements of the homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or (v) any combination of (e)(i)-(e)(iv); f. any combination of (a)-(e), and a promoter, wherein the promoter is operably linked to the sequence of any one of (a)-(f). Furthermore, there is provided a method of producing a papillomavirus protein, or parts thereof, comprising the steps of: 1) contacting a plant cell with a vector and/or construct as claimed herein; 2) cultivating the plant cell under conditions suitable for the production of a protein or fragment of a protein of: (a) the isolated nucleotide sequences set out in any one of FIGS. 4 to 7 , or parts thereof; or (b) homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or (c) complements of the isolated nucleotide sequences, or parts thereof; or (d) complements of the homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or (e) a nucleotide sequence that is capable of hybridising to: i. the isolated nucleotide sequences, or parts thereof; or ii. the homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or iii. the complements of the isolated nucleotide sequences, or parts thereof; or iv. the complements of the homologues, variants or derivatives of the isolated nucleotide sequences, or parts thereof; or v. any combination of (e)(i)-(e)(iv); (f) any combination of (a)-(e), wherein, preferably, the plant cell is a Nicotiana benthamiana plant cell and, preferably, the conditions permit the spontaneous assembly of the proteins or fragments of proteins into immunogenic-response eliciting virus-like particles. Furthermore, there is provided a method of isolating and purifying proteins or fragments of the proteins of the isolated nucleotide sequences set out in any one of FIGS. 4 to 7 , or parts thereof. Furthermore, in accordance with the invention there is provided a use of a papillomavirus protein, or fragment of a papillomavirus protein, which comprises: a. the amino acid sequence of the nucleotide sequence set out in any one of FIGS. 4 to 7 ; or b. the amino acid sequence of a nucleotide sequence having at least 80%, preferably 90%, more preferably 95% homology, after optimal alignment, with the nucleotide sequence set out in any one of FIGS. 4 to 7 ; or c. an amino acid sequence of a fragment of a papillomavirus protein as defined in a), in a process for the manufacture of a pharmaceutical composition for use in a prophylactic treatment against causative agents of lesions and carcinomas, more particularly cervical lesions and carcinomas. According to the present invention the pharmaceutical composition will preferably be contained in a pharmaceutically acceptable medium, preferably a medium which can be injected into humans or given orally, which may, preferably, consist of water, or an aqueous saline solution, or of an aqueous solution based on dextrose and/or glycerol. According to the present invention the pharmaceutical compositions as claimed may contain a pharmaceutically acceptable surfactant, such as for example anionic, cationic, non-ionic or amphoteric surfactants. According to the invention the pharmaceutical compositions as claimed may contain pharmaceutically acceptable adjuvants and/or excipients which, preferably, may be administered by subcutaneous, intradermal or transmucousal route. |
Roller folding head and method for folding a flange |
The invention relates to a roller folding head. Roller folding heads are used, for instance, when fixing the frame of a sliding roof in an opening in the roof of a vehicle. In order to do this quickly and automatically, the roller folding head is provided with at least two pairs of rollers (8, 10; 9, 11 or 20, 22; 21, 23), each pair of rollers (8, 10; 9, 11 or 20, 22; 21, 23) consisting of a pressing roller and a counter roller which interact in order to bend a flange (32) along a folding line at a specific angle. The folding process is carried out in several stages, one flange being crimped around another flange in a series of partial steps at an angle of 180°. One pair of rollers is provided for each step, at least two thereof being arranged on a roller folding head. |
1. A roller folding head for fastening to a robotic arm comprising a base body, a carriage adapted to slide along said base body, and two pairs of rollers, denoted as a first pair and a second pair, each pair of rollers, comprising a pressure roller and a counter-roller, both of which interact to bend a flange at a specific angle along a folding line, one of said pressure rollers being rotatably mounted on the carriage. 2. A roller folding head, as claimed in claim 1, further comprising a second carriage adapted to be moved relative to said base body, wherein the counter-roller of the first pair of rollers is mounted immoveably on the base body; and the counter-roller of the second pair of rollers is mounted rotatably on said second carriage. 3. A roller folding head, as claimed in claim 1, wherein both counter-rollers are mounted rotatably in immoveable bearings on the base body. 4. A roller folding head, as claimed in claim 2, wherein both carriages are arranged on the base body such that they can be moved perpendicularly in opposite directions. 5. A roller folding head, as claimed in any one of the preceding claims, wherein one pressure roller is adapted to cooperate with a plurality of counter-rollers. 6. A roller folding head, as claimed in claim 5, further comprising a revolver head connected to the base body wherein the counter-rollers are coupled with said revolver head. 7. A four-step method for folding a flange 180° around another flange running parallel to the first flange to form a bead, comprising pushing the two flanges against each other by means of one pressure roller and one counter-roller; wherein a conically tapering segment of the pressure roller provides for one portion of the bead, and producing subsequent portions of the bead by respective correspondingly oriented counter-rollers. 8. A four-step method for folding a flange at 180°, wherein at least three different pairs of rollers are used that are carried by a roller folding head, according to one of the claims 1 to 4. 9. A four-step method for folding a flange at 180°, wherein at least three different pairs of rollers are used that are carried by a roller folding head, according to claim 5. 10. A four-step method for folding a flange at 180° C., wherein at least three different pairs of rollers are used that are carried by a roller folding head, according to claim 6. |
Communication type navigation system and navigation method |
A communication type navigation system which presents information supporting judgement of a route selection when a user selects a guidance route from a plurality of searched and recommended routes. In response to a route search request from a navigation terminal (60), a navigation information providing server (10) performs a route search and selects a plurality of recommended routes. By using the information (traffic information, weather information, facility information and user profile information) managed and held by the navigation information providing server (10), evaluation information is generated for the plurality of selected and recommended routes. A user at the navigation terminal (6) is made to refer to this evaluation information and to select a desired recommended route from the plurality of recommended routes. |
1. A communication type navigation system in which a navigation server searches a route in response to a route search request from a terminal, wherein: said navigation server comprises: reception means for receiving from said terminal; search means for searching a route between a departure place and a destination place contained in said route search request and selecting a plurality of recommended routes; evaluation means for forming evaluation information of comparison between estimated running times of the plurality of recommended routes selected by said search means; and presentation means for presenting said terminal transmitted said route search request with route information of the plurality of recommended routes selected by said search means along with the evaluation information formed by said evaluation means; said terminal comprises: transmission means for transmitting said route search request containing the departure place and the destination place to said navigation server; reception means for receiving the route information of the plurality of recommended routes from said navigation server along with said evaluation information for the plurality of recommended routes; and presentation means for presenting a user with the route information of the plurality of recommended routes along with said evaluation information received at said reception means; and wherein said presentation means outputs said evaluation information as voice information. 2. A navigation server in a communication type navigation system in which a route is searched in response to a route search request from a terminal, wherein: said navigation server comprises: reception means for receiving from said terminal; search means for searching a route between a departure place and a destination place contained in said route search request and selecting a plurality of recommended routes; evaluation means for forming evaluation information of comparison between the plurality of recommended routes selected by said search means; and presentation means for presenting said terminal transmitted said route search request with route information of the plurality of recommended routes selected by said search means along with the evaluation information formed by said evaluation means; and wherein said evaluation means forms the evaluation information of comparison of estimated running times between the plurality of recommended routes selected by said search means. 3. The navigation server in the communication type navigation system according to claim 2, wherein said evaluation means further forms the evaluation information of the estimated running times of the plurality of recommended route in accordance with traffic congestion at each of the plurality of recommended routes selected by said search means. 4. The navigation server in the communication type navigation system according to claim 2, wherein said evaluation means further forms the evaluation information of a road toll at each of the plurality of recommended routes selected by said search means, in accordance with information of a road toll at each road section. 5. The navigation server in the communication type navigation system according to claim 2, wherein said evaluation means further forms the evaluation information of weather at each of the plurality of recommended route in accordance with weather information in a district where the plurality of recommended routes selected by said search means pass through. 6. The navigation server in the communication type navigation system according to claim 2, wherein said evaluation means further forms the evaluation information of running environment by obtaining a road width and the number of right and left turns of each of the plurality of recommended routes selected by said search means. 7. The navigation server in the communication type navigation system according to claim 2, wherein said evaluation means further forms the evaluation information of a distance to a facility from any one of the plurality of recommended routes selected by said search means, by using information of said facility registered beforehand in correspondence with a user at said terminal. 8. The navigation server in the communication type navigation system according to claim 2, wherein said evaluation means further forms the evaluation information of an adoption record of each of the plurality of recommended routes selected by said search means, adopted to route guidance, in accordance with information of a recommended route adopted in the past. 9. A navigation method for a terminal connected to a said navigation server comprises steps of: receiving a route search request from said terminal; searching a route between a departure place and a destination place contained in said route search request and selecting a plurality of recommended routes; forming evaluation information of the plurality of recommended routes; and presenting route information of the plurality of recommended routes along with the evaluation information to said terminal transmitted said route search request; and said terminal comprises steps of: transmitting the route search request to said navigation server; and presenting a user with the route information and evaluation information of the plurality of recommended routes received from said navigation server in response to said route search request. |
<SOH> BACKGROUND ART <EOH>A navigation system (hereinafter called a communication type navigation system) has been proposed which is of the type that a navigation information providing server performs a route search or the like and supplies the search results to a navigation terminal mounted on a vehicle. In a general route search, routes satisfying predetermined conditions and/or conditions set by a user are searched from routes interconnecting a departure place and a destination place by using the Dijkstra's algorithm or the like, and the searched routes are presented as recommended routes. In a communication type navigation system, a navigation information providing server collectively manages general information such as traffic information and weather information, user profiles such as user preferences at navigation terminals, history information of route guidance adopted at navigation terminals, and other information. An object of the invention is to allow a user at a navigation terminal in a communication type navigation system to select a useful recommended route by using information under the management by a navigation information providing server. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a schematic diagram showing a communication type navigation system according to a preferred embodiment of the invention. FIG. 2 is a schematic diagram showing the structure of a navigation terminal of the communication type navigation system. FIG. 3 is a schematic diagram showing the structure of an information providing server. FIG. 4 is a schematic diagram showing the structure of a route search server. FIG. 5 is a schematic diagram showing the structure of a portal server. FIG. 6 is a diagram showing an example of the contents registered in a user profile file DB 208 of the portal server. FIG. 7 is a diagram illustrating the operation procedure to be executed by the communication type navigation system according to the preferred embodiment of the invention. FIG. 8 is a diagram illustrating the operation procedure to be executed by the communication type navigation system, following the operation illustrated in FIG. 7 . FIG. 9 is a diagram illustrating the operation procedure to be executed by the communication type navigation system, following the operation illustrated in FIG. 8 . FIG. 10 is a diagram showing an example of a selection screen for recommended routes displayed on a monitor of a navigation terminal of the communication type navigation system according to the preferred embodiment of the invention. FIG. 11 is a diagram showing an example of a display screen to be used when a recommended route displayed on the monitor is selected. FIG. 12 is a diagram showing the structure of a command/object convertor unit of the portal server of the communication type navigation system according to the preferred embodiment of the invention. FIG. 13 is a diagram showing the structure of a dialog processor unit. FIG. 14 is a flow chart illustrating the operation of a command correction reception process to be executed by the portal server of the communication type navigation system according to the preferred embodiment of the invention. FIG. 15 is a diagram illustrating the operation sequence of a voice recognition system to be used when a navigation terminal requests an information providing server for a route search process. FIG. 16 is a diagram illustrating the operation sequence of the voice recognition system to be used when the navigation terminal requests the information providing server for the route search process. detailed-description description="Detailed Description" end="lead"? |
Optical storage medium and method of manufacturing same |
The invention describes an optical data storage medium comprising a substrate (2), a spacer layer (5) and a cover layer (8). The medium comprises two information layers (3, 4) and (6, 7). The invention further describes methods of manufacturing such an information medium (1), which relate to the manner in which the information layers are provided with data patterns. The invention further discloses a method wherein an UV light (10)-transparent stamper (9) is used in combination with a photopolymerization lacquer and/or pressure-sensitive adhesives. |
1. An optical storage medium (1) which successively comprises i) a substrate (2), ii) a first data pattern (3) of pits/grooves iii) a first stack (4) of layers adjoining the first data pattern (3) iv) a spacer (5) v) a second stack (6) of layers vi) a second data pattern (7) of pits/grooves, and vii) a cover (8), characterized in that the storage medium (1) can be considered to be composed of elements that are interconnected, said elements successively being: a) a substrate (2) provided with the first data pattern (3) of pits/grooves over which the first stack (4) of layers is provided, b) a spacer (5), c) a cover (8) provided with the second data pattern (7) of pits/grooves over which the second stack (6) of layers is provided, said second stack (6) of layers lying against the spacer (b), (5). 2. An optical storage medium (1) that successively comprises i) a substrate (2), ii) a first data pattern (3) of pits/grooves, iii) a first stack (4) of layers adjoining the first data pattern (3) iv) a spacer (5) v) a second stack (6) of layers, vi) a second data pattern (7) of pits/grooves, vii) a cover (8), characterized in that the storage medium can be considered to be composed of elements that are interconnected, which elements successively are: a) a substrate (2) provided with the first data pattern (3) of pits/grooves over which the first stack (4) of layers is provided d) a spacer (5) provided with the second stack (6) of layers over which the second data pattern (7) of pits/grooves is provided, said second data pattern (7) being separated from the first data pattern (3) by the spacer (5), e) a cover (8). 3. An optical storage medium (1) that successively comprises i) a substrate (2), ii) a first data pattern (3) of pits/grooves iii) a first stack (4) of layers adjoining the first data pattern (3) iv) a spacer (5) v) a second stack (6) of layers, vi) a second data pattern (7) of pits/grooves, and vii) a cover (5), characterized in that the storage medium (1) can be considered to be composed of elements that are interconnected, which elements successively are: f) a substrate (2), g) a spacer (5) provided with the first stack (4) of layers over which the first data pattern (3) of pits/grooves is provided, said first data pattern lying against the substrate (2), c) a cover (5) provided with the second data pattern (7) of pits/grooves over which the second stack (6) of layers is provided, said second stack (6) of layers lying against the spacer (g), 5). 4. An optical storage medium (1), which successively comprises i) a substrate (2) ii) a first data pattern (3) of pits/grooves, iii) a first stack (4) of layers adjoining the first data pattern (3), iv) a spacer (5) v) a second stack (6) of layers, vi) a second data pattern (7) of pits/grooves, and vii) a cover (5), characterized in that the storage medium (1) can be considered to be composed of elements that are interconnected, which elements successively are: f) a substrate (2), h) a spacer (5) which is provided, on one side, with the first data pattern (3) of pits/grooves over which the first stack (4) of layers is provided, and, on the other side, with the second data pattern (7) of pits/grooves over which the second stack (6) of layers is provided, and e) a cover (8). 5. A method of manufacturing an optical storage medium (1) as claimed in claims 1, 3, characterized in that the element c) is obtained by means of the following steps: c1) providing the cover material with an auxiliary substrate, c2) applying a photopolymerizable lacquer to the cover material, c30 placing a stamper on the photopolymerizable lacquer, c40 carrying out a polymerization reaction and removing the stamper, c5) sputtering a stack of layers onto the structured photopolymerizable lacquer, and c6) removing the auxiliary substrate. 6. A method of manufacturing an optical storage medium (1) as claimed in claim 2, characterized in that element d) is obtained by means of the following steps: d1) applying a photopolymerizable lacquer to a stamper, d2) carrying out a polymerization treatment, and d3) connecting the lacquer polymerized in step d2) to a spacer (5), d4) removing the stamper, d5) sputtering a stack of layers onto the structured photopolymerizable lacquer. 7. A method of manufacturing an optical storage medium (1) as claimed in claim 2, characterized in that element d) or g) is obtained by means of the following steps: d6) applying a stack (6) of layers to a stamper, d7) applying a photopolymerizable lacquer to the stack (6) of layers, and d8) carrying out a polymerization reaction so as to form a structured photopolymerizable lacquer that is provided with a stack of layers (6). 8. A method of manufacturing an optical storage medium (1) as claimed in claim 2, characterized in that element d) is obtained by means of the following steps: d9) providing the spacer (5) on the element a), d10) placing an UV radiation (10)-transparent stamper (9) on the spacer (5) of step d9), d11) carrying out a polymerization reaction, d12) removing the transparent stamper (9), and finally d13) sputtering a stack (6) of layers onto the structured photopolymerizable lacquer as obtained after step d11). 9. A method of manufacturing an optical storage medium (1) as claimed in claim 8, characterized in that steps d9)-d10) are divided into four sub-steps, i.e.: d14) providing the spacer (5) on element a) d15) carrying out a polymerization reaction, d16) providing an additional spacer on the polymerized spacer of d15), and d17) placing the UV radiation (10)-transparent stamper (9) on said additional spacer of d16). 10. A method of manufacturing an optical storage medium (1) as claimed in claim 1, characterized in that one or more of the elements a)-g) are interconnected by means of a pressure-sensitive adhesive or a photopolymerization lacquer. |
Automatic question formulation from a user selection in multimedia content |
The invention notably has for its object to permit a user who uses multimedia content to make a search for an object of interest evoked in said content, without having to formulate the question himself. For this purpose, a selection tool (for example, a key) permits the user to select a passage of the content while he is using it. When the user makes a selection, a context data is extracted from the content (for example, the current reading time). This context data is then used for recovering one or more descriptions in a document (for example, an MPEG-7 document) which describes said content. The recovered descriptions are finally used for automatically formulating a question intended to be transmitted to a search engine. |
1. Electronic equipment comprising reading means for reading a multimedia content which is described in a document containing descriptions, characterized in that it comprises a user command which permits a user to make a selection in said multimedia content, extraction means for extracting from said multimedia content one or more context data relating to said selection, means for recovering one or more descriptions in said document from said context data, and automatic formulation means based on recovered descriptions, of a question intended to be transmitted to a search engine. 2. Electronic equipment as claimed in claim 1, characterized in that said multimedia content contains a plurality of multimedia entities associated with a reading time, said document comprises descriptions relating to one or more multimedia entities which can be recovered from a reading time, and the current reading time (T) at the moment of the selection forms a context data. 3. Electronic equipment as claimed in claim 1, characterized in that said multimedia content contains objects identified by an object identifier, said document comprises descriptions relating to one or more objects that can be recovered by an object identifier, said user command comprises an object selection tool and the object identifier of the selected object forms a context data. 4. Electronic equipment as claimed in claim 1, characterized in that said document is a tree-like structure of father and son nodes (N0, N1, N2, N3, N21, N22, N31, N32, N33) containing one or various descriptions that are instances of one or more descriptors, a content description in a father node being valid for the son node when no other node from said father to said son node contains another description that is an instance of the same descriptor, and said description recovery means compare the context data with instances of one or more descriptors called recovery descriptors for selecting a node in the tree-like structure and recovering other descriptions which are also valid for this node. 5. A method of formulating a question intended to be transmitted to a search engine while a user is using a multimedia content, said multimedia content being described in a document that contains descriptions, characterized in that it comprises: a selection step (1) by the user in said multimedia content, an extraction step (2) for extracting from the multimedia content one or more context data relating to said selection, a recovery step (3; 4) of one or more descriptions in said document from said context data and an automatic formulation step (5) of said question from recovered descriptions. 6. A method as claimed in claim 5 of formulating a question, characterized in that said multimedia content contains a plurality of multimedia entities associated with a reading time, said document comprises descriptions relating to one or more of the media entities, which may be recovered from a reading time and in that the current reading time (T) constitutes context data at the moment of the selection (S). 7. A method as claimed in claim 5 of formulating a question, characterized in that said multimedia content contains objects identified by an object identifier, said document comprises descriptions relating to one or more objects which may be recovered by an object identifier, said selection step comprises an object selection and in that the object identifier of the selected object constitutes a context data. 8. A method as claimed in claim 5 of formulating a question, characterized in that said document is a tree-like structure of father and son nodes (N0, N1, N2, N3, N21, N22, N31, N32, N33) containing one or various descriptions that are instances of one or more descriptors, a content description in a father node being valid for the son node when no other node from said father to said son node contains another description that is an instance of the same descriptor, and said description recovery means compare the context data with instances of one or more descriptors called recovery descriptors for selecting a node in the tree-like structure and recovering other descriptions which are also valid for this node. 9. A program comprising program code instructions for implementing a method as claimed in claim 5, when it is executed by a processor. 10. A system comprising equipment (EQT) as claimed in claim 5 which comprises transceiver means (EX/RX) for transmitting said question to a remote search engine (SE) and for receiving a response (R) to said question coming from said remote search engine, a search engine (R) and transmission means (TR) for transmitting said question from the equipment to the search engine and for transmitting said response from the search engine to said equipment. |
Method of etching copper on cards |
Etching of copper on a card is achieved by applying an electrical voltage between a cathode (102) and the card (42), the card (42) thereby forming an anode. The cathode (102) and the card (42) are immersed in an electrolyte comprising a first component, which may be reduced from a first state in the form of an ion having a metal atom with a first positive oxidation number to a second state in the form of an ion having said metal atom with a second positive oxidation number, which is less than said first positive oxidation number. A first redox potential in the electrolyte for reduction from the first to the second state is larger than a second redox potential in the electrolyte for reduction of divalent copper ions to metallic copper. During the etching metallic copper on the card is oxidised and transferred into positively charged copper ions while the first component is reduced from its first state to its second state. The quality of the etched structures on the card is improved since no metallic copper is precipitated on the cathode. |
1. A method of etching copper on boards (2; 42), in which method an electric voltage is applied between a cathode (12; 58, 66; 102, 104) and a board (2; 42), which constitutes an anode, the cathode (12; 58, 66; 102, 104) and the board (2; 42) being immersed in an electrolyte, c h a r a c t e r i s e d .in that the electrolyte contains at least a first component, which can be reduced from a first state in the form of an ion having a metal atom with a first positive oxidation number, to a second state in the form of an ion comprising said metal atom with a second positive oxidation number, which is smaller than said first positive oxidation number, a first redox potential in the electrolyte for reduction from the first to the second state being greater than a second redox potential in the electrolyte .for reduction of divalent copper ions to metallic copper, metallic copper on the board (2; 42) being oxidised and converted into positively charged copper ions while the first component is reduced from its first to its second state. 2. A method as claimed in claim 1, in which the first component is selected from the group consisting of Fe, Mn, Ce and inorganic and organic complexes thereof 3. A method as claimed in claim 2, in which the first component is Fe, which is added to the electrolyte in the form of Fe30+ or a precursor thereof. 4. A method as claimed in any one of the preceding claims, in which the capacity of the electrolyte to etch the copper on the board, in the absence of an applied voltage, corresponds to an etching rate of 6-100 nm/s. 5. A method as claimed in any one of the preceding claims, in which a second component is also added to the electrolyte, which second component can be reduced from a first state in the form of an ion having an atom with a first positive oxidation number, to a second state in the form of an ion comprising said atom with a second positive oxidation number, which is smaller than said first positive oxidation number, a third redox potential in the electrolyte for reduction of the second component from its first state to its second state being lower than the second redox potential in the electrolyte for reduction of divalent copper ions to metallic copper. 6. A method as claimed in claim 5, in which the second component is Sn; which is preferably present in the electrolyte in a concentration of 0.005-0.4 mol/l. 7. A method as claimed in any one of the preceding claims, in which the electrolyte also contains fluoride ions, F, in a concentration of 0.010.5 mol/l. 8. A method as claimed in claim 7, in which the electrolyte also contains chloride ions, Cl−, in a concentration of 0.03-1.5 mol/l. 9. A method as claimed in any one of the preceding claims, in which the voltage applied is 0.5-6 V. 10. A method as claimed in any one of the preceding claims, in which the concentration of the first component is 0.02-0.7 mol/l. 11. A method as claimed in any one of the preceding claims, in which at least two different first components are added to the electrolyte. 12. A method as claimed in any one of the preceding claims, in which the pH of the electrolyte is 0.5 to 4.0. 13. A method as claimed in any one of the preceding claims, in which the electrolyte is treated with a regenerant, which is a strong oxidising agent, such as Na2S2O8, for converting the first component from its second to its first state. 14. A method as claimed in any one of the preceding claims, in which a fourth redox potential in the electrolyte for reduction of said second state of the first component to a third state is lower than the redox potential of hydrogen gas. 15. A device for etching copper on boards (2; 42), which device comprises a voltage generator (604), a cathode (12; 58, 66; 102, 104), which is arranged to be connected to the negative pole of the voltage generator (604), and a container (600), which is arranged to contain an electrolyte and, immersed in the electrolyte, the cathode (12; 58, 66; 102, 104) and a board (2; 42), which is arranged to be connected to the positive pole of the voltage generator (604) to constitute an anode, c h a r a c t e r i s e d in that the container (600) is arranged to receive an electrolyte containing at least a first component, which can be reduced from a first state in the form of an ion having a metal atom with a first positive oxidation number, to a second state in the form of an ion is comprising said metal atom with a second positive oxidation number, which,is smaller than said first positive oxidation number, a first redox potential in the electrolyte for reduction from the first to the second state being greater than a second redox potential in the electrolyte for reduction of divalent copper ions to metallic copper. 16. A device as claimed in claim 15, in which a regeneration container (612) is arranged to receive electrolyte from the container (600), the regeneration container (612) having means (620, 622, 624, 626, 628) for adding a regenerant to convert the first component from its second state to its first state and means (616) for recirculating the thus regenerated electrolyte to said container (600). 17. A device as claimed in claim 15 or 16, in which the distance (H; H1, H2) between a first flat side (6; 46, 52) of the board (2; 42) and a first flat side (202) of the cathode (12; 58, 66; 102, 104) facing the board (2; 42) is less than 10 mm. 18. An electrolyte for use in electrochemical etching of copper on boards (2; 42), in which etching an electric voltage is applied between a cathode (12; 58, 66; 102, 104) and a board (2; 42), which constitutes an anode, the cathode (12; 58, 66; 102, 104) and the board (2; 42). being immersed in the electrolyte, c h a r a c t e r i s e d in that the electrolyte contains at least a first component, which can be reduced from a first state in the form of an ion having a metal atom with a first positive oxidation number, to a second state in the form of an ion comprising said metal atom with a second positive oxidation number, which is smaller than said first positive oxidation number, a first redox potential in the electrolyte for reduction from the first to the second state being greater than a second redox potential in the electrolyte for reduction of divalent copper ions to metallic copper. 19. An electrolyte as claimed in claim 18, which comprises Fe in a concentration of 0.02-0.7 mol/l. 20. An electrolyte as claimed in claim 18 or 19, which comprises Sn in a concentration of 0.005-0.4 mol/l. 21. An electrolyte as claimed in any one of claims 18-20, which comprises fluoride ions, F−, in a concentration of 0.01-0.5 mol/l. 22. An electrolyte as claimed in claim 21, which comprises chloride ions, Cl−, in a concentration of 0.03-1.5 mol/l. |
<SOH> BACKGROUND ART <EOH>In the manufacture of patterns, such as electric conductors, on boards, for example copper laminates intended for the manufacture of circuit boards, use is often made of etching to remove portions of an electrically conductive layer. The board usually has a sheet of a nonconductive material, which sheet is provided with-a copper layer. on one or on both flat sides. Usually a photosensitive coating is applied on the copper layer. A photographic process is used to transfer a desired pattern from a master or a template to the coating, after which the portions of the coating surrounding the pattern are removed. In the etching operation, the copper layer is etched away where the coating has been removed, whereas the copper layer remains in the portions of the copper layer which are still covered by the coating. Etching is often carried out as electrochemical etching, in which a voltage is applied between a cathode and the board, which constitutes an anode, both the anode and the cathode being immersed in an electrically conductive electrolyte. This method of etching is disclosed, for example, in EP 0 889 680 and WO 98/10121. One problem of electrochemical etching is that it is necessary for the portions of the copper layer which are to be etched away to be in continuous electrical contact with the voltage source. If, during etching, a portion of the copper layer loses this electrical contact, this portion will form an “isalet” which cannot be etched away. Another problem is that the copper in the copper layer which is oxidised and converted into copper ions will be reduced on the cathode and form metallic copper anew. Since this precipitation is not uniformly distributed over the surface of the cathode, the distance between the cathode and the board will vary over the flat side of the board. Owing to this, also the etching effect will vary over the flat side of the board, implying that some portions of the board are excessively etched and some portions are not sufficiently. etched. To obviate these problems, the distance between the board and the cathode has to be so long that a change of this distance, due to precipitation of copper, will be insignificant in relation to its magnitude. If a long distance is provided between the cathode and the board, it is necessary to apply a high voltage between the board and the cathode to obtain the desired current density and thus a sufficient etching rate. However, if a high voltage is applied, the electric conductors do not obtain the desired dimensions. This in turn prevents the electric conductors on the board from obtaining the intended conductive and resistive properties, which makes it necessary to reject a great deal of the etched boards. |
<SOH> SUMMARY OF THE INVENTION <EOH>The object of the present invention is to obviate or reduce the above-mentioned disadvantages and provide a method of etching boards, said method being efficient and resulting in a low defect rate for the etched boards. More specifically, the invention provides a method of etching copper on boards, in which method an electric voltage is applied between a cathode and a board, which constitutes an anode, the cathode and the board being immersed in an electrolyte, the method being characterised in that the electrolyte contains at least a first component, which can be reduced from a first state in the form of an ion having a metal atom with a first positive oxidation number, to a second state in the form of an ion comprising said metal atom with a second positive oxidation number, which is smaller than said first positive oxidation number, a first redox potential in the electrolyte for reduction from the first to the second state being greater than a second redox potential in the electrolyte for reduction of divalent copper ions to metallic copper, metallic copper on the board being oxidised and converted into positively charged copper ions while the first component is reduced from its first to its second state. Another object of the present invention is to obviate or reduce the above-mentioned disadvantages and provide a device for etching boards, which device allows efficient etching of boards with a low defect rate for the etched boards. More specifically, the invention provides a device for etching copper on boards, which device comprises a voltage generator, a cathode, which is arranged to be connected to the negative pole of the voltage generator, and a container, which is arranged to contain an electrolyte and, immersed in the electrolyte, the cathode and a board, which is arranged to be connected to the positive pole of the voltage generator to constitute an anode, said device being characterised in that the container is arranged to receive an electrolyte containing at least a first component, which can be reduced from a first state in the form of an ion having a metal atom with a first positive oxidation number, to a second state in the form of an ion comprising said metal atom with a second positive oxidation number, which is smaller than said first positive oxidation number, a first redox potential in the electrolyte for reduction from the first to the second state being greater than a second redox potential in the electrolyte for reduction of divalent copper ions to metallic copper. It is also an object of the present invention to obviate or reduce the above-mentioned disadvantages and provide an electrolyte for etching boards, which electrolyte allows efficient etching of boards with a low defect rate for the etched boards. More specifically, the invention provides an electrolyte for use in electrochemical etching of copper on boards, in which etching an electric voltage is applied between a cathode and a board, which constitutes an anode, the cathode and the board being immersed in the electrolyte, the electrolyte being characterised in that it contains at least a first component, which can be reduced from a first state in the form of an ion having a metal atom with a first positive oxidation number, to a second state in the form of an ion comprising said metal atom with a second positive oxidation number, which is smaller than said first positive oxidation number, a first redox potential in the electrolyte for reduction from the first to the second state being greater than a second redox potential in the electrolyte for reduction of divalent copper ions to metallic copper. Further advantages and characteristics of the invention will appear from the description below and the appended claims. |
Plasmids expressing human insulin and the preparation method for human insuling thereby |
The present invention relates to human insulin expression plasmids and a method for producing insulin using the same. The plasmids comprise a sequence encoding a compound of the formula R—B—X-A, in which R is a leader peptide of the formula of Met-Thr-Met-Ile-Thr-Y (SEQ ID NO: 36), in which Y is one selected from lysine, arginine, a peptide containing lysine as an amino acid at its C-terminal, or a peptide containing arginine as an amino acid at its C-terminal; B is human insulin B-chain or analogue thereof; X is a peptide connecting B with A; and A is human insulin A-chain or analogue thereof. The method for preparing insulin using the plasmids according to the present invention converts the proinsulin fusion protein into human insulin in a single enzymatic cleavage process and minimizes the generation of by-products after the enzymatic cleavage, thereby producing insulin at a high yield. Therefore, the plasmids according to the present invention and the method for preparing insulin using the same can be usefully applied to the industrial mass-production of human insulin. |
1. A plasmid comprising a sequence encoding a compound of the following formula (I): R—B—X-A (I) in which, (i) R is a leader peptide represented by the following formula (II): Met-Thr-Met-Ile-Thr-Y (II) (SEQ ID NO: 36) in which Y is one selected from lysine, arginine, a peptide containing lysine as an amino acid at its C-terminal, or a peptide containing arginine as an amino acid at its C-terminal; (ii) B is human insulin B-chain or analogue thereof; (iii) X is a peptide connecting B with A; and (iv) A is human insulin A-chain or analogue thereof. 2. The plasmid according to claim 1, in which the R of the formula (I) is selected from the following peptide sequences of SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7 or 8: Met-Thr-Met-Ile-Thr-Lys: SEQ ID NO. 1 Met-Thr-Met-Ile-Thr-Asp-Ser-Leu-Ala- SEQ ID NO. 2 Lys: Met-Thr-Met-Ile-Thr-Asp-Ser-Leu-Ala- SEQ ID NO. 3 Val-Val-Leu-Gln-Lys: Met-Thr-Met-Ile-Thr-Asp-Ser-Leu-Ala- SEQ ID NO. 4 Val-Val-Leu-Gln-Gly-Ser-Leu-Gln-Lys: Met-Thr-Met-Ile-Thr-Arg: SEQ ID NO. 5 Met-Thr-Met-Ile-Thr-Asp-Ser-Leu-Ala- SEQ ID NO. 6 Arg: Met-Thr-Met-Ile-Thr-Asp-Ser-Leu-Ala- SEQ ID NO. 7 Val-Val-Leu-Gln-Arg: Met-Thr-Met-Ile-Thr-Asp-Ser-Leu-Ala- SEQ ID NO. 8 Val-Val-Leu-Gln-Gly-Ser-Leu-Gln-Arg: 3. The plasmid according to claim 1, in which the peptide sequences of SEQ ID NOs. 1 to 8 are encoded by the following sequences of SEQ ID NOs. 9 to 16: ATG ACC ATG ATT ACG AAG: SEQ ID NO. 9 ATG ACC ATG ATT ACG GAT TCA CTG GCC SEQ ID NO. 10 AAG: ATG ACC ATG ATT ACG GAT TCA CTG GCC SEQ ID NO. 11 GTC GTT TTA CAA AAG: ATG ACC ATG ATT ACG GAT TCA CTG GCA SEQ ID NO. 12 GTC GTT TTA CAA GGT TCT CTG CAG AAG: ATG ACC ATG ATT ACG CGT: SEQ ID NO. 13 ATG ACC ATG ATT ACG GAT TCA CTG GCC SEQ ID NO. 14 CGT: ATG ACC ATG ATT ACG GAT TCA CTG GCC SEQ ID NO. 15 GTC GTT TTA CAA CGT: ATG ACC ATG ATT ACG GAT TCA CTG GCA SEQ ID NO. 16 GTC GTT TTA CAA GGT TCT CTG CAG CGT: 4. The plasmid according to claim 2 or 3, in which the B, X and A of the formula (I) are human insulin B-chain, C-chain and A-chain, respectively. 5. The plasmid according to claim 2 or 3, in which the B of the formula (I) is a peptide having the residues Nos. 28 and 29 of human insulin B-chain exchanged to each other, and X and A are human insulin C-chain and A-chain, respectively. 6. The plasmid according to claim 4, which the plasmid has the structure of FIG. 1c. 7. The plasmid according to claim 6, in which the plasmid is selected from pK-B5Kpi, pK-B9Kpi, pK-B13Kpi, pK-B5Rpi, pK-B9Rpi and pK-B13Rpi. 8. The plasmid according to claim 7, in which the plasmid is pK-B5Kpi plasmid deposited under accession No. KCTC 10363BP. 9. The plasmid according to claim 4, in which the plasmid has the structure of FIG. 3c. 10. The plasmid according to claim 9, in which the plasmid is selected from pPT-B5Kpi, pPT-B9Kpi, pPT-B13Kpi, pPT-B5Rpi, pPT-B9Rpi, pPT-B13Rpi, pPT-17Kpi and pPT-17Rpi. 11. The plasmid according to claim 4, in which the plasmid has the structure of FIG. 4c. 12. The plasmid according to claim 11, in which the plasmid selected from pPL-B5Kpi, pPL-B9Kpi, pPL-B13Kpi, pPLD-B5Kpi, pPLD-B9Kpi, pPLD-B13Kpi, pPL-B5Rpi, pPL-B9Rpi, pPL-B13Rpi, pPLD-B5Rpi, pPLD-B9Rpi and pPLD-B13Rpi. 13. The plasmid according to claim 5, in which the plasmid has the structure of FIG. 3c. 14. The plasmid according to claim 13, in which the plasmid is selected from pPT-B5KpiKP, pPT-B9KpiKP, pPT-B13KpiKP, pPT-B5RpiKP, pPT-B9RpiKP and pPT-B13RpiKP. 15. A microorganism transformed with the plasmid according to any one of claims 6 to 14. 16. A method for preparing human insulin or a analogue thereof comprising: (a) a step to induce the expression of a compound of the following formula (I) by fermenting the microorganism of claim 15: R—B—X-A (I) in which (i) R is a leader peptide represented by the following formula (II): Met-Thr-Met-Ile-Thr-Y (II) (SEQ ID NO: 36) in which, Y is one selected from lysine, arginine, a peptide containing lysine as an amino acid at its C-terminal, or a peptide containing arginine as an amino acid at its C-terminal; (ii) B is human insulin B-chain or analogue thereof; (iii) X is a peptide connecting B with A; and (iv) A is human insulin A-chain or analogue thereof; (b) a step of cell disruption and dissolution; (c) a step of refolding; (d) a step of co-cleavage of R and X by an enzymatic reaction; and (e) a step of purification of active insulin by chromatography. 17. The method according to claim 16, in which the (d) step is performed at pH 7 to 8, a reaction temperature of 4° to 28°, trypsin level per protein 1 mg of 0.1 u to 0.5 u, carboxypeptidase B level per protein 1 mg of 0.1 u to 0.3 u, a reaction time of 12 to 24 hours. 18. The method according to claim 16, in which the (d) step is performed using both immobilized trypsin and immobilized carboxypeptidase B. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates to plasmids for expression of human insulin and a method for preparing insulin using the same. 2. Description of the Related Art Insulin is a hormone secreted in the pancreas to regulate the glucose level in blood and binds to insulin receptors on the cell surfaces, thereby promoting the use of glucose and reducing the blood glucose level. Now, it is widely used as a therapeutic agent of diabetes. Insulin is produced as a precursor form in the pancreas. Proinsulin comprises an A-chain, a B-chain, and a C-chain connecting the two chains. When the C-chain is cut off in the cell, proinsulin is converted into active insulin comprising only the A-chain and the B-chain. As the genetic engineering technology develops, various recombinant proteins can be mass-produced using E. coli transformed with recombinant plasmids. One of the most important problems in the production of the recombinant proteins is that the proteins have short half life in the host cells (Talmadge K, et al. Proc Natl Acad Sci USA. 1982;79:1830-3, Shen S H. Proc Natl Acad Sci USA. 1984;81:4627-31). For example, the half life of rat proinsulin in E. coli has been reported to be about 2 minutes (Talmadge K, et al. Proc Natl Acad Sci USA. 1982;79:1830-3). The degradation of expressed proteins is closely related to the folding of the proteins. Cells degrade proteins with an incomplete tertiary structure or damaged, and convert them into amino acids, whereby the intracellular composition can be efficiently used. In the cytoplasm of E. coli, the initial protein degradation is performed by HSPs (heat shock proteins) using ATP. A method that includes expressing a protein in the form of inclusion body, followed by refolding it to recover its activity may be used to increase the stability of the recombinant protein. Generally, the inclusion body is not affected by proteases and can be accumulated to a high concentration up to 50% of intracellular proteins. Accordingly, the expression of a target protein in the form of inclusion body would be a very excellent method which can economically produce the target protein, if an efficient refolding process for the formation of the correct tertiary structure of the protein is developed (Mukhopadhyay A. Adv Biochem Eng Biotechnol. 1997;56:61-109). In the production of human insulin in E. coli, the above-described method has been broadly applied. The commonly used methods are expressing recombinant insulin in the form of a fusion protein to increase stability, followed by chemical cleavage. For example, proinsulin gene is inserted into a plasmid, containing a gene of a protein having a high stability in E. coli such as β-galactosidase, to construct a recombinant plasmid and the proinsulin fusion protein is expressed in E. coli transformed with the plasmid. According to the above-described method, in order to prepare human insulin by purification of the proinsulin fusion protein, the bodies are purified to increase the purity of the target protein and the washed inclusion bodies are dissolved by a treatment with a denaturant and subjected to sulfonation to minimize the formation of hydrophobic interaction and the wrong disulfide bonding between molecules. Then, the proinsulin fusion protein is treated with cyanogen bromide (hereinafter referred to as ‘CNBr’) to cleave methionine residue connecting the leader peptide with proinsulin. After completion of the cleavage, CNBr is removed and the resulting proinsulin is separated, purified, and refolded with an oxidation and reduction system. Proinsulin is converted into active insulin by removing C-chain between A-chain and B-chain using trypsin and carboxypeptidase B. Insulin is purified by ion exchange chromatography and reverse phase high performance chromatography and zinc-crystallized in the final step. The above-described method includes complex purification processes, and thus the conversion of the proinsulin fusion protein into insulin has a low yield and has problems requiring considerable expenses and time in terms of industrial production. Also, though the expression level of the fusion protein may be increased by the above-described method, the final yield of the recombinant human insulin does not reach a satisfactory level (Goeddel D V, et al. Proc Natl Acad Sci USA. 1979;76:106-10, Talmadge K, et al. Proc Natl Acad Sci USA. 1980;77:3988-92, Sung W L, et al. Proc Natl Acad Sci USA. 1986;83:561-5). Further, in terms of industrial production, the use of toxic CNBr is attended with danger in handling a toxic substance and brings about problems associated with much expense required to dispose of the used CNBr. Therefore, the leader peptide is preferably cleaved by a protease. As enzymatic cleavage methods, the following have been developed. Evans et al. fused a peptide comprising 8 amino acids, containing a metal binding site, and a renin cleavage site to the N-terminal of a target protein to cleave the leader peptide with renin as a protease (Evans D B, et al. Protein Expr Purif. 1991;2:205-13). Sharma et al. used a peptide comprising 9 amino acids, containing 6 successive histidines, and a renin cleavage site as a leader peptide (Sharma S K, et al. Biotechnol Appl Biochem. 1991; 14:69-81). For production of insulin, a method is developed, in which a proinsulin precursor having a recognition site which can be cleaved by a protease is expressed in E. coli, and the obtained inclusion bodies are subjected to refolding and other purification process. For example, U.S. Pat. Nos. 5,126,249 and 5,378,613 disclose a method for preparing a gene encoding methionine-tyrosine or arginine-proinsulin by inserting only one amino acid between methionine, left only at the translational initiation site in E. coli, and the target protein. Generally, expression of a non-fusion protein results in a low level or the product is readily degraded, and the transcription and the translation may be damaged. But, it is possible to obtain a high expression level by this method. However, in this method, cathepsin C or dipeptidyl-aminopeptidase should be used to remove two amino acids in front of proinsulin prior to the cleavage of C-chain by a protease. Consequently, an additional enzymatic reaction should be further included, complicating the purification process. As another example, U.S. Pat. Nos. 5,227,293 and 5,358,857 disclose methods for expressing a protein comprising methionine as a translational initiation site, a peptide encoded by a short nucleotide sequence of (DCD)x, an enzyme cleavage site and a proinsulin analogue, which are sequentially fused together, in a microorganism. In the (DCD)x sequence, D represents adenine, guanine or thymine, C represents cytosine, and x represents 4 to 12. Therefore, amino acids encoded by the sequence are limited to serine, threonine or alanine. In this method, intact proinsulin is not used as a target protein and the mini-proinsulin having the C-chain composed of only one arginine is fused to the leader peptide. However, in the above patent, there is no description of an example to convert the proinsulin fusion protein into insulin using trypsin and carboxypeptidase B simultaneously, and thus it is not considered that the complexity of the purification process is solved. Also, Chen et al. expressed methionine-lysine-proinsulin composite in E. coli, thereby improving the expression level and simplifying the purification process (Chen J Q, et al. Appl Biochem Biotechnol. 1995;55:5-15). However, the method has problems in that a large amount of insulin by-products are generated when the methionine-lysine-proinsulin is cleaved with trypsin and carboxypeptidase B to produce active insulin (Yang Z H, et al. Appl Biochem Biotechnol. 1999;76:107-14). Korean Patent Registration No. 1002029580000 discloses a method for improving the efficiency of refolding and facilitating enzymatic cleavage by expressing a leader peptide-proinsulin composite. In this method, the leader peptide is composed of the N-terminal fragment of β-galactosidase, 6 successive threonines, and two amino acids comprising lysine or arginine. The leader peptide shows hydrophilic property as a whole, and thus it exerts a little influence on the refolding of proinsulin and a protease can readily recognize it and react. However, in the above patent, there is no description of the generation of insulin by-products when the fusion protein is converted into human insulin through enzymatic cleavage, and thus it is not sure whether the problems associated with the generation of the by-products are solved. Indeed, it has been shown that a large amount of insulin by-products is generated upon conversion into insulin. Also, since the efficiency of the enzymatic cleavage is low, though the expression level of the fusion protein is high, the separation of insulin from the by-products in the subsequent processes becomes difficult. Consequently, the yield of the insulin production is low. Meanwhile, Jonasson et al. succeeded in enzymatic cleavage with trypsin by expressing two IgG binding domain (hereinafter referred to as ‘ZZ’)-a linker comprising one or more amino acids of lysine or arginine-proinsulin composite (Jonasson P, et al. Eur J Biochem. 1996;236:656-61). In this method, proinsulin is refolded in the form of the ZZ leader peptide fused thereto, then the leader peptide and C-chain of proinsulin is concomitantly cleaved by trypsin and carboxypeptidase B, which simplifies the enzymatic treatment process. However, in this method, the number of amino acids forming the ZZ leader peptide is greater than the number of amino acids forming proinsulin, and thus more than half polypeptide should be removed from the expressed recombinant protein in the purification process, which relatively reduces the yield. Also, the use of the lysine-arginine linker has a problem of the generation of a by-product with one arginine attached to B-chain of insulin. As a similar example, U.S. Pat. No. 6,001,604 discloses a method for expressing SOD (superoxide dismutase)-arginine-proinsulin composite. In this method, the C-chain of the proinsulin comprises one or two amino acids, the proinsulin is refolded in the form of the SOD leader peptide fused thereto and the amino acids of the C-chain and the SOD are concomitantly cleaved by trypsin and carboxypeptidase B. However, this method also has a problem in that the number of amino acids forming the leader peptide is greater than the number of amino acids forming the modified proinsulin. U.S. Pat. No. 6,068,993 discloses a method for expressing a fusion protein of a leader peptide comprising 11 amino acids, containing 6 successive histidines and an arginine as the C-terminal amino acid, and proinsulin. In this method, the fusion protein is converted into insulin by enzymatic reaction after metal ion adsorption process and refolding. Then, the produced insulin is purified by ion exchange chromatography and reverse phase chromatography. According to this method, the chromatography using Ni-chelating Sepharose FF resin and the buffer solution exchange using Sephadex G-25 resin should be performed prior to the refolding, which makes the purification process complex. Therefore, there are demands for a recombinant plasmid and a preparation method which can produce human insulin at a high yield in a simple process. |
<SOH> SUMMARY OF THE INVENTION <EOH>In order to accomplish the above demands, it is an object of the present invention to provide recombinant plasmids which can stably express a fusion protein of a leader peptide and proinsulin or analogue thereof in microorganisms, in which the leader peptide has a site which can be selectively cleaved by an enzyme, and can be easily isolated from the target protein. Also, in another aspect of the present invention, it is an object to provide a method for preparing insulin in a large amount by a simple process, in which a proinsulin fusion protein is converted into active insulin while minimizing generation of by-products, using the recombinant plasmid according to the present invention. The plasmid according to the present invention comprises a sequence encoding a compound of the following formula (I): in-line-formulae description="In-line Formulae" end="lead"? R—B—X-A (I) in-line-formulae description="In-line Formulae" end="tail"? In the formula (I), R is a leader peptide represented by the following formula (II). in-line-formulae description="In-line Formulae" end="lead"? Met-Thr-Met-Ile-Thr-Y (II) in-line-formulae description="In-line Formulae" end="tail"? In the formula (II), Y is one selected from lysine, arginine, a peptide containing lysine as an amino acid at its C-terminal, or a peptide containing arginine as an amino acid at its C-terminal. In the formula (I), B is human insulin B-chain or analogue thereof, X is a peptide connecting B with A, A is human insulin A-chain or analogue thereof. In the plasmid according to the present invention, where Y is lysine, R of the formula (I) may be the following amino acid sequence. Met-Thr-Met-Ile-Thr-Lys: SEQ ID NO. 1 In the plasmid according to the present invention, where Y is a peptide containing lysine as an amino acid at its C-terminal, R of the formula (I) may be the following amino acid sequences. Met-Thr-Met-Ile-Thr-Asp-Ser-Leu-Ala- SEQ ID NO. 2 Lys Met-Thr-Met-Ile-Thr-Asp-Ser-Leu-Ala- SEQ ID NO. 3 Val-Val-Leu-Gln-Lys Met-Thr-Met-Ile-Thr-Asp-Ser-Leu-Ala- SEQ ID NO. 4 Val-Val-Leu-Gln-Gly-Ser-Leu-Gln-Lys In the plasmid according to the present invention, where Y is arginine, R of the formula (I) may be the following amino acid sequence. Met-Thr-Met-Ile-Thr-Arg SEQ ID NO. 5 In the plasmid according to the present invention, where Y is a peptide containing argnine as an amino acid at its C-terminal, R of the formula (I) may be the following amino acid sequences. Met-Thr-Met-Ile-Thr-Asp-Ser-Leu-Ala- SEQ ID NO. 6 Arg Met-Thr-Met-Ile-Thr-Asp-Ser-Leu-Ala- SEQ ID NO. 7 Val-Val-Leu-Gln-Arg Met-Thr-Met-Ile-Thr-Asp-Ser-Leu-Ala- SEQ ID NO. 8 Val-Val-Leu-Gln-Gly-Ser-Leu-Gln-Arg According to the present invention, preferred examples of nucleotide sequences encoding the amino acid sequences of SEQ ID NO. 1 to 8 are as follows. ATG ACC ATG ATT ACG AAG SEQ ID NO. 9 ATG ACC ATG ATT ACG GAT TCA CTG GCC SEQ ID NO. 10 AAG ATG ACC ATG ATT ACG GAT TCA CTG GCC SEQ ID NO. 11 GTC GTT TTA CAA AAG ATG ACC ATG ATT ACG GAT TCA CTG GCA SEQ ID NO. 12 GTC GTT TTA CAA GGT TCT CTG CAG AAG ATG ACC ATG ATT ACG CGT SEQ ID NO. 13 ATG ACC ATG ATT ACG GAT TCA CTG GCC SEQ ID NO. 14 CGT ATG ACC ATG ATT ACG GAT TCA CTG GCC SEQ ID NO. 15 GTC GTT TTA CAA CGT ATG ACC ATG ATT ACG GAT TCA CTG GCA SEQ ID NO. 16 GTC GTT TTA CAA GGT TCT CTG CAG GGT The leader peptide according to the present invention acts as a mask to help proinsulin or analogue thereof stably exist and be expressed since it can be stably expressed in E. coli. In the present invention, the short leader peptide is used, and thus the ratio of the target protein to the leader peptide is relatively high and the target protein is readily isolated and purified by cleaving the fusion protein. Also, in the leader peptide according to the present invention, lysine or arginine at C-terminal provides a site which can be selectively cleaved by trypsin. Therefore, it is possible to convert the proinsulin fusion protein into active insulin by enzymatic cleavage without toxic CNBr treatment which has been conventionally used. The plasmid according to the present invention has a sequence encoding proinsulin or analogue thereof (B—X—Y) connected with 3′-end of the above sequence. Representative example of the proinsulin analogue according to the present invention is a protein having positions of residue No. 28 and residue No. 29 of B-chain exchanged with each other (hereinafter referred to as ‘LysB 28 ProB 29 analogue’). The LysB 28 ProB 29 insulin analogue has effect equal to that of human insulin and can be more rapidly absorbed from a subcutaneous injection site. In the preparation of the plasmid according to the present invention, the gene encoding the proinsulin fusion protein is obtained by cleaving pPRO plasmid (Korean Patent Registration No. 1000766010000) with restriction enzymes of EcoRI and Bg1II, ligating the product with ligase to construct pHHI plasmid, and performing Polymerase Chain Reaction (PCR) using the resulting plasmid as a template. The pHHI plasmid used in the present invention has a gene at 3′-end of tac promoter. The gene encodes a fusion protein which is sequentially expressed in the order of a peptide comprising 28 amino acids, containing a histidine tag, a methionine, and proinsulin. In the preparation of the plasmid according to the present invention, expression vectors used in cloning include any vectors which can show a high expression level in E. coli. Preferred examples include pET-24a(+) vector containing strong T7 promoter (Novagen, Catalogue No. 69749-3) and pHHI-derived vectors containing E. coli rrnB P2 promoter (Lukacsovich T, et al. Gene. 1989;78:189-94) or rac promoter (Boros I, et al. Gene. 1986;42:97-100). Representative examples of the plasmid according to the present invention include pK-BKpi type, pK-BRpi type, pPT-BKpi type, pPT-BRpi type, pPL-BKpi type, pPL-BRpi type, pPLD-BKpi type, pPLD-BRpi type, pPT-BKpiKP type, and pPT-BRpiKP type plasmids, which are classified by vectors, peptide types and target proteins to be expressed. |
3-Hydroxymethylglutaryl coenzyme a reductase and diagnosis and prognostication of dementia |
The invention relates to methods and commercial packages for the diagnosis and/or prognostication of a dementia. The methods are based upon the assessment of a feature or features relating to 3-hydroxymethylglutaryl coenzyme A reductase (HMGR) gene, a polymorphism associated with the gene, the nature of mRNA transcripts produced, and/or HMGR protein activity. Applicants have determined that a decrease in HMGR activity correlates with Alzheimer disease. Applicants have further determined that the presence of a polymorphism in the HMGR gene correlates with Alzheimer disease. Applicants have identified two abnormal HMGR mRNA transcripts and have shown that their presence correlates with Alzheimer disease. |
1. A method of diagnosing or prognosticating a dementia in a subject, said method comprising: (a) obtaining a sample from said subject, wherein said sample comprises nucleic acid comprising a 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene; and (b) determining whether said nucleic acid comprises a polymorphism relative to a corresponding control sample obtained from a control subject; wherein the presence of said polymorphism is used to diagnose or prognosticate a dementia. 2. The method of claim 1, wherein said polymorphism is localized in intron B of said HMGR gene. 3. The method of claim 2, wherein step (b) comprises: (i) amplifying a nucleic acid sequence comprising said intron B by polymerase chain reaction (PCR) to obtain a PCR product; (ii) digesting said PCR product with a restriction enzyme to obtain a restriction digest product; and (iii) determining a size of said restriction digest product. 4. The method of claim 3 wherein said restriction enzyme is ScrFI. 5. The method of claim 1, wherein said sample is a tissue or body fluid of said subject. 6. The method of claim 5, wherein said tissue or body fluid is neural tissue or fluid. 7. The method of claim 5 wherein said tissue or body fluid is selected from saliva, hair, blood, plasma, lymphocytes, cerebrospinal fluid, epithelia and fibroblasts. 8. The method of claim 1, wherein said control subject is a normal age-matched subject. 9. The method of claim 1, wherein the method is used to prognosticate a dementia and wherein the control sample was obtained from the subject at another time. 10. A method of diagnosing or prognosticating a dementia in a subject, said method comprising: (a) measuring a first level of HMGR activity in a sample obtained from said subject; and (b) comparing said first level to a second level which is an average HMGR activity measured in at least one corresponding control sample obtained from at least one control subject, whereby if said first level is significantly less than said second level then said subject suffers from a dementia; wherein said method is used to diagnose or prognosticate a dementia. 11. The method of claim 10, wherein said sample is a tissue or body fluid of said subject. 12. The method of claim 11, wherein said tissue or body fluid is neural tissue or fluid. 13. The method of claim 11 wherein said tissue or body fluid is selected from saliva, hair, blood, plasma, lymphocytes, cerebrospinal fluid, epithelia and fibroblasts. 14. The method of claim 10, wherein said control subject is a normal age-matched subject. 15. The method of claim 10, wherein the method is used to prognosticate a dementia and wherein the control sample was obtained from the subject at another time. 16. A method of diagnosing or prognosticating a dementia in a subject, said method comprising: (a) obtaining a sample from said subject, wherein said sample comprises ribonucleic acid encoded by an HMGR gene; and (b) determining whether said sample comprises at least one alteration relative to a corresponding control sample obtained from a control subject, wherein said alteration is selected from the group consisting of: (i) an increase in a level of a first ribonucleic acid encoded by an HMGR gene, wherein said first ribonucleic acid has a deletion of exon 13; (ii) an increase in a level of a second ribonucleic encoded by an HMGR gene, wherein said second ribonucleic acid has an insertion of intron M; and (iii) a decrease in a level of a third ribonucleic acid comprising a normal HMGR transcript; wherein the presence of said at least one alteration is used to diagnose or prognosticate a dementia. 17. The method of claim 16 wherein said alteration is determined using reverse transcriptase-polymerase chain reaction (RT-PCR). 18. The method of claim 16, wherein said sample is a tissue or body fluid of said subject. 19. The method of claim 18, wherein said tissue or body fluid is neural tissue or fluid. 20. The method of claim 18 wherein said tissue or body fluid is selected from saliva, hair, blood, plasma, lymphocytes, cerebrospinal fluid, epithelia and fibroblasts. 21. The method of claim 16, wherein said control subject is a normal age-matched subject. 22. The method of claim 16, wherein the method is used to prognosticate a dementia and wherein the control sample was obtained from the subject at another time. 23. A commercial package for the diagnosis and/or the prognostication of a dementia, said commercial package comprising at least one of: (a) means for detecting a polymorphism in an HMGR gene in a sample together with instructions for assessing said polymorphism relative to a corresponding control sample; (b) means for determining a level of HMGR activity in a sample together with instructions for comparing said level with an established standard or a control level measured in a corresponding control sample; and (c) means for determining the presence of at least one feature selected from the group consisting of (i) a first HMGR ribonucleic acid having a deletion of exon 13, (ii) a second HMGR ribonucleic acid having an insertion of intron M, (iii) a decrease in a level of a third ribonucleic acid comprising a normal HMGR transcript; and/or an increase in said first and/or second ribonucleic acid relative to said third ribonucleic acid, together with instructions for comparing said feature with a control feature in a corresponding control sample. 24. The method according to claim 1, wherein said dementia is an Alzheimer disease. 25. The commercial package of claim 23, wherein said dementia is an Alzheimer disease. 26. The method according to claim 10, wherein said dementia is an Alzheimer disease. 27. The method according to claim 16, wherein said dementia is an Alzheimer disease. |
<SOH> BACKGROUND OF THE INVENTION <EOH>An example of a dementia is Alzheimer disease (AD). AD is a progressive neurodegenerative disorder with clinical characteristics and pathological features. AD is etiologically heterogenous and can be produced by mutations on genes localized on chromosomes 1, 14 and 21. Major risk factors have been identified for the common form of AD (also refer to as sporadic AD). These include the apolipoprotein E4 allele (chromosome 19), butirylcholinesterase K (chromosome 3), alpha 2 -macroglobulin (chromosome 12), lipoprotein lipase (Baum et al., 1999) and two of the apoE receptors called LRP (Beffert et al., 1999a [chromosome 12]) and VLDL receptor (Okuizumi et al., 1995). The apolipoprotein E4 polymorphism was also shown to affect age of onset, rate of progression, cholinergic function and therapeutic response in AD. Several other genetic risk factors have been identified in sporadic AD but replication has proven difficult for those novel risk markers. To date, there is no known genetic mutation responsible for the common form of AD. These observations, combined with several independent genome scans, indicate that AD has a genetic etiology that includes several genetic loci, of which only a minority have been identified so far (Pericak-Vance et al., 1998; Kehoe et al. 1999). Alzheimer disease (AD) is considered today to be a multifactorial disease with a strong genetic component. It is generally agreed that the disease can be subdivided into two distinct categories: the [so-called] familial and sporadic forms of the disease. The familial form of AD accounts for roughly 10-15% of all cases worldwide, whereas the sporadic form of AD represents 85-90% of the remaining cases and is generally believed to be of late onset, occurring after 65 years of age. Familial Forms of Alzheimer Disease Molecular genetic studies have identified several different genetic loci which are believed to be linked to the presence of AD in the general population. The known genetic causes of AD, which include mutation in the amyloid precursor protein gene and two presenilin genes, are rare and account for about 5% of all cases worldwide. These rare mutations are transmitted as autosomal dominant traits in certain families from Europe, North America and Asia. Although a lot has been learned from the familial studies of the mutation in the amyloid protein precursor gene and the presenilin, the molecular mechanism(s) behind the sporadic form of AD is much more complex and requires a different approach. One polymorphism called apolipoprotein E4 on chromosome 19 has been linked to both the late onset familial form, as well as to the sporadic form of AD. The majority of patients referred to as sporadic cases probably arise as the result of several genetic anomalies, each making an independent contribution to the overall phenotype and pathophysiological process. It is suspected that at least one, and most probably several, additional mutations 1, remain to be identified since only 50% of all AD cases have been linked to specific genetic anomalies in case/control studies. The Amyloid Precursor Protein: The first gene ever identified in association with familial AD was the amyloid precursor protein (APP) (Chartier-Harlin et al., 1991). The APP gene encodes a transcript which, once translated, encodes a single trans-membrane spanning polypeptide of roughly 750 amino acids. Alternative splicing of exon 7 and exon 8 results in a polypeptide of 695 amino acids, which is expressed at very high concentration in the central nervous system. APP is known to undergo a series of proteolytic cleavages which result in the production of a small 40 to 42-amino acid long peptide referred to as the A beta peptide (Sisodia et al., 1990). The exact function of the amyloid precursor protein is currently unknown. Onset at around the age of 50 years is characteristic of familial AD pedigrees associated with mutation in the amyloid precursor protein gene: several mutations (including those at positions 665, 670, 673, 692, 693, 713, 716, 717) have been identified as mutations causing early to late onset familial AD. It has been proposed that these mutations in the APP gene cause an overproduction of the so-called neurotoxic form of beta amyloid referred as the 1-42/1-43 beta peptides. Polymerization of these fibres could then result in the development of senile plaque in the brain of AD patients with a concomitant impact on the brain integrity. Presenilin 1: Following the discovery that only a portion of the familial cases of AD could be explained by the presence of a mutation in the amyloid precursor protein gene, several independent investigators pursued the hunt for other candidate genes that might be involved in the remaining familial forms of the disease. The presenilin 1 gene localised on chromosome 14 (St-George-Hyslop et al., 1992) was then isolated using positional cloning strategy and more than 35 different mutations were identified by several independent groups as anomalies causing the familial form of the disease. The presenilin 1 gene is transcribed in several organs and in several cell types. There is a concentration of mutations located near or in the highly conserved transmembrane domain of presenilin 1 (Hutton et al., 1996). No deletions, nonsense mutation nor genomic rearrangements have yet been found in the sporadic (common) form of AD. Mutations in the presenilin 1 gene are associated with families where the age of onset is in the late 30s, early 40s. Presenilin 2: Following the cloning of the presenilin 1 gene on chromosome 14, a very similar sequence has been identified and subsequently localized on chromosome 1 (Levy-Lahad et al., 1995). This polypeptide, referred to as presenilin 2, has an open reading frame of about 448 amino acids with substantial amino acid sequence identity with the presenilin 1 protein. Presenilin 2 appears to be more ubiquitously expressed but less abundant than presenilin 1. It has been proposed that presenilins may be involved in the intracellular trafficking and/or transport of specific proteins inside the cells. Mutational analysis in familial cases of AD uncovered two different missense mutations in the presenilin 2 gene in families segregating for early onset AD. Sporadic (common) Alzheimer Disease: Polymorphic genetic markers and the risk of developing Alzheimer disease: The inheritance of common forms of AD appears considerably more complex than familial AD and probably reflects the co-action or interaction of several genes with environmental factors. One gene that is clearly implicated in this form of the disease is that encoding apolipoprotein E (apoE). The E4 allele of apoE, although neither necessary nor sufficient to cause AD, is strongly associated with increased risk, rate of progression and severity of the neuropathology. The effect of apoE4 appears additive such that heterozygotes and homozygotes are, three and eight times more likely, respectively, to be affected than controls. However, the variation at the apoE locus accounts for at most 50% of the genetic variation in liability (Pericak-Vance and Haines, 1995) to develop the disorder and there must be other genetic variants that account for remaining risk. A number of candidate gene association studies have been performed in sporadic AD since the identification of the apoE locus. Some positive findings have been claimed but none of these have been consistently confirmed. These include alpha 1 -antichymotrypsin (Morgan et al., 1997; Schwab et al., 1999), bleomycin hydrolase (Farrer et al., 1998), lipoprotein lipase (Baum et al., 1999) (Brandi et al., 1999) and LRP (Beffert et al., 1999a). These inconsistencies are likely due to a number of factors such as genetic heterogeneity, ethnicity, issues of statistical power, multiple testing and population stratification (Owen et al., 1997). Therefore, prior to applicants' work presented herein, most of the positive association studies have been essentially based on testing of genes whose candidature is suggested by existing understanding of the pathophysiology of AD. Apolipoprotein E and Cholesterol Homeostasis in Alzheimer Disease: Apolipoproteins are protein components of lipoprotein particles. The latter are macromolecular complexes that carry lipids such as cholesterol and phospholipids from one cell to another within a tissue or between organs. Some apolipoproteins regulate extracellular enzymatic reactions related to lipid homeostasis while others are ligands for cell surface receptors that mediate lipoprotein uptake into cells and their subsequent metabolism. ApoE is a component of several classes of plasma and cerebrospinal fluid lipoproteins. ApoE was shown to be synthesized and secreted by glial cells, predominantly astrocytes. Neurons appear to contribute very little to steady state levels of apoE in the brain. Several cell surface receptors for apoE are known to be expressed on many of the different cell types that constitute the brain parenchyma. These receptors are members of a single family and include the low-density lipoprotein (LDL) receptor, the very low-density (VLDL) receptor, the apoER2 receptor, the LDL receptor-related protein (LRP), and the megalin/gp330 receptor. The importance of apoE in lipid homeostasis in the brain is underscored by the fact that major plasma apolipoproteins such as apoB and apoAI are not synthesized in the central nervous system. Early data from animal lesion paradigms such as sciatic nerve crush and entorhinal cortex lesioning indicate that apoE plays a role in the coordinated storage and redistribution of cholesterol and phospholipids among cells within the remodeling area. FIG. 1 illustrates the role of apoE and its major receptors in the transport and recycling of cholesterol from dead or dying neurons to intact neurons undergoing synaptic remodeling and compensatory terminal outgrowth. It was shown that following neuronal cell loss and terminal differentiation in the CNS, large amounts of lipids-are released from degenerating axon membranes and myelin ( FIG. 1 , # 1 ). In response, astrocytes ( FIG. 1 , # 2 ) and macrophages synthesize apoE within the lesion to scavenge lipids from both cellular and myelin debris. During that critical phase, cholesterol synthesis [as monitored by the activity of the 3,3-hydroxy-methylglutaryl-CoA reductase (HMGR)] is progressively repressed in response to a massive increase in intracellular cholesterol concentration through receptor-mediated internalization. It was demonstrated in different cell culture systems that eukaryote cells obtain their cholesterol from two distinct sources: a) it is synthesized directly from acetyl-CoA through the so-called HMGR pathway or, b) it is imported through the apoE/apoB (LDL) receptor family via lipoprotein-complex internalization (for a review see Beffert et al., 1998b). These two different pathways are tightly coupled: i.e. a reduction of cholesterol internalization through the receptor pathway rapidly causes increases in HMGR activity (cholesterol synthesis), whereas inhibition of intracellular cholesterol synthesis induces expression of the LDL receptor and lipoprotein internalization. Much of the free cholesterol generated during synapse degradation is stored in astrocytes in the CNS and, in macrophages in the PNS where it is eventually reused during PNS regeneration and CNS reinnervation. Following binding of the apoE/lipoprotein complexes with neuronal LDL receptors, the apoE/Lipoprotein/LDL receptor complex is internalized, degraded and the cholesterol released inside neurons where it is used for membrane synthesis and synaptic remodelling ( FIG. 1 , # 3 and #4). The intra cellular rise in cholesterol causes a down-regulation of HMG-COA reductase activity and mRNA prevalence in granule cell neurons undergoing dendritic and synaptic remodeling ( FIG. 1 , # 7 ). In humans, three alleles (2, 3, and 4) at a single gene locus on the long arm of chromosome 19 code for the common isoforms of apoE, namely apoE2, apoE3, and apoE4. This allelic heterogeneity gives rise to a protein polymorphism at two positions: residues 112 and 158 on the mature protein. In 1993, the apoE 4 allele was found to be over-represented in groups of both familial and sporadic case's of late-onset AD. The 4 allele frequency was shown to be significantly higher (˜3-fold i.e. 40%-50%) in the Alzheimer population (Corder et al., 1993; Owen et al., 1997; Poirier et al., 1993a; Farrer et al., 1997′). Interestingly, a sharp decline in the prevalence of the 4 allele was observed in very old subjects (>85 years), suggesting the presence of a very late onset form of AD and consistent with the increased risk of coronary heart disease in apoE4 subjects. A meta-analysis of 40 studies representing nearly 30,000 apoE alleles concluded that the E4 allele represents a major risk factor for AD in all ethnic groups, across all ages between 40 and 90 years (Farrer et al., 1997). Interestingly, careful analysis of regenerative markers in the brain of AD subjects indicates that the E4 sub-population clearly show impaired synaptic plasticity and marked loss of regenerative capacity when compared to age-matched controls or apoE3/3 AD subjects, highlighting the crucial role played by cholesterol transport during compensatory remodelling in the CNS (Arendt et al., 1997). Apolipoprotein E, Cholesterol levels and the Amyloid Hypothesis of AD: While the abnormal processing of the APP into toxic forms of beta amyloid appears to underlie the pathophysiological process that characterizes chromosome 1, 14 and 21 familial cases, the role of apoE as a potent scavenger of beta amyloid in the brain is certainly consistent with this working hypothesis (Beffert and Poirier, 1998; Beffert et al., 1998b; Beffert et al., 1999b). For a while, it was generally believed that mutations in the apoE gene on chromosome 19 and in the APP gene on chromosome 21 represented independent biochemical pathways with similar outcomes; i.e. dementia of the Alzheimer type. However, recent evidence suggests a direct link between these two apparently separate metabolic pathways. ApoE4 allele dosage was shown to modulate the age of onset of AD in families with the amyloid precursor protein (APP) mutation (Farrer et al., 1997; Hardy, 1994). Strittmatter et al. (1993) and Wisniewski et al. (1993) demonstrated that purified, non-reconstituted, human apolipoprotein E binds avidly to beta amyloid fragments in vitro. Furthermore, apo E4 allele dose was shown to positively correlate with the density of beta A4 immunopositive plaques and neurofibrillary tangles in the cortex and hippocampus of AD subjects. Howland et al. (1998) reported decreased processing of APP in gene-targeted APP mice (humanized for beta amyloid and containing the Swedish familial AD mutation) in response to high dietary cholesterol as evidenced by concomitant decrease in secreted APP (alpha and beta), beta amyloid 1-40 and 1-42. The reduction in beta amyloid peptides (1-40 and 1-42) in the brain inversely correlated with increased concentration of brain apoE. In recent months, there has been a surprising convergence of the beta amyloid cascade hypothesis with the apoE/cholesterol metabolism. It has been shown that breeding of an apoE knockout mouse with an APP transgenic mouse showing amyloid plaques completely abolished amyloid deposition in the hybrid mice, without affecting the steady state levels of beta amyloid 1-40 and 1-42 in the brain (Bales et al, 1997). Expression of the human apoE3 or apoE4 gene in apoE knockout mice drastically reduces beta amyloid deposition in hybrid human apoE/human APP mice (Holtzman et al., 1999). Cholesterol Synthesis, HMGR activity and beta amyloid Production: Simons and colleagues found that blocking cholesterol synthesis with the HMGR inhibitor simvastatin in absence of external lipoproteins caused a marked inhibition of beta amyloid formation in primary neuronal cell cultures derived from the hippocampus (Simons et al., 1998). The 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase: Rate Limiting Step in Cholesterol Synthesis Mammalian cells, particularly astrocytes and neurons, cultured in vitro synthesize cholesterol at a rate which is inversely proportional to the cholesterol content in the growth medium. Cholesterol requirements of most mammalian cells are met by two separate but interrelated processes. One process is the endogenous synthesis of cholesterol. This synthesis pathway which involves over 20 reactions is regulated primarily by the activity of the 3-Hydroxy-3-MethylGlutaryl Coenzyme A Reductase (HMGR) which catalyzes the formation of mevalonate, the key precursor molecule in the synthesis of cholesterol. The other process involves the utilization of lipoprotein-derived cholesterol following internalization of the lipoprotein bound to its surface receptor (usually, an apoE-rich lipoprotein complex). Cholesterol homeostasis in brain cells is controlled by the perfect balance between cholesterol influx through the apoE receptors pathway and synthesis via the HMGR pathway, the rate limiting step in cholesterol biosynthesis. However, the brain differs significantly from peripheral organs where a multitude of apolipoproteins such as apoB, apoH, apoA1, A2, apoCI and apoCII are playing a pro-active-role in lipid transport and homeostasis. The brain is entirely devoid of apoB (apbE's main back-up system in the blood) and contains only trace amounts of the other apos described above. For some unknown reason, the brain is extremely dependent on apoE and its accessory proteins to deliver and/or produce cholesterol in the intact or injured brain cells. Under normal circumstances, cholesterol synthesis via the HMGR pathway is required only when lipoprotein internalization by apoE/apoE receptor pathway is insufficient to meet the cholesterol requirement of the cell (Brown et al., 1973; Rodwell et al., 2000). The endoplasmic reticulum-bound HMGR is regarded as the rate limiting step in the synthesis of cholesterol, a critical membrane lipid, precursor of steroid hormones (glucocorticoids and estrogen) and a signaling molecule involved in embryogenesis (Ness and Chambers, 2000). The other shorter form of HMGR localized in the peroxisomal compartment does not appear to play an important role in cholesterol homeostasis and is far more resistant to commonly used HMGR inhibitors such as simvastatin (Aboushadi et al., 2000). In cells grown in excess of cholesterol-rich lipoproteins, the HMGR activity is down regulated in favor of uptake via apoE receptors (Sato and Takano, 1995). A similar process was reported in the PNS and CNS during the acute phase of regeneration that ensue degradation of dead cells following experimental injury (Boyles et al., 1990) (Poirier et al., 1993b; Poirier, 1994). To maintain cellular cholesterol homeostasis, there exists a rather potent negative feed-back system on the HMGR activity and gene expression which results in decrease in synthesis of cholesterol in response to excess intracellular sterol internalization via the apoE receptors family (Ness and Chambers, 2000). This first and most important feedback regulation of the HMGR activity is through decrease in gene transcription (Reynolds et al., 1984; Chin et al., 1984). The factor that has been shown to regulate the expression of the reductase is the controlled degradation of the HMGR protein (Gardner and Hampton, 1999; Cronin et al., 2000). Lastly, there is evidence from hamster for a modulation in translation efficiency of mRNA for HMGR resulting in decreased or increased reductase protein and activity (Choi and Choi, 2000). |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides methods and commercial packages for the diagnosis and prognostication of a dementia based on an HMGR gene, its transcripts, and activity of HMGR protein. In an embodiment, such a dementia is an Alzheimer disease. Accordingly, the invention provides a method of diagnosing or prognosticating a dementia in a subject, said method comprising: (a) obtaining a sample from said subject, wherein said sample comprises nucleic acid comprising a 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene; and (b) determining whether said nucleic acid comprises a polymorphism relative to a corresponding control sample obtained from a control subject; wherein the presence of said polymorphism is used to diagnose or prognosticate a dementia. In an embodiment, the above-mentioned polymorphism is localized in intron B (also known as intron 2, eg in hamsters) of said HMGR gene. In an embodiment, the above-mentioned step (b) comprises: (i) amplifying a nucleic acid sequence comprising said intron B (also known as intron 2, eg in hamsters) by polymerase chain reaction (PCR) to obtain a PCR product; (ii) digesting said PCR product with a restriction enzyme to obtain a restriction digest product; and (iii) determining a size of said restriction digest product. In an embodiment, the above-mentioned restriction enzyme is ScrFI. The invention further provides a method of diagnosing or prognosticating a dementia in a subject, said method comprising: (a) measuring a first level of HMGR activity in a sample obtained from said subject; and (b) comparing said first level to a second level which is an average HMGR activity measured in at least one corresponding control sample obtained from at least one control subject, whereby if said first level is significantly less than said second level then said subject suffers from a dementia; wherein said method is used to diagnose or prognosticate a dementia. The invention further provides a method of diagnosing or prognosticating a dementia in a subject, said method comprising: (a) obtaining a sample from said subject, wherein said sample comprises ribonucleic acid encoded by an HMGR gene; and (b) determining whether said sample comprises at least one alteration relative to a corresponding control sample obtained from a control subject, wherein said alteration is selected from the group consisting of: (i) an increase in a level of a first ribonucleic acid encoded by an HMGR gene, wherein said first ribonucleic acid has a deletion of exon 13; (ii) an increase in a level of a second ribonucleic encoded by an HMGR gene, wherein said second ribonucleic acid has an insertion of intron M; and (iii) a decrease in a level of a third ribonucleic acid comprising a normal HMGR transcript; wherein the presence of said at least one alteration is used to diagnose or prognosticate a dementia. In an embodiment, the above-mentioned alteration is determined using reverse transcriptase-polymerase chain reaction (RT-PCR). The invention further provides a commercial package for the diagnosis and/or the prognostication of a dementia, said commercial package comprising at least one of: (a) means for detecting a polymorphism in an HMGR gene in a sample together with instructions for assessing said polymorphism relative to a corresponding control sample; (b) means for determining a level of HMGR activity in a sample together with instructions for comparing said level with an established standard or a control level measured in at least one corresponding control sample; and (c) means for determining the presence of at least one feature selected from the group consisting of (i) a first HMGR ribonucleic acid having a deletion of exon 13, (ii) a second HMGR ribonucleic acid having an insertion of intron M, (iii) a decrease in a level of a third ribonucleic acid comprising a normal HMGR transcript; and/or an increase in said first and/or second ribonucleic acid relative to said third ribonucleic acid, together with instructions for comparing said feature with a corresponding control feature in a corresponding control sample. In an embodiment, the above-mentioned sample is a tissue or body fluid of said subject. In an embodiment, the above-mentioned tissue or body fluid is neural tissue or fluid. In an embodiment, the above-mentioned tissue or body fluid is selected from saliva, hair, blood, plasma, lymphocytes, cerebrospinal fluid, epithelia and fibroblasts. In an embodiment, the above-mentioned control subject is a normal age-matched subject. In an embodiment, the above-mentioned method is used to prognosticate a dementia and wherein the control sample was obtained from the subject at another time. In an embodiment, the above-mentioned dementia is an Alzheimer disease. |
Fluorescent multiplex hpv pcr assays using multiple fluorophores |
The present invention relates a fluorescent multiplex PCR assay for detecting the presence of an HPV subtype in a sample using multiple fluorophores to simultaneously detect a plurality of HPV genes of the same HPV subtype. The present invention also relates to primer pairs and probes specific to HPV subtypes for use in the methods of the present invention. |
1. A method for detecting the presence of a human papillomavirus (HPV) subtype in a nucleic acid-containing sample comprising: (a) amplifying the nucleic acid in the presence of a nucleic acid polymerase and a plurality of oligonucleotide sets; wherein each oligonucleotide set consists of (i) a forward discriminatory PCR primer hybridizing to a first location of an HPV subtype, (ii) a reverse discriminatory PCR primer hybridizing to a second location of the HPV subtype downstream of the first location, (iii) a fluorescent probe labeled with a quencher molecule and a fluorophore which emits energy at a unique emission maxima; said probe hybridizing to a location of the HPV subtype between the first and the second locations; wherein each oligonucleotide set specifically hybridizes to a different HPV amplicon derived from the same HPV subtype; (b) allowing said nucleic acid polymerase to digest each fluorescent probe during amplification to dissociate said fluorophore from said quencher molecule; (c) detecting a change of fluorescence upon dissociation of the fluorophore and the quencher molecule, the change of fluorescence corresponding to the occurrence of nucleic acid amplification; and (d) determining that the sample is positive for the HPV subtype if a change of fluorescence is detected in at least two emission maxima. 2. The method of claim 1 wherein the number of oligonucleotide sets is odd and wherein the sample is positive for the HPV subtype if a change of fluorescence is detected in a majority of emission maxima. 3. The method of claim 2, wherein the number of oligonucleotide sets is three. 4. The method of claim 3, wherein the oligonucleotide sets specifically hybridize to the E6, E7 and L1 genes. 5. The method of claim 1, wherein the quencher is non-fluorescent. 6. The method of claim 3, wherein the fluorophores are FAM, JOE and TET and the quencher is BHQ1. 7. The method of claim 1, wherein the HPV subtype is selected from the group consisting of: HPV6, HPV11, HPV16 and HPV18. 8. The method of claim 3, wherein the HPV subtype is selected from the group consisting of: HPV6, HPV11, HPV16 and HPV18. 9. An oligonucleotide probe comprising a sequence of nucleotides selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO: 30, SEQ ID NO: 33 and SEQ ID NO:36. 10. The oligonucleotide probe of claim 9 further comprising a fluorophore and a quencher molecule. 11. The oligonucleotide probe of claim 10, wherein the fluorophore is attached to a 5′ terminal nucleotide of the sequence of nucleotides and the quencher is attached to a 3′ terminal nucleotide of the sequence of nucleotides. 12. The oligonucleotide probe of claim 10, wherein the fluorophore is selected from the group consisting of: FAM, JOE and TET. 13. The oligonucleotide probe of claim 10, wherein the quencher molecule is non-fluorescent. 14. The oligonucleotide probe of claim 13, wherein the quencher molecule is BHQ1. 15. (canceled) 16. A method for detecting the presence of HPV6 in a nucleic acid-containing sample comprising: (a) amplifying the nucleic acid in the presence of a nucleic acid polymerase and three oligonucleotide sets; the first oligonucleotide set consisting of a forward discriminatory PCR primer as set forth in SEQ ID NO:1, a reverse discriminatory PCR primer as set forth in SEQ ID NO:2, and a probe as set forth in SEQ ID NO:3, said probe labeled with BHQ1 on the 3′ end and a fluorophore on the 5′ end, said fluorophore selected from the group consisting of FAM, JOE and TET; the second oligonucleotide set consisting of a forward discriminatory PCR primer as set forth in SEQ ID NO:4, a reverse discriminatory PCR primer as set forth in SEQ ID NO:5, and a probe as set forth in SEQ ID NO:6, said probe labeled with BHQ1 on the 3′ end and a fluorophore on the 5′ end, said fluorophore selected from the group consisting of FAM, JOE and TET; the third oligonucleotide set consisting of a forward discriminatory PCR primer as set forth in SEQ ID NO:7, a reverse discriminatory PCR primer as set forth in SEQ ID NO:8, and a probe as set forth in SEQ ID NO:9, said probe labeled with BHQ1 on the 3′ end and a fluorophore on the 5′ end, said fluorophore selected from the group consisting of FAM, JOE and TET; (b) allowing said nucleic acid polymerase to digest each probe during amplification to dissociate said fluorophore from said quencher molecule; (c) detecting a change of fluorescence upon dissociation of the fluorophore and the quencher, the change of fluorescence corresponding to the occurrence of nucleic acid amplification; and (d) determining that the sample is positive for the HPV6 subtype if a change of fluorescence is detected with at least two probes. 17. The method of claim 16 wherein the fluorophore of the first oligonucleotide set is FAM, the fluorophore of the second oligonucleotide set is JOE and the fluorophore of the third oligonucleotide set is TET. 18. A method for detecting the presence of HPV11 in a nucleic acid-containing sample comprising: (a) amplifying the nucleic acid in the presence of a nucleic acid polymerase and three oligonucleotide sets; the first oligonucleotide set consisting of a forward discriminatory PCR primer as set forth in SEQ ID NO:10, a reverse discriminatory PCR primer as set forth in SEQ ID NO:11, and a probe as set forth in SEQ ID NO:12, said probe labeled with BHQ1 on the 3′ end and a fluorophore on the 5′ end, said fluorophore selected from the group consisting of FAM, JOE and TET; the second oligonucleotide set consisting of a forward discriminatory PCR primer as set forth in SEQ ID NO:13, a reverse discriminatory PCR primer as set forth in SEQ ID NO:14, and a probe as set forth in SEQ ID NO:15, said probe labeled with BHQ1 on the 3′ end and a fluorophore on the 5′ end, said fluorophore selected from the group consisting of FAM, JOE and TET; the third oligonucleotide set consisting of a forward discriminatory PCR primer as set forth in SEQ ID NO:16, a reverse discriminatory PCR primer as set forth in SEQ ID NO:17, and a probe as set forth in SEQ ID NO:18, said probe labeled with BHQ1 on the 3′ end and a fluorophore on the 5′ end, said fluorophore selected from the group consisting of FAM, JOE and TET; (b) allowing said nucleic acid polymerase to digest each probe during amplification to dissociate said fluorophore from said quencher molecule; (c) detecting a change of fluorescence upon dissociation of the fluorophore and the quencher, the change of fluorescence corresponding to the occurrence of nucleic acid amplification; and (d) determining that the sample is positive for the HPV11 subtype if a change of fluorescence is detected with at least two probes. 19. The method of claim 18 wherein the fluorophore of the first oligonucleotide set is FAM, the fluorophore of the second oligonucleotide set is JOE and the fluorophore of the third oligonucleotide set is TET. 20. A method for detecting the presence of HPV16 in a nucleic acid-containing sample comprising: (a) amplifying the nucleic acid in the presence of a nucleic acid polymerase and three oligonucleotide sets; the first oligonucleotide set consisting of a forward discriminatory PCR primer as set forth in SEQ ID NO:19, a reverse discriminatory PCR primer as set forth in SEQ ID NO:20, and a probe as set forth in SEQ ID NO:21, said probe labeled with BHQ1 on the 3′ end and a fluorophore on the 5′ end, said fluorophore selected from the group consisting of FAM, JOE and TET; the second oligonucleotide set consisting of a forward discriminatory PCR primer as set forth in SEQ ID NO:22, a reverse discriminatory PCR primer as set forth in SEQ ID NO:23, and a probe as set forth in SEQ ID NO:24, said probe labeled with BHQ1 on the 3′ end and a fluorophore on the 5′ end, said fluorophore selected from the group consisting of FAM, JOE and TET; the third oligonucleotide set consisting of a forward discriminatory PCR primer as set forth in SEQ ID NO:25, a reverse discriminatory PCR primer as set forth in SEQ ID NO:26 and a probe as set forth in SEQ ID NO:27, said probe labeled with BHQ1 on the 3′ end and a fluorophore on the 5′ end, said fluorophore selected from the group consisting of FAM, JOE and TET; (b) allowing said nucleic acid polymerase to digest each probe during amplification to dissociate said fluorophore from said quencher molecule; (c) detecting a change of fluorescence upon dissociation of the fluorophore and the quencher, the change of fluorescence corresponding to the occurrence of nucleic acid amplification; and (d) determining that the sample is positive for the HPV16 subtype if a change of fluorescence is detected with at least two probes. 21. The method of claim 20, wherein the fluorophore of the first oligonucleotide set is FAM, the fluorophore of the second oligonucleotide set is JOE and the fluorophore of the third oligonucleotide set TET. 22. A method for detecting the presence of HPV18 in a nucleic acid-containing sample comprising: (a) amplifying the nucleic acid in the presence of a nucleic acid polymerase and three oligonucleotide sets; the first oligonucleotide set consisting of a forward discriminatory PCR primer as set forth in SEQ ID NO:28, a reverse discriminatory PCR primer as set forth in SEQ ID NO:29, and a probe as set forth in SEQ ID NO:30, said probe labeled with BHQ1 on the 3′ end and a fluorophore on the 5′ end, said fluorophore selected from the group consisting of FAM, JOE and TET the second oligonucleotide set consisting of a forward discriminatory PCR primer as set forth in SEQ ID NO:31, a reverse discriminatory PCR primer as set forth in SEQ ID NO:32, and a probe as set forth in SEQ ID NO:33, said probe labeled with BHQ1 on the 3′ end and a fluorophore on the 5′ end, said fluorophore selected from the group consisting of FAM, JOE and TET; the third oligonucleotide set consisting of a forward discriminatory PCR primer as set forth in SEQ ID NO:34, a reverse discriminatory PCR primer as set forth in SEQ ID NO:35, and a probe as set forth in SEQ ID NO:36, said probe labeled with BHQ1 on the 3′ end and a fluorophore on the 5′ end, said fluorophore selected from the group consisting of FAM, JOE and TET; (b) allowing said nucleic acid polymerase to digest each probe during amplification to dissociate said fluorophore from said quencher molecule; (c) detecting a change of fluorescence upon dissociation of the fluorophore and the quencher, the change of fluorescence corresponding to the occurrence of nucleic acid amplification; and (d) determining that the sample is positive for the HPV18 subtype if a change of fluorescence is detected with at least two probes. 23. The method of claim 22, wherein the fluorophore of the first oligonucleotide set is FAM, the fluorophore of the second oligonucleotide set is JOE and the fluorophore of the third oligonucleotide set is TET. |
<SOH> BACKGROUND OF THE INVENTION <EOH>There are more than 80 types of human papillomavirus (HPV) that cause a wide variety of biological phenotypes, from benign proliferative warts to malignant carcinomas (for review, see McMurray et al., Int. J. Exp. Pathol. 82(1): 15-33 (2001)). HPV6 and HPV11 are the types most commonly associated with benign warts, whereas HPV16 and HPV18 are the high-risk types most frequently associated with malignant lesions. Determination of the specific type of HPV in a clinical sample is, therefore, critical for predicting risk of developing HPV-associated disease. Several nucleic acid-based methods have been utilized to identify and quantify specific HPV types in clinical samples, such as detection of viral nucleic acid by in situ hybridization, Southern blot analysis, hybrid capture or polymerase chain reaction (PCR). PCR-based methods often involve amplification of a single specific HPV target sequence followed by blotting the resulting amplicon to a membrane and probing with a radioactively labeled oligonucleotide probe. Other methods exploit the high homology between specific HPV genes of different subtypes through the use of commercially available consensus primers capable of PCR amplifying numerous HPV subtypes present in a sample. The presence of a specific HPV subtype is then identified using a subtype-specific oligonucleotide probe. See, e.g., Kleter et al., Journal of Clinical Microbiology 37(8): 2508-2517 (1999); Gravitt et al., Journal of Clinical Microbiology 38(1): 357-361 (2000). Similarly, assays that utilize degenerate PCR primers take advantage of the homology between HPV subtypes, allowing detection of a greater number of HPV types than methods utilizing specific primer sets. See, e.g. Harwood et al., Journal of Clinical Microbiology 37(11): 3545-3555 (1999). Such assays also require additional experimentation to identify specific HPV subtypes. The PCR methods described above can be associated with several problems. For example, differences in reaction efficiencies among HPV subtypes can result in disproportionate amplification of some subtypes relative to others. Additionally, the equilibrium for amplification will be driven towards those subtypes that exist at higher copy numbers in a sample, which will consume the PCR reaction components, thus making amplification of the minor HPV subtypes less likely. Also described in the art is a 5′ exonuclease fluorogenic PCR-based assay (Taq-Man PCR) which allows detection of PCR products in real-time and eliminates the need for radioactivity. See, e.g., U.S. Pat. No. 5,538,848; Holland et al, Proc. Natl. Acad. Sci. USA 88: 7276-7280 (1991). This method utilizes a labeled probe, comprising a fluorescent reporter (fluorophore) and a quencher, that hybridizes to the target DNA between the PCR primers. Excitation of the fluorophore results in the release of a fluorescent signal by the fluorophore which is quenched by the quencher. Amplicons can be detected by the 5′-3′ exonuclease activity of the TAQ DNA polymerase, which degrades double-stranded DNA encountered during extension of the PCR primer, thus releasing the fluorophore from the probe. Thereafter, the fluorescent signal is no longer quenched and accumulation of the fluorescent signal, which is directly correlated with the amount of target DNA, can be detected in real-time with an automated fluorometer. Taq-Man PCR assays have been adapted for HPV subtype detection. Swan et al. ( Journal of Clinical Microbiology 35(4): 886-891 (1997)) disclose a fluorogenic probe assay that utilizes type-specific HPV primers that amplify a portion of the L1 gene in conjunction with type-specific probes. The Swan et al. assay measures fluorescent signal at the end of a fixed number of PCR cycles (endpoint reading) and not in real-time. Josefsson et al. ( Journal of Clinical Microbiology 37(3): 490-96 (1999)) report a Taq-Man assay that targets a highly conserved portion of the E1 gene in conjunction with type-specific probes labeled with different fluorescent dyes. A number of HPV types were amplified by utilizing a mixture of specific and degenerate primers. Josefsson et al. utilized up to three type-specific probes per assay, which were designed to detect a portion of the E1 gene from different HPV subtypes. Unlike the Swan et al. assay, Josefsson et al. measured the accumulation of fluorescence in real-time. Tucker et al. ( Molecular Diagnosis 6(1): 3947 (2001)) describe an assay that targets a conserved region spanning the E6/E7 junction. Like the Josefsson assay, Tucker et al. employed real-time detection and type-specific fluorescent probes. Tucker et al. also utilized multiplex PCR to simultaneously detect HPV target sequences and either the actin or globin cellular loci in the same reaction tube. The methods described above typically involve testing for the presence of a single viral locus in a DNA sample such as the L1 locus. A disadvantage of single-locus assays is that the high degree of homology among specific HPV genes from one HPV type to another leads to an excessive occurrence of false positive results. This level of homology makes it difficult to design a PCR assay that is specific for a single HPV type. It is therefore necessary to confirm positive results by testing for the presence of several loci of a single HPV-type. The further experimentation required to verify positive results is cumbersome and time-consuming. Establishment of the HPV status of a clinical sample for four different HPV types typically consumes 26-30 man-hours. Single-locus assays may also lead to false negative results. It is well established that the relationship between the HPV genome and chromosomal host DNA may change during the multistage tumorigenic process (For review, see McMurray et al., Int. J. Exp. Path. 82: 15-33 (2001)). Premalignant lesions are often associated with episomal forms of HPV DNA while later-stage tumors typically have integrated HPV sequences. As a result of the integration correlated with advanced stages of disease progression, the open reading frame of specific HPV genes, such as the L1 gene, may become disrupted. Such disruption of HPV gene sequences may lead to false negative results in assays that target the disrupted sequence. Despite the development of the HPV assays described above, it would be advantageous to develop an assay that is highly sensitive and reproducible, and that requires reduced man-hours compared to methods disclosed in the art. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to a fluorescent multiplex PCR assay for detecting the presence of an HPV subtype in a sample which uses multiple fluorophores to simultaneously detect a plurality of HPV loci of the same HPV subtype. More specifically, the present invention relates to a method for detecting the presence of a human papillomavirus (HPV) subtype in a nucleic acid-containing sample comprising: amplifying the nucleic acid in the presence of a nucleic acid polymerase and a plurality of oligonucleotide sets to produce a plurality of PCR amplicons; wherein each oligonucleotide set consists of (a) a forward discriminatory PCR primer hybridizing to a first location of an HPV subtype, (b) a reverse discriminatory PCR primer hybridizing to a second location of the HPV subtype downstream of the first location, and (c) a fluorescent probe labeled with a quencher molecule and a fluorophore which emits energy at a unique emission maxima; said probe hybridizing to a location of the HPV subtype between the first and the second locations; wherein each oligonucleotide set specifically hybridizes to a different HPV amplicon derived from the same HPV subtype; allowing said nucleic acid polymerase to digest each fluorescent probe during amplification to dissociate said fluorophore from said quencher molecule; detecting a change of fluorescence upon dissociation of the fluorophore and the quencher, the change of fluorescence corresponding to the occurrence of nucleic acid amplification; and determining that the sample is positive for the HPV subtype if a change of fluorescence is detected in at least two emission maxima. In a preferred embodiment of this invention, each oligonucleotide set of the plurality of oligonucleotide sets is specific to a single gene of the HPV subtype to be detected. In other words, each oligonucleotide set of the method of the present invention hybridizes to nucleotide sequences derived from a single HPV gene of the same subtype. For example, the oligonucleotide primers and probe of a first oligonucleotide set hybridize to the E6 gene, the oligonucleotide primers and probe of a second oligonucleotide set hybridize to the E7 gene and the oligonucleotide primers and probe of a third oligonucleotide set hybridize to the L1 gene. As a result, a plurality of PCR amplicons is created wherein each PCR amplicon is specific to a single HPV gene of the HPV subtype to be detected. In an alternative embodiment of this invention, the forward discriminatory PCR primer and the reverse discriminatory PCR primer of at least one oligonucleotide set are specific to a different gene of the same HPV subtype. For example, a forward discriminatory primer hybridizes to the E6 gene and a reverse discriminatory primer hybridizes to the E7 gene. As a result, at least one PCR amplicon comprises a sequence of nucleotides derived from more than one gene. The oligonucleotide probe specific to said amplicon may hybridize, for example, to a sequence of nucleotides derived from the E6 gene, a sequence of nucleotides derived from the E7 gene, or a sequence of nucleotides that crosses the E6/E7 boundary. In a preferred embodiment of this invention, the HPV subtype is selected from the group consisting of: HPV6, HPV11, HPV16 and HPV18. In a further preferred embodiment of the method of the present invention, the number of oligonucleotide sets is three and the oligonucleotide sets specifically hybridize to the E6, E7 and L1 genes of HPV. A sample is positive for the HPV subtype being tested if two or three of the E6, E7 or L1 genes are detected. Another embodiment of this invention relates to an oligonucleotide probe comprising a sequence of nucleotides specific to a single HPV type. Said oligonucleotide probe can bind to specific HPV amplicons resulting from PCR amplification of viral DNA using specific oligonucleotide primers. In a further embodiment of this invention, said oligonucleotide probe comprises a sequence of nucleotides selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO: 30, SEQ ID NO: 33 and SEQ ID NO:36. The present invention also relates to said oligonucleotide probes further comprising a fluorophore and a quencher molecule. In a preferred embodiment of this invention, the fluorophore is selected from the group consisting of: FAM, JOE and TET and the quencher is non-fluorescent. In an especially preferred embodiment of this invention, the quencher is BHQ1. The present invention further relates to a primer pair for the PCR amplification of HPV nucleic acid, wherein both the forward and reverse PCR primers are discriminatory. In a preferred embodiment of the invention, the nucleotide sequences of the primer pair are selected from the group consisting of: SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:29, SEQ ID NO:31 and SEQ ID NO:32, and SEQ ID NO:34 and SEQ ID NO:35. As used herein, the term “oligonucleotide” refers to linear oligomers of natural or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, and the like, capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type base pairing. For purposes of this invention, the term oligonucleotide includes both oligonucleotide probes and oligonucleotide primers. As used herein, the term “primer” refers to an oligonucleotide that is capable of acting as a point of initiation of synthesis along a complementary strand when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is catalyzed. Such conditions include the presence of four different deoxyribonucleoside triphosphates and a polymerization-inducing agent such as DNA polymerase or reverse transcriptase, in a suitable buffer (“buffer” includes components which are cofactors, or which affect ionic strength, pH, etc.), and at a suitable temperature. As employed herein, an oligonucleotide primer can be naturally occurring, as in a purified restriction digest, or be produced synthetically. The primer is preferably single-stranded for maximum efficiency in amplification. As used herein, “primer pair” refers to two primers, a forward primer and a reverse primer, that are capable of participating in PCR amplification of a segment of nucleic acid in the presence of a nucleic acid polymerase to produce a PCR amplicon. The primers that comprise a primer pair can be specific to the same HPV gene, resulting in an amplicon that consists of a sequence of nucleotides derived from a single HPV gene. Alternatively, the primers that comprise a primer pair can be specific to different HPV genes that reside within close proximity to each other within the HPV genome, thereby producing amplicons that consist of a sequence of nucleotides derived from more than one gene. As used herein, “unique,” in reference to the fluorophores of the present invention, means that each fluorophore emits energy at a differing emission maxima relative to all other fluorophores used in the particular assay. The use of fluorophores with unique emission maxima allows the simultaneous detection of the fluorescent energy emitted by each of the plurality of fluorophores used in the particular assay. As used herein, the term “discriminatory,” used in reference to the oligonucleotide primers and probes of the present invention, means that said primers and probes are specific to a single HPV subtype. It includes HPV primers and probes specific to a single HPV subtype, but that share some homology with other HPV subtypes. “Discriminatory” primers and probes of the present invention include those oligonucleotides that lack 3′ homology with other HPV subtypes in at least one nucleotide or more. Such a residue that is unique for the specific HPV subtype at the specific position and acts to discriminate the HPV subtype from the others in the alignment referred to as a “discriminatory base”. The term “discriminatory,” in reference to oligonucleotides, does not include primers and probes that are specific to more than one HPV subtype, i.e. those that share full homology with greater than one HPV subtype. As used herein, “amplicon” refers to a specific product of a PCR reaction, which is produced by PCR amplification of a sample comprising nucleic acid in the presence of a nucleic acid polymerase and a specific primer pair. An amplicon can consist of a nucleotide sequence derived from a single gene of a single HPV subtype or an amplicon can consist of a nucleotide sequence derived from more than one gene of a single HPV subtype. As used herein, “oligonucleotide set” refers to a grouping of a pair of oligonucleotide primers and an oligonucleotide probe that hybridize to a specific nucleotide sequence of a single HPV subtype. Said oligonucleotide set consists of: (a) a forward discriminatory primer that hybridizes to a first location of an HPV subtype; (b) a reverse discriminatory primer that hybridizes to a second location of the HPV subtype downstream of the first location and (c) a fluorescent probe labeled with a fluorophore and a quencher, which hybridizes to a location of the HPV subtype between the primers. In other words, an oligonucleotide set consists of a set of specific PCR primers capable of initiating synthesis of an amplicon specific to a single HPV subtype, and a fluorescent probe which hybridizes to the amplicon. As used herein, “plurality” means two or more. As used herein, “specifically hybridizes,” in reference to oligonucleotide sets, oligonucleotide primers or oligonucleotide probes, means that said oligonucleotide sets, primers or probes hybridize to a single HPV subtype. As used herein, “gene” means a segment of nucleic acid involved in producing a polypeptide chain. It includes both translated sequences (coding region) and 5′ and 3′ untranslated sequences (non-coding regions) as well as intervening sequences (introns) between individual coding segments (exons). For purposes of this invention, the HPV genome has nine genes: L1, L2, and E1-E7. As used herein, “locus” refers to the position on a chromosome at which the gene for a trait resides. The term locus includes any one of the alleles of a specific gene. It also includes homologous genes from different HPV subtypes. For example, PCR assays that detect the L1 gene in HPV16 and HPV6 are single-locus assays, despite the detection of sequences from different HPV subtypes. Contrarily, for example, assays that detect the L1 gene and the E1 gene of a single HPV type are multiple locus assays, even though a single HPV subtype is detected. As used herein, “HPV” means human papillomavirus. “HPV” is a general term used to refer to any subtype of HPV, whether currently known or subsequently described. As used herein, “fluorophore” refers to a fluorescent reporter molecule which, upon excitation with a laser, tungsten, mercury or xenon lamp, or a light emitting diode, releases energy in the form of light with a defined spectrum. Through the process of fluorescence resonance energy transfer (FRET), the light emitted from the fluorophore can excite a second molecule whose excitation spectrum overlaps the emission spectrum of the fluorophore. The transfer of emission energy of the fluorophore to another molecule quenches the emission of the fluorophore. The second molecule is known as a quencher molecule. The term “fluorophore” is used interchangeably herein with the term “fluorescent reporter”. As used herein “quencher” or “quencher molecule” refers to a molecule that, when linked to a fluorescent probe comprising a fluorophore, is capable of accepting the energy emitted by the fluorophore, thereby quenching the emission of the fluorophore. A quencher can be fluorescent, which releases the accepted energy as light, or non-fluorescent, which releases the accepted energy as heat, and can be attached at any location along the length of the probe. As used herein “dark quencher” refers to a non-fluorescent quencher. As used herein, “probe” refers to an oligonucleotide that is capable of forming a duplex structure with a sequence in a target nucleic acid, due to complementarity of at least one sequence of the probe with a sequence in the target region, or region to be detected. The term “probe” includes an oligonucleotide as described above, with or without a fluorophore and a quencher molecule attached. The term “fluorescent probe” refers to a probe comprising a fluorophore and a quencher molecule. As used herein, “FAM” refers to the fluorophore 6-carboxy-fluorescein. As used herein “JOE” refers to the fluorophore 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein. As used herein, “TET” refers to the fluorophore 5-tetrachloro-fluorescein. |
Catalysis of the cis/trans-isomerisation of secondary amide peptide compounds |
The present invention is based on the finding that the cis/trans isomerisation of secondary amide peptide bonds in oligo- and polypeptides can be catalytically promoted. This catalysis is effected by enzymes which are hereinafter called “secondary amide peptide bond cis/trans isomerases (APIases). It can be assumed that the APIase activity plays a central role in a number of pathophysiological processes. Thus, the invention relates to pharmaceutical compositions comprising substances which inhibit APIase activity. |
1. The use of a compound as inhibitor of a secondary amide peptide bond-specific cis/trans isomerase (APIase) which assumes the structure and conformation of a peptide motif R2—R3 if it is bound as a substrate to the active centre of this isomerase, wherein R2 comprises all natural amino acids and R3 comprises the amino acids methionine, alanine, serine, glutamic acid, leucine, lysine, isoleucine and/or glycine or their mimetics. 2. The use according to claim 1, wherein the residues flanking the peptide motif are characterised on the one side by hydrophobic properties and on the other side by hydrophobic or positively charged residues, wherein these flanking residues are in contact with the active centre of the isomerase. 3. The use according to claim 1, wherein the compound is a peptide, an oligopeptide or a peptide mimetic. 4. The use according to claim 3 with an inhibition constant of 100 micro molar or less. 5. The use according to claim 2, wherein the peptide, oligopeptide or peptide mimetic can be linear or cyclic. 6. The use according to claim 1, wherein the inhibitor represents a peptide and/or a peptide mimetic with the basic structure Xa—R2—R3Xb, wherein X comprises any L-amino or D-amino acid, R2 comprises any L-amino acid or mimetics thereof and R3 comprises the following amino acids: methionine, alanine, serine, glutamic acid, leucine, lysine, isoleucine and glycine or mimetics thereof and wherein a and b represent the number of flanking amino acids and are selected so that, while maintaining the structure R2—R3, the length of the peptide is larger than 2 amino acids or the corresponding amino acid mimetics and shorter than 10 amino acids or the corresponding amino acid mimetics. 7. The use according to claim 6, wherein the compound has a molecular weight of 200 to a maximum of 2,000. 8. The use according to claim 6, wherein single amino acids or mimetics thereof have a molecular weight of not less than 75 and of not more than 500 and are combined in such a way that either only each of the terminal residues Xa, Xb individually or both of them or at least one of the neighbouring R2-R3 amino acids or amino acid mimetics or several of the neighbouring R2-R3 amino acids or amino acid mimetics have a hydrophilic functional group either formed by —OH, —SH, —NH, or NH2 or by acid groups such as phosphatyl, sulfatyl or carboxyl. 9. A peptide or peptide mimetic with the general basic structure Y—R2—R3 wherein Y is a saturated or unsaturated, linear or branched chain fatty acid which is linked to the amino acid or the amino acid mimetic R2 by a C(O)NH bond and wherein the amino acid or the amino acid mimetic R3, via its amino group, is linked to R2 by a C(O)NH bond, wherein R2 and R3 have the meanings as indicated above. 10. The compounds according to claim 6, wherein the peptide mimetic R3 consists of at least 5 and not more than 29 atoms and has to be able to form a chemical bond with R2 which corresponds to the structure of a peptide bond, wherein the basic structure of R3 is described as H2N—R-Z, wherein Z represents a functional group selected from nitro, sulfoxy, phospho, amino, carboxy, sulfhydryl, wherein R represents a linear or branched hydrocarbon chain, whose length is a maximum of 14 carbon atoms and a minimum of 1 carbon atom, wherein the distance between H2N—-Z via R is a maximum of 4 carbon atoms and a minimum of 1 carbon atom, wherein R itself can optionally have one or up to three hydroxyl, carboxylic acid, carboxylic acid ester, amide, aldehyde, ether, iminoether, hydrazide, izidoether, thiol or sulfoxime groups. 11. The compound according to claim 9, wherein the fatty acid has 8 to 24 carbon atoms. 12. The compound according to claim 9, wherein the fatty acid has 14 to 16 carbon atoms. 13. The compound according to claim 9, wherein the fatty acid is mono-hydroxylated. 14. The compound according to claim 9, wherein the fatty acid is poly-hydroxylated. 15. The embodiment according to claim 6, wherein the amino acid mimetics R2 and R3 are the same or different and have up to 34 carbon atoms and a linear structure, which can optionally be branched, a C5- to C34-carbycyclic structure or a heterocyclic structure, containing S, N and O as hetero atoms and having ring sizes of 5 to 8, wherein these structures can be completely saturated or can have mono- or poly-unsaturated carbon moieties. 16. The embodiment according to claim 15, wherein R2 and R3 include benzoic or non-benzoic aromatic compounds which can optionally be mono- or poly-substituted with functional groups selected from nitro, sulfoxy, phospho, amino, carboxy, sulflhydryl groups. 17. The embodiment according to claim 15, wherein R2 and R3 are mono- or poly-halogenated, wherein halogen is selected from fluoro, chloro, bromine or iodine. 18. The compound according to claim 9, wherein Y is a saturated fatty acid. 19. The compound according to claim 9, wherein Y is an unsaturated fatty acid. 20. The compound according to claim 18, wherein the saturated fatty acid is selected from: propionic acid, butyric acid, iso butyric acid, valeric acid, iso-valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, iso tuberculostearic acid, arachic acid, behenic acid, lignoceric acid, cerotic acid and melissic acid. 21. The compound according to claim 19, wherein the unsaturated fatty acid is selected from: acrylic acid, crotonic acid, palmitoleic acid, oleic acid, erucic acid, sorbic acid, elaeosteric acid, arachidonic acid, clupanodonic acid, docosahexaenoic acid, elaidic acid, linolic acid and linolenic acid. 22. The embodiment according to claim 6, wherein R2 and R3 together represent a peptide mimetic. 23. The compound according to claim 9, with the formula: 24. The embodiment according to claim 6, wherein the inhibitor has an inhibition constant of 100 micro molar or less. 25. Mixtures comprising an inhibitor according to claim 1 and optionally a pharmaceutical acceptable carrier. 26. A method for inhibiting cell growth by inhibiting a APIase activity by contacting the corresponding cells with an effective amount of an inhibitor according to claim 1. 27. A method for inhibiting the formation of incorrectly folded proteins, wherein the specific APIase which catalyses this folding is inhibited with an effective amount of an inhibitor according to claim 1. 28. The method according to claim 26, wherein the specific APIase is DnaK. 29. The use of an inhibitor according to claim 1 for the preparation of a pharmaceutical composition for inhibiting APIase. 30. The use of claim 29, wherein the APIase is DnaK. 31. The use according to claim 29 for inhibiting the cell growth of hyperplastic or neoplastic disease processes. 32. The method according to claim 26, wherein the cells are eukaryotic cells. 33. The method according to claim 26, wherein the cells are selected from the following group: mammalian cells, yeast cells, fungal cells. 34. The method according to claim 27, wherein the incorrect folding of proteins leads to diseases. 35. A method for identifying an inhibitor of a secondary amide peptide bond-specific cis/trans isomerase (APIase) comprising the following steps: a. preparing a library of compounds with the sequence X1X2X3R3-R4X4X5X6, wherein in each peptide X represents an amino acid; b. adding an APIase of choice to the library indicated under a) for a corresponding time and under such conditions which are sufficient for binding the APIase to peptides; c. determining the amino acid sequence of the peptide bound to the APIase; d. synthesizing the peptide found under c; and e. determining the inhibitor constant of the peptide, wherein an inhibition constant of 100 micro molar or less shows an inhibitor of this APIase. 36. A method for locating an inhibitor of a secondary amide peptide bond-specific cis/trans isomerase (APIase) comprising the following steps: a. preparing a secondary amide peptide bond-specific cis/trans isomerase (APIase), b. mixing the APIase with: i. a possible inhibitor molecule and ii. a specific APIase substrate of this enzyme in order to iii. obtain a mixture of APIase, possible inhibitor molecule and substrate, c. incubating this mixture under conditions which are necessary for the APIase to catalyse the cis/trans isomerisation of the substrate; and d. determining the inhibitor constant of the peptide, wherein a Ki of 100 micro molar or less shows an inhibitor of this APIase. |
Soap bar wrapper |
The package with reinforced regions can be produced from a single sheet of material, preferably in-line with the packaging of an item. Gusset-like folds are made to form the reinforced regions of the film material that is to form the package. The reinforcement will be through at least a three-ply section between a center panel and side edge panels. The gusset-like folds provide a significant reinforcement due to the multi-ply nature of such folds. These folds can be made in any part of a film that is subsequently used to make a package. |
1. A packaged soap bar comprising a soap bar, said soap bar surrounded by a plastic film, said plastic film folded inward on itself in a multi-layer gusset-like fold adjacent the longitudinal edge of said film, a margin of plastic film extending beyond said gusset-like fold. 2. A packaged soap bar as in claim 1 wherein the layers if plastic film in said gusset-like multi-layer fold are at least partially attached. 3. A packaged soap bar as in claim 2 wherein said layers of plastic film are adhesively attached. 4. A packaged soap bar as in claim 2 wherein said layers of plastic film are attached by heat welding. 5. A packaged soap bar as in claim 1 wherein said margin of plastic film is sufficient to form the sidewalls of said packaged soap bar. 6. A packaged soap bar as in claim 5 wherein said margin of plastic film is sealed by heat welding. 7. A packaged soap bar as in claim 5 wherein said margin is adhesively sealed. 8. A packaged soap bar as in claim 1 wherein said gusset-like fold is a fold of three layers of said film. 9. A packaged soap bar as in claim 1 wherein said gusset-like fold has a width of about 1 cm to about 5 cm. 10. A packaged soap bar as in claim 9 wherein said gusset-like fold has a width of about 1.5 cm to about 3 cm. 11. A packaged soap bar as in claim 11 wherein said film has a thickness of about 10 micron to about 300 micron. 12. A packaged soap bar as in claim 11 wherein said film has a thickness of about 25 micron to about 200 micron. 13. A method of packaging a soap bar and a plastic film having a length with a longitudinal edge and a width, forming a gusset-like fold adjacent each longitudinal edge to provide at least three layers in said gusset-like fold, a margin of plastic extending beyond the gusset-like fold, wrapping said film around said soap bar to fully envelop said soap bar, and folding and sealing said film to form said packaged soap bar wherein said soap bar is fully enclosed within said film. 14. A method of packaging a soap bar as in claim 13 wherein said gusset-like multi-layer fold is a three-layer fold. 15. A method of packaging a soap bar as in claim 13 wherein the plastic film in gusset-like layers are at least partially attached. 16. A method of packaging a soap bar as in claim 13 wherein the gusset-like folds are adhesively attached. 17. A method of packaging a soap bar as in claim 13 wherein the gusset-like folds are heat welded. 18. A method of packaging a soap bar as in claim 13 wherein the margin is sufficient to form the end walls of said soap bar package. |
<SOH> BACKGROUND OF THE INVENTION <EOH>In many soap bar wrappers there is a need for a two piece wrapping. This particularly is the case when he soap bar is not in a substantially rectangular shape. This two piece wrapping is comprised of an inner partial wrapper called a stiffener and an outer wrapper which fully encircles and encloses the stiffener and the soap bar. The stiffener laterally substantially encircles the soap bar and shapes an outer wrapper into a substantially rectangular shape on an outer wrapper. The ends are open, the outer wrapper then fully surrounds the stiffener and soap bar. This dual wrapping uses more wrapping material than a single layer of material such as when the soap bar is substantially rectangular or in the use of flow wrapping techniques. This use of material in two piece wrapping can be decreased if the wrapping technique similar to that disclosed in U.S. Pat. No. 2,009,511 is used. Here reinforcement bands adjacent each end of a package reinforce the end regions of a package. This is shown in FIGS. 1 to 3 . This package concept saves packaging material and can be used to package soap bars with a generally planar bottom to the soap bar. However, this package of U.S. Pat. No. 2,009,511 can be improved. It can be made from a single sheet of material rather than three separate pieces which reduces the cost of making the package. In the process of U.S. Pat. No. 2,009,511 reinforcing bands are adhesively attached to the outer wrapper. This requires the attaching of two materials together. The result is a package with reinforced end areas. This is an effective package for soap bars. There is an effective reinforcing of the end edges to form a generally rectangular package. However, it has been found that a soap bar package with essentially the same advantages can be made from a single sheet of material in place of a sheet of material and two tapes. |
<SOH> BRIEF DESCRIPTIONS OF THE INVENTION <EOH>It has been found that a package similar to that described in FIGS. 1 to 3 of U.S. Pat. No. 2,009,511 can be made from a single film. This is produced by forming gusset-like folds in the area where reinforcement is desired. The gusset-like folds will produce a three layer structure for the reinforcement bands. All or some of the walls of the folds can be adhesively bonded, one to the other. Such a three layer structure will provide a significant reinforcement of the area of the gusset-like fold. The gusset-like folds will be adjacent each of the longitudinal edges of the sheet of film. The gusset-like folds can extend inwardly to about any depth. However, it is preferred that they do not extend inwardly to the extent that the gusset-like folds overlap. The folds which will provide a three-ply structure results in significant reinforcement of the part of the package with the folds. The gusset-like folds are made continuously by means of plows that extend inwardly and that are usually used to form a gusset. Any gusset forming equipment and processes can be used. The forming of gusset folds is well-known in the bag making art. |
Handlebar assembly |
A handlebar assembly (100) is disclosed comprising a lower base portion (101) which is adapted to support a handlebar (202) through a guide means which is so constructed and arranged to permit the handlebar (202) to move along a path relative to the base portion (101) whereby upon movement of the handlebar (202) along the path to the left or the right relative to a central position the handlebar (202) tilts and the centre of the handlebar (202) moves downwards relative to the base portion (101) as well as horizontally from the central position. The assembly allows the handlebars to move in a way which mimics the movement of the handlebars of a bicycle. In a modification, the guide means may restrain the handlebar for movement up/down or forwards/backwards or side to side, again to provide a more realistic range of movement. |
1. Apparatus, having a frame, a seat and a handlebar assembly comprising a lower base portion (101) which is adapted to support a handlebar (202) through a guide means which is so constructed and arranged to permit the handlebar (202) to move along a path relative to the base portion (101) whereby upon movement of the handlebar (202) along the path to the left or the right relative to a central position the handlebar (202) tilts and the centre of the handlebar (202) moves downwards relative to the base portion (101) as well as horizontally from the central position, in which the frame supports the handlebar assembly and the seat above a floor. 2. The apparatus of claim 1, in which the apparatus comprises a stationary exercise bicycle, a motorbike, a JetSki or a bicycle mounted on rollers or a Turbotrainer. 3. A handlebar assembly (100) comprising a lower base portion (101) which is adapted to support a handlebar (202) through a guide means which is so constructed and arranged to permit the handlebar (202) to move along a path relative to the base portion (101) whereby upon movement of the handlebar (202) along the path to the left or the right relative to a central position the handlebar (202) tilts and the centre of the handlebar (202) moves downwards relative to the base portion (101) as well as horizontally from the central position, in which the base portion comprises a stem shaft (101) which is adapted to be at least partially received within the headtube of a bicycle or stationary exercise bike. 4. A handlebar assembly (100) comprising a lower base portion (101) which is adapted to support a handlebar (202) through a guide means which is so constructed and arranged to permit the handlebar (202) to move along a path relative to the base portion (101) whereby upon movement of the handlebar (202) along the path to the left or the right relative to a central position the handlebar (202) tilts and the centre of the handlebar (202) moves downwards relative to the base portion (101) as well as horizontally from the central position, in which the guiding means comprises one or more links (112; 113) defining a linkage, the linkage comprising at least two non-parallel links (112; 113), each link being connected at one point along the link to the base portion and at a different point along the link to the handlebar. 5. A handlebar assembly (100) comprising a lower base portion (101) which is adapted to support a handlebar (202) through a guide means which is so constructed and arranged to permit the handlebar (202) to move along a path relative to the base portion (101) whereby upon movement of the handlebar (202) along the path to the left or the right relative to a central position the handlebar (202) tilts and the centre of the handlebar (202) moves downwards relative to the base portion (101) as well as horizontally from the central position, in which the handlebar assembly includes at least one output device which is dependent on the portion of the handlebar relative to the base portion, the output devices and any buttons or switches are adapted to be connected to a PC or games console through one or more electrical cables and industry standard electronics, and in which resistance means (215; 217) are provided to oppose the movement of the handlebars against any or all of its degrees of freedom, the resistance means including one or more dampers which damp the movement of the handlebar. 6. A handlebar assembly according to any one of claims 3 to 5, in which the handlebar is adapted to move forwards or backwards relative to the base portion. 7. A handlebar assembly according to claim 6 in which the handlebar is adapted to move forwards and backwards by a trapezoidal linkage set up in a substantially horizontal forward facing plane. 8. A handlebar assembly according to any one of claims 3 to 7, in which the handlebar assembly is adapted to be incorporated into a stationary exercise bicycle. 9. A handlebar assembly according to claim 3 or claim 5, or any one of claims 6 to 8 when dependent upon claim 3 or claim 5 in which the guiding means comprises one or more links (112; 113) defining a linkage. 10. A handlebar assembly according to claim 9, in which the linkage comprises at least two non-parallel links (112; 113), each link being connected at one point along the link to the base portion and at a different point along the link to the handlebar. 11. A handlebar assembly according to claim 4 or claim 10, in which the two points where the links join the base portion lie in a horizontal plane for all positions of the handlebar, whilst the two points where they join the handlebar lie in a horizontal plane only when the handlebar is in its central position. 12. A handlebar assembly according to any one of claims 4, 10 or 11, in which the two points at which the links join the handlebar are spaced apart further than the points further than the points where they join the base portion, both links leaning outwardly away from one another when the handlebar is in its normal rest position. 13. A handlebar assembly according to any one of claims 4 or 10 to 12, in which the distance between the two points on at least one of the links of the linkage is adjustable such that the location of the virtual pivot point relative to the handlebar may be varied to move it closer to or further from the handlebar. 14. A handlebar assembly according to any one of claims 4 or 10 to 13 in which the spacing between the point where the two links join the top link may be varied in order to accommodate different feels and to simulate different types and sizes of bicycle. 15. A handlebar assembly according to any one of claims 3 or 5, or any one of claims 6 to 8 when dependent upon claim 3 or claim 5, in which the guiding means comprises one or more tracks defining a guide rail. 16. A handlebar assembly according to claim 15, in which the or each track is secured to one of the base and the handlebar and co-operates with one or more runners, attached to the other of the handlebar and the base portion. 17. A handlebar assembly according to claim 15 or claim 16, in which the guide rail provides the path for the handlebars to follow, with the handlebar connected to a runner assembly which permits the handlebar to move along the guide rail and in doing so, tilt and drop as moved away from the central position. 18. A handlebar assembly according to any one of claims 3 to 17, in which the base portion includes a pivot (203) adapted to be located close to a floor, and there is provided an upright link (204) which is connected to and pivotally extends from the pivot to the handlebar such that the handlebar when moving along the path away from the central position leans through an arc centred on the pivot, of the upright link, the pivot defining the pivot point. 19. A handlebar assembly according to claim 18, in which the upright link, or a member attached to it spaced from the pivot, passes through a slot in a control plate (219), the slot constraining the movement of the handlebar. 20. A handlebar assembly according to claim 18 or claim 19 in which the pivot point is adapted to, in use, be at floor level or just above or at the same height above the floor as the bottom bracket of a normal bicycle. 21. A handlebar assembly according to any of claims 4 to 20, in which the base portion comprises a stem shaft (101), which is adapted to be at least partially received within the headtube of a bicycle or stationary exercise bike. 22. A handlebar assembly according to any one of claims 3 to 21, in which the path comprises an arc centred about a pivot point located below the centre of the handlebar. 23. A handlebar assembly according to any one of claims 3 to 22, in which height adjustment means (208a; 208b) is provided to permit the handlebar to be raised and lowered relative to base portion. 24. A handlebar assembly according to claim 23, in which the height adjustment means constrains the upward and downward movement of the handlebar relative to the base portion to move about a point which would correspond to the point of contact of the rear wheel of a bicycle with the ground. 25. A handlebar assembly according to any one of claims 3 to 24, in which the handlebar is adapted to be rotated in a plane relative to the base. 26. A handlebar assembly according to claim 3, claim 4 or any one of claims 6 to 25 when dependent upon claim 3 or claim 4, in which resistance means (215; 217) are provided to oppose the movement of the handlebars against any or all of its degrees of freedom. 27. A handlebar assembly according to any one of claims 3 to 26, in which locking means are provided to enable the handlebars to be locked against movement about any or all of its degrees of freedom. 28. A handlebar assembly according to any of one claims 3 to 27, in which the handlebar assembly is adapted to be used as a replacement for the conventional handlebars on a normal bicycle. 29. A handlebar assembly according to claim 3, claim 4 or any one of claims 6 to 28 when dependent upon claim 3 or claim 4, in which the handlebar assembly includes at least one output device which is dependent on the portion of the handlebar relative to the base portion. 30. A handlebar assembly according to claim 29, in which the output devices and any buttons or switches are adapted to be connected to a PC or games console through one or more electrical cables and industry standard electronics. 31. A handlebar assembly according to claim 5, claim 29 or claim 30, in which the handlebar assembly further includes one or more switches or buttons which can be operated by a user. 32. A handlebar assembly according to any one of claims 3 to 31, in which the handlebar assembly further includes a display upon which information can be presented to the user of the handlebars. 33. Apparatus having a frame, a seat and a handlebar assembly in accordance with any one of claims 3 to 32, in which the frame supports the handlebar assembly and the seat above a floor. 34. The apparatus of claim 33, in which the apparatus comprises a stationary exercise bicycle, a motorbike, a JetSki or a bicycle mounted on rollers or a Turbotrainer. 35. Apparatus according to any one of claims 1, 2, 33 or 34, in which the handlebar moves about a point aligned with the centre of the handlebar which would be equivalent to the contact point of a front wheel of the bicycle, or a point which is the same height above the floor as the bottom bracket of the bicycle. 36. Apparatus according to any one of claims 33 to 35, when dependent from claim 4 or claim 9, in which the linkage comprises a non-parallel trapezoidal linkage comprising two links which form the sides of a trapezoid with the handlebar forming the top of the trapezoid and the frame forming the bottom of the trapezoid. 37. Apparatus according to any one of claims 1, 2 or 33 to 36, in which the handlebar assembly includes a resistance which may provide a variable resistance to movement as a function of the speed at which a rider is pedalling the cycle. 38. Apparatus according to claim 37, in which the bicycle provides variable resistance to movement of the handlebars is dependent upon the resistance provided by the bicycle pedalling. 39. Apparatus according to any one of claims 1, 2 or 33 to 38 in which the, seat is also supported by a guide means which permits movement of the seat from side to side and/or up/down and/or forwards/backwards about a curved path. 40. Apparatus according to claim 39 in which the seat guide means comprises two non-parallel links connected at a first point to the seat (or a seat support) and at a second portion connected directly or indirectly to the frame (or any point fixed relative to the frame). |
Pyrrolo pyrimidines as agents for the inhibition of cystein proteases |
The invention provides compounds of Formula I or a pharmaceutically acceptable salt or ester thereof wherein the symbols have meaning as defined, which are inhibitors of cathepsin K and find use pharmaceutically for treatment of diseases and medical conditions in which cathepsin K is implicated, e.g. various disorders including inflammation, rheumatoid arthritis, osteoarthritis, osteoporosis and tumors. |
1. A compound of formula I, or a pharmaceutically acceptable salt or ester thereof wherein R is H, —R2, —OR2 or NR1R2, wherein R1 is H, lower alkyl or C3 to C10 cycloalkyl, and R2 is lower alkyl or C3 to C10 cycloalkyl, and wherein R1 and R2 are independently, optionally substituted by halo, hydroxy, lower alkoxy, CN, NO2, or optionally mono- or di-lower alkyl substituted amino; X is ═N— or ═C(Z)-, wherein Z is H, —C(O)—NR3R4-, —NH—C(O)—R3, —CH2—NH—C(O)—R3, —C(O)—R3, —S(O)—R3, —S(O)2—R3, —CH2—C(O)—R3, —CH2—NR3R4-, —R4-, —C≡C—CH2—R5, N-heterocyclyl, N-heterocyclyl-carbonyl, or —C(P)═C(Q)-R4 wherein P and Q independently are H, lower alkyl or aryl, R3 is aryl, aryl-lower alkyl, C3-C10cycloalkyl, C3-C10cycloalkyl-lower alkyl, heterocyclyl or heterocyclyl-lower alkyl, R4 is H, aryl, aryl-lower alkyl, aryl-lower-alkenyl, C3-C10cycloalkyl, C3-C10cycloalkyl-lower alkyl, heterocyclyl or heterocyclyl-lower alkyl, or wherein R3 and R4 together with the nitrogen atom to which they are joined to form an N-heterocyclyl group, wherein N-heterocyclyl denotes a saturated, partially unsaturated or aromatic nitrogen containing heterocyclic moiety attached via a nitrogen atom thereof having from 3 to 8 ring atoms optionally containing a further 1, 2 or 3 heteroatoms selected from N, NR6, O, S, S(O) or S(O)2 wherein R6 is H or optionally substituted (lower alkyl, carboxy, acyl (including both lower alkyl acyl e.g. formyl, acetyl or propionyl, or aryl acyl, e.g. benzoyl), amido, aryl, S(O) or S(O)2, and wherein the N-heterocyclyl is optionally fused in a bicyclic structure, e.g. with a benzene or pyridine ring, and wherein the N-heterocyclyl is optionally linked in a spiro structure with a 3 to 8 membered cycloalkyl or heterocyclic ring wherein the heterocyclic ring has from 3 to 10 ring members and contains from 1 to 3 heteroatoms selected from N, NR6, O, S, S(O) or S(O)2 wherein R6 is as defined above), and wherein heterocyclyl denotes a ring having from 3 to 10 ring members and containing from 1 to 3 heteroatoms selected from N, NR6, O, S, S(O) or S(O)2 wherein R6 is as defined above), and wherein R3 and R4 are independently, optionally substituted by one or more groups, e.g. 1-3 groups, selected from halo, hydroxy, oxo, lower alkoxy, CN or NO2, or optionally substituted (optionally mono- or di-lower alkyl substituted amino, aryl, aryl-lower alkyl, N-heterocyclyl or N-heterocyclyl-lower alkyl (wherein the optional substitution comprises from 1 to 3 substituents selected from halo, hydroxy, lower alkoxy, CN, NO2, or optionally mono- or di-lower alkyl substituted amino)), and wherein R5 is aryl, aryl-lower alkyl, aryloxy, aroyl or N-heterocyclyl as defined above, and wherein R5 is optionally substituted by R7 which represents from 1 to 5 substituents selected from halo, hydroxy, CN, NO2 or oxo, or optionally substituted (lower-alkoxy, lower-alkyl, aryl, aryloxy, aroyl, lower-alkylsulphonyl, arylsulphonyl, optionally mono- or di-lower alkyl substituted amino, or N-heterocyclyl, or N-heterocyclyl-lower alkyl (wherein N-heterocyclyl is as defined above), and wherein R7 is optionally substituted by from 1 to 3 substituents selected from halo, hydroxy, optionally mono- or di- lower-alkyl substituted amino, lower-alkyl carbonyl, lower-alkoxy or lower-alkylamido; R13 is lower alkyl, C3 to C10 cycloalkyl or C3-C10cycloalkyl-lower alkyl, all of which are independently optionally substituted by halo, hydroxy, CN, NO2 or optionally mono- or di-lower alkyl-substituted amino; and R14 is H or optionally substituted (aryl, aryl-W—, aryl-lower alkyl-W—, C3 to C10 cycloalkyl, C3 to C10 cycloalkyl-W—, N-heterocyclyl or N-heterocyclyl-W— (wherein N-heterocyclyl is as defined above), phthalimide, hydantoin, oxazolidinone, or 2,6-dioxo-piperazine), wherein —W— is —O—, —C(O)—, —NH(R6)-, —NH(R6)-C(O)—, —NH(R6)-C(O)—O—, (where R6 is as defined above), —S(O)—, —S(O)2— or —S—, wherein R14 is optionally substituted by R18 which represents from 1 to 10 substituents selected from halo, hydroxy, CN, NO2, oxo, amido, carbonyl, sulphonamido, lower-alkyldioxymethylene, or optionally substituted (lower-alkoxy, lower-alkyl, lower-alkenyl, lower alkynyl, lower alkoxy carbonyl, optionally mono- or di-lower alkyl substituted amino, aryl, aryl-lower alkyl, aryl-lower alkenyl, aryloxy, aroyl, lower-alkylsulphonyl, arylsulphonyl, N-heterocyclyl, N-heterocyclyl-lower alkyl (wherein N-heterocyclyl is as defined above), heterocyclyl or R14 comprising aryl has aryl fused with a hetero-atom containing ring, and wherein R18 is optionally substituted by R19 which represents from 1 to 4 substituents selected from halo, hydroxy, CN, NO2 or oxo, or optionally substituted (lower-alkoxy, lower-alkyl, lower-alkoxy-lower-alkyl, C3-C10cycloalkyl, lower-alkoxy carbonyl, halo-lower alkyl, optionally mono- or di-lower alkyl substituted amino, aryl, aryloxy, aroyl (e.g. benzoyl), acyl (e.g. lower-alkyl carbonyl), lower-alkylsulphonyl, arylsulphonyl or N-heterocyclyl, or N-heterocyclyl-lower alkyl (wherein N-heterocyclyl is as defined above)), wherein R19 is optionally substituted by from 1 to 4 substituents selected from halo, hydroxy, CN, NO2, oxo, optionally mono- or di-lower alkyl substituted amino, lower-alkyl, or lower-alkoxy. 2. A compound according to claim 1 of formula II or a pharmaceutically acceptable salt or ester thereof wherein R13 and R14 are as defined in claim 1. 3. A compound according to claim 1, or a pharmaceutically acceptable salt or ester thereof, selected from any one of the Examples 2 to 10. 4. A compound according to claim 1 for use as a pharmaceutical. 5. A pharmaceutical composition comprising a compound according to claim 1 as an active ingredient. 6. A method of treating a patient suffering from or susceptible to a disease or medical condition in which cathepsin K is implicated, comprising administering an effective amount of a compound according to claim 1 to the patient. 7. The use of a compound according to claim 1 for the preparation of a medicament for therapeutic or prophylactic treatment of a disease or medical condition in which cathepsin K is implicated. 8. A process for the preparation of a compound of formula I or a salt or ester thereof which comprises i) for the preparation of compounds of formula V′ or a pharmaceutically acceptable salts or esters thereof wherein R13 is as defined above and R14′ is as defined above for R14, except that R14′ is not optionally substituted carbocyclic aryl, coupling of a halo precursor of formula XI R13 is as defined above and Halo is preferably bromo, with an R14′ precursor, or ii) for the prearation of compounds of formula V″ or a pharmaceutically acceptable salts or esters thereof wherein R13 is as defined above and R14″ is optionally substituted (carbocylic aryl or azole), cyclising a corresponding carbocyclic arly-1-prop-2-yne, or azole-1-prop-2-yne of formula XII with a 5-halo-pyrimidine-2-carbonitrile precursor of formula XIII wherein Halo is preferably Br, and R13 and R14″ are as defined above, and thereafter, if desired, converting the product obtained into a further compound of formula I, or into a salt or ester thereof. |
Integrated circuit for controlling a laser diode |
The invention relates to an integrated circuit for controlling a laser diode, comprising a signal input for receiving a data signal; a signal output for connection to the laser diode; a modulator for modulation of the data signal; a current infeed connected to the signal output for the supply of a bias current; a coupling capacitor between the output of the modulator and the signal output in order to form a high pass with the differential resistor of the laser diode. Preferably, the circuit also comprises an active compensation circuit with a low pass connected to the output of the modulator; and a circuit which is connected to the low pass for producing a signal which is inversely proportional to the low pass output voltage and which is added to the output signal or subtracted from the output signal. The low pass can be controlled by a circuit guiding the data signal. |
1. An integrated circuit for controlling a laser diode, having: a signal input for receiving a data signal; a signal output for connection to the laser diode; a modulator for modulation the data signal; a current infeed connected to the signal output and serving for the supply of a bias current; and a coupling capacitor between the output of the modulator and the signal output, for forming a high-pass filter with the differential resistor of the laser diode connected to the signal output. 2. The integrated circuit as claimed in claim 1, having an active compensation circuit having: a low-pass filter connected to the output of the modulator; and a circuit connected to the low-pass filter and serving for generating a signal which is inversely proportional to the low-pass filter output voltage, which signal is subtracted from the output signal. 3. The integrated circuit as claimed in claim 1, having an active compensation circuit having: a low-pass filter connected to the output of the modulator; and a circuit connected to the low-pass filter and serving for generating a signal which is proportional to the low-pass filter output voltage, which signal is added to the output signal. 4. The integrated circuit as claimed in claim 2 or 3, the low-pass filter being controlled by a circuit carrying the data signal. 5. The integrated circuit as claimed in claim 1, having an active compensation circuit having: a low-pass filter connected to the output of the modulator, a voltage inverter connected to the low-pass filter and serving for inverting the voltage generated by the low-pass filter in accordance with the low-pass filter transfer function; a voltage follower connected to the output of the voltage inverter; and a converter between the output of the voltage follower and the signal output, for generating a current which is inversely proportional to the low-pass filter output voltage. 6. The integrated circuit as claimed in one of the preceding claims, the supply voltage essentially being 3.3V. 7. An integrated circuit for differentially controlling a laser diode, having a signal input for receiving a data signal; a first signal output for connection to the anode of the laser diode; a second signal output for connection to the cathode of the laser diode; a modulator for modulating the data signal; a bias current supply connected to the cathode; a reference-ground potential connected to the anode; a first and a second coupling capacitor between a first and second output, respectively of the modulator and the first and second signal output, respectively; and a first and a second compensation circuit connected in parallel with the first and second coupling capacitors, respectively, the first and second compensation circuits in each case having a low-pass filter, which is connected to the first and second output, respectively, of the modulator and can be controlled by a circuit carrying the data signal; and a circuit connected to the low-pass filter and serving for generating a signal which is inversely proportional and proportional, respectively, to the low-pass filter output voltage, which signal is subtracted from the output signal and, respectively, added to the output signal. 8. The integrated circuit as claimed in claim 7, the first and second compensation circuits in each case having a low-pass filter connected to the first and second output, respectively, of the modulator; a voltage inverter connected to the low-pass filter and serving for inverting the voltage generated by the low-pass filter in accordance with the low-pass filter transfer function; a voltage follower connected to the output of the voltage inverter; and a converter between the output of the voltage follower and the first and second signal output, especially, for generating a current which is inversely proportional to the low-pass filter output voltage. 9. The integrated circuit as claimed in claim 7 or 8, the cathode being inductively isolated from the reference-ground potential. 10. The integrated circuit as claimed in claim 7 or 8, the cathode and the reference-ground potential being internally coupled. 11. The integrated circuit as claimed in one of claims 7 to 10, the anode being inductively isolated from the bias current supply. 12. The integrated circuit as claimed in one of claims 7 to 10, the anode and the bias current supply being internally coupled. 13. The integrated circuit as claimed in one of the preceding claims, the laser diode being formed by a VCSEL. 14. An integrated circuit for controlling a laser diode array having a multiplicity of laser diodes, having; a supply voltage feed; one or a plurality of signal inputs for receiving data signals; a multiplicity of signal outputs for connection to the laser diodes, a signal output in each case being assigned to a laser diode; a multiplicity of modulation stages for modulating the data signals, a modulation stage in each case being assigned to a signal output; and a respective coupling capacitor between the output of one of the modulation stages and the assigned signal output, for the formation of a high-pass filter together with the differential resistor of the assigned laser diode. 15. The integrated circuit as claimed in claim 14, an active compensation circuit being connected in parallel with each coupling capacitor, having: a low-pass filter connected to the output of the assigned modulation stage; and a circuit connected to the low-pass filter and serving for generating a signal which is inversely proportional to the low-pass filter and serving for generating a signal which is inversely proportional to the low-pass filter output voltage, which signal is subtracted from the output signal. 16. The integrated circuit as claimed in claim 14, an active compensation circuit being connected in parallel with each coupling capacitor, having: a low-pass filter connected to the output of the assigned modulation stage; and a circuit connected to the low-pass filter and serving for generating a signal which is proportional to the low-pass filter and serving for generating a signal which is inversely proportional to the low-pass filter output voltage, which signal is added to the output signal. 17. The integrated circuit as claimed in claim 15 or 16, the low-pass filter being controlled by a circuit carrying the data signal. 18. The integrated circuit as claimed in claim 14, an active compensation circuit being connected in parallel with each coupling capacitor, having: a low-pass filter connected to the output of the assigned modulation stage; a voltage inverter connected to the low-pass filter and serving for inverting the voltage generated by the low-pass filter in accordance with the low-pass filter transfer function; a voltage follower connected to the output of the voltage inverter; and a converter between the output of the voltage follower and the assigned signal output, for generating a current which is inversely proportional to the low-pass filter output voltage. 19. The integrated circuit as claimed in one of claims 14 to 18, the laser diodes being formed by VCSELs. 20. The integrated circuit as claimed in one of claims 14 to 19, the supply voltage essentially being 3.3 V. |
Polycarbonate-based oriented film and retardation film |
A uniaxially or biaxially oriented film which is made from a specific polycarbonate having a fluorene ring and a glass transition temperature of 165° C. or higher and which has a heat shrinkage factor when it is heated at 90° C. for 500 hours of 0.1% or less, and a ratio of retardation R(450) within the film plane at a wavelength of 450 nm to retardation R(550) within the film plane at a wavelength of 550 nm of 1 to 1.06. This film can be used in a high-quality liquid crystal display device such as a vertical alignment liquid crystal display device and is useful as a retardation film which almost solves a frame problem. |
1. A uni- or bi-axially oriented film (A) which comprises a polymer or polymer mixture containing a recurring unit represented by the following formula (I): wherein R1 to R8 are each independently a member selected from the group consisting of hydrogen atom, halogen atom, hydrocarbon group having 1 to 6 carbon atoms and hydrocarbon-O-group having 1 to 6 carbon atoms, and X is represented by the following formula (i)-1: wherein R30 and R31 are each independently a halogen atom or alkyl group having 1 to 3 carbon atoms, and n and m are each independently an integer of 0 to 4, each of the polymer and the polymer mixture containing the recurring unit represented by the above formula (I) in an amount of 30 to 60 mol % based on the total of all the recurring units of the polymer or polymer mixture and having a glass transition temperature of 165° C. or higher, (B) which has a heat shrinkage factor when it is heated at 90° C. for 500 hours under no load of 0.1% or less, and (C) which satisfies the following formula (1): 1≦R(450)/R(550)≦1.06 (1) wherein R(450) and R(550) are retardations within the film plane at wavelengths of 450 nm and 550 nm, respectively. 2. The film of claim 1, wherein the amount of the recurring unit represented by the formula (I) is larger than 30 mol % based on the total of all the recurring units. 3. The film of claim 1 which is a uniaxially oriented film further satisfying the following expressions (2) and (3) at the same time: R(550)>K(550) (2) R(550)>20 nm (3) wherein R(550) is as defined in the above expression (1) and K(550) is a value (nm) defined by the following expression (4) at a wavelength of 550 nm: K=[(nx+ny)/2−nx]×d (4) wherein nx, ny and nz are refractive indices in x axis, y axis and z axis directions of the film, respectively, and d is the thickness (nm) of the film. 4. The film of claim 1 which is a biaxially oriented film further satisfying the following expression (2′) and the above expression (3) at the same time: R(550)≦K(550) (2′) wherein R(550) and K(550) are as defined in the above expressions. 5. The film of claim 4 which satisfies the following expression (1′): 1≦R(450)/R(550)≦1.05 (1′) wherein R(450) and R(550) are as defined in the above expressions. 6. The film of claim 1 which is a biaxially oriented film further satisfying the above expression (2′), the following expression (3′) and the following expression (5) at the same time: R(550)≦20 nm (3′) K(550)≧50 nm (5) wherein R(550) and K(550) are as defined in the above expressions. 7. The film of claim 1, wherein the polymer or polymer mixture has a glass transition temperature of 200° C. or higher. 8. The film of claim 1 which is a retardation film. 9. A laminated polarizer film which comprises the film of any one of claims 1 to 6 and a polarizer film in a laminated form. 10. The laminated polarizer film of claim 9 which is laminated in such a manner that the transmission axis of the polarizer film becomes parallel to the slow axis within the plane of the film. 11. A liquid crystal display device comprising the laminated polarizer film of claim 9 or 10. 12. The liquid crystal display device of claim 11 which is in a vertical alignment mode. |
<SOH> BACKGROUND ART <EOH>A retardation film is used in an STN (Super Twisted Nematic) liquid crystal display device or the like to solve problems such as color compensation and the expansion of viewing angle. As the material of a retardation film for color compensation has been used a polycarbonate, polyvinyl alcohol, polysulfone, polyether sulfone or amorphous polyolefin. Liquid crystalline polymer and discotic liquid crystals have also been used as the material of a retardation film for the expansion of viewing angle in addition to the above materials. A vertical alignment liquid crystal display device in which liquid crystals are aligned almost vertically to a substrate when voltage is off has already been used in monitors and TVs due to its high contrast and wide viewing angle. It is described in the 1997 Society for information display international symposium digest of technical papers at pages 845 to 848 that the use of a retardation film is important to obtain a wide viewing angle. A retardation film made from a polycarbonate homopolymer produced from bisphenol A as a starting material has been widely used in the above STN liquid crystal display device. However, as especially a vertical alignment liquid crystal display device has higher quality than an STN liquid crystal display device, it has been found that a retardation film made from a polycarbonate material which has been used in the conventional STN liquid crystal display device cannot obtain sufficiently high display quality. That is, the retardation value and the optical axis of a retardation film are changed by stress in the step of joining together a retardation film made from a polycarbonate homopolymer and a polarizer film, stress in the step of joining the laminated polarizer film obtained in the above step to a liquid crystal display device, or the shrinkage stress of a polarizer film which is produced during a durability test at a high temperature or at a high temperature and a high humidity, with the result that the brightness of the screen of the liquid crystal display device becomes nonuniform particularly when black is displayed on the entire screen, thereby deteriorating display quality. The place where this brightness nonuniformity appears which depends on the mode of the liquid crystal display device is around the edges of the four sides of the screen of the liquid crystal display device in most cases. Therefore, this phenomenon will be referred to as “frame phenomenon” and this problem will be referred to as “frame problem” in this description hereinafter. Cellulose acetate, polyolefin and polycarbonate are known as the material of the retardation film. However, a retardation film made from cellulose acetate has poor stability of molecular orientation as cellulose acetate has a high water absorption coefficient, thereby making it difficult to use it when a high degree of orientation is required within the plane and to suppress variations in anisotropy within the plane for the same reason. Since a polyolefin having a cyclic skeleton such as a norbornene skeleton has a low photoelastic constant and low intrinsic birefringence, it must be stretched at a high draw ratio to obtain a retardation required for a retardation film. Since a bulky molecular structure such as a norbornene skeleton is employed to obtain a high glass transition point, a retardation film made from the polyolefin has low impact resistance, handling ease and stretchability, easily breaks and often ruptures. Therefore, this film has a lot of problems to be solved when it is produced or used as a retardation film. Meanwhile, a polycarbonate comprising an aromatic dihydroxy compound (bisphenol) having two aromatic rings through a bond group out of aromatic polycarbonates has appropriate flexibility and a high glass transition point. However, a homopolymer having a bisphenol A skeleton which is widely used in an STN mode has no problem with handling ease and stretchability but does have the above frame problem. Therefore, it is difficult to use it in a vertical alignment liquid crystal display device which must have high quality. There are many kinds of polycarbonates and there are a large number of examples in which the polycarbonates are used as retardation films. JP-A 7-246661 and JP-A 6-82624 (the term “JP-A” as used herein means an “unexamined published Japanese patent application”) propose retardation films made from a polycarbonate comprising a dihydroxy component other than a bisphenol A skeleton. Polycarbonates are divided into aliphatic and aromatic polycarbonates. In general, aliphatic polycarbonates are not used as the material of a retardation film because they have a low glass transition temperature and poor productivity though they have a low photoelastic constant. One of the causes of the frame phenomenon is that stress generated by the shrinkage of a polarizer spreads to a retardation film through an adhesive layer to change the retardation of the retardation film. Therefore, a retardation film having a lower photoelastic constant is considered to be preferred because a change in retardation caused by stress becomes smaller, which is not a necessary and sufficient condition. Meanwhile, aromatic polycarbonates have high production ease and their glass transition temperatures can be easily raised by the existence of an aromatic ring. As described above, they are actually used as the material of a retardation film but they have a problem that their photoelastic constants are relatively high. Attempts have already been made to reduce the photoelastic constant of an aromatic polycarbonate film, and some homopolymers and copolymers are proposed. However, in the case of these aromatic polycarbonates, though the reason is not clear, probably due to the existence of an aromatic ring, it is difficult to reduce the photoelastic constants of the aromatic polycarbonates to the level of a commercially available optical film made from a polyolefin having a bulky functional group such as the above norbornene skeleton. That is, although polycarbonates are superior to the above polyolefin in handling ease and moldability, it is difficult to reduce their photoelastic constants while realizing a high glass transition temperature. JP-A 6-25398 discloses a polycarbonate resin having a high refractive index and low birefringence which comprises a structural unit represented by the following formula (a): wherein R 1 to R 4 are each a hydrogen atom, halogen atom, phenyl group or alkyl group having 1 to 3 carbon atoms, and a structural unit represented by the following formula (b): wherein w is a single bond, alkylidene group, cycloalkylidene group, phenyl substituted alkylidene group, sulfone group, sulfide group or oxide group, R 5 and R 6 are each a hydrogen atom, halogen atom, phenyl group or alkyl group having 1 to 3 carbon atoms, and m and n are each an integer of 1 to 4, and which contains the structural unit (b) in an amount of 41 to 95 mol %. It is disclosed in Examples of the above publication that polycarbonates (powders) produced by the solution polymerization of 9,9-bis(4-hydroxyphenyl)fluorene and bisphenol A in molar ratios of 85/15 (Example 1), 75/25 (Example 2) and 50/50 (Example 3) are dissolved in methylene chloride to obtain films. However, the publication is silent about uniaxially oriented or biaxially oriented films made from the above polycarbonates and therefore about retardation films composed of these films as well. JP-A 2001-318232 discloses an optical film which is made from a polycarbonate containing 1 mol % or more of a recurring unit represented by the following formula (c): and having a glass transition temperature of 160° C. or higher, and which has a heat shrinkage factor when heated at 80° C. for 500 hours of 0.07% or less, a hardness measured by a super microhardness meter of 16 kg/mm 2 or more, a thickness of 10 to 200 μm and a retardation (R(550)) at a wavelength of 550 nm satisfying |R(550)|≦20 nm. It is disclosed in Example 7 of the above publication that a polycarbonate copolymer produced by the solution polymerization of 30 mol % of a bisphenol compound represented by the following formula: and 70 mol % of bisphenol A is dissolved in methylene chloride to obtain a cast film which is then stretched uniaxially to 1.5 times at 196° C. to obtain an optical film having an R(550) of 5.0 nm. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a schematic sectional view of an example of a vertical alignment liquid crystal display device comprising the oriented film of the present invention as a retardation film; FIG. 2 is a schematic sectional view of another example of a vertical alignment liquid crystal display device comprising the oriented film of the present invention as a retardation film; and FIG. 3 is a schematic sectional view of still another example of a vertical alignment liquid crystal display device comprising the oriented film of the present invention as a retardation film. detailed-description description="Detailed Description" end="lead"? |
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