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Modulating apparatus and method, and dsv control bit producing method |
The present invention relates to a modulation apparatus and method and a DSV-control-bit generating method for suppressing an increase in circuit size of the modulation apparatus. When an input data stream is supplied to a DSV control bit determination unit 31, the DSV control bit determination unit 31 determines a DSV control bit to be inserted into the input data stream. Upon supplying the input data stream to the DSV control bit determination unit 31, the input data stream is simultaneously supplied to a delay processor 32. The input data stream is delayed for a predetermined delay time and supplied to a determined-DSV-control-bit insertion unit 33. The determined-DSV-control-bit insertion unit 33 inserts the DSV control bit determined by the DSV control bit determination unit 3.1 into a predetermined position of the input data stream supplied by the delay means and supplies the input data stream containing the DSV control bit to a modulator 34. The modulator 34 modulates the input data stream containing the DSV control bit into a code string in accordance with a predetermined coding rule (for example, 1,7PP modulation). |
1. A modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string, comprising: DSV-control-bit generating means for generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; timing adjusting means for adjusting transmission timing for transmitting the input bit string; DSV-control-bit-inserted bit string generating means for generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated by the DSV-control-bit generating means into a predetermined position of the input bit string whose transmission timing is adjusted by the timing adjusting means; and first modulation means for modulating the DSV-control-bit-inserted bit string generated by the DSV-control-bit-inserted bit string generating means-into the channel bit string on the basis of a conversion rule (d, k; m, n; r). 2. The modulation apparatus according to claim 1, further comprising NRZI means for performing NRZI modulation of the channel bit string generated by modulation by the first modulation means to generate the recording code string or the transmission code string. 3. The modulation apparatus according to claim 1, wherein the conversion rule states that the remainder of the number of β1sβ in a predetermined block of the input bit string or the DSV-control-bit-inserted bit string divided by two equals the remainder of the number of β1sβ in the corresponding block of the channel bit string divided by two. 4. The modulation apparatus according to claim 1, wherein the conversion rule states that the number of consecutive minimum run length d of the channel bit string is limited to a predetermined number or less. 5. The modulation apparatus according to claim 1, wherein the conversion rule states a variable-length code (d, k; m, n; r), where the minimum run length d=1, the maximum run length k=7, the length of basic data prior to conversion m=2, and the length of basic channel bits subsequent to conversion n=3. 6. The modulation apparatus according to claim 1, wherein data of m length, which is the length of basic data, is input for a time period in which the channel bit string of n length, which is the length of the basic channel bits, is output. 7. The modulation apparatus according to claim 1, wherein the DSV-control-bit generating mean includes: first-candidate-bit-inserted bit string generating means for generating a first-candidate-bit-inserted bit string, which is a candidate for the DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into the predetermined position of the input bit string; second-candidate-bit-inserted bit string generating means for generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; second modulation means for modulating, on the basis of the same conversion rule as the conversion rule used by the first modulation means, the first-candidate-bit-inserted bit string generated by the first-candidate-bit-inserted bit string generating means into a first candidate channel bit string, which is a candidate for the channel bit string, and for modulating the second-candidate-bit-inserted bit string generated by the second-candidate-bit-inserted bit string generating means into a second candidate channel bit string, which is another candidate for the channel bit string; DSV calculating means for calculating a DSV of each of the first and second candidate channel bit strings generated by modulation by the second modulation means; and DSV control bit determining means for determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated by the DSV calculating means. 8. The modulation apparatus according to claim 7, wherein the DSV calculating means includes: section DSV calculating means for calculating a section DSV of a current DSV control section of each of the first and second candidate channel bit strings; cumulative DSV calculating means for calculating a cumulative DSV on the basis of the determination result by the DSV control bit determining means; and adding means for calculating the DSV by adding each section DSV calculated by the section DSV calculating means and the cumulative DSV immediately before the current DSV control section, the cumulative DSV being calculated by the cumulative DSV calculating means. 9. The modulation apparatus according to claim 7, wherein the first and second modulation means each have a minimum number of registers required to perform modulation based on the coding rule. 10. The modulation apparatus according to claim 1, further comprising: first sync signal inserting means for inserting a sync pattern including a preset unique pattern into the channel bit string, wherein the DSV-control-bit generating means includes second sync signal inserting means for inserting the same sync pattern as the sync pattern inserted by the first sync signal inserting means into each of the first and second candidate channel bit strings generated by modulating the first- and second-candidate-bit-inserted bit strings generated by inserting the first and second candidate bits, respectively, into the input bit string, and wherein the DSV calculating means calculates the DSV on the basis of each of the first and second candidate channel bit strings, each of which includes the sync pattern inserted by the second sync signal inserting means. 11. The modulation apparatus according to claim 1, wherein the timing adjusting means adjusts the transmission timing by adding a delay time to the input bit string. 12. The modulation apparatus according to claim 1, wherein the timing adjusting means inserts a temporary value prior to determination of the DSV control bit into the input bit string at predetermined intervals. 13. The modulation apparatus according to claim 1, further comprising checking information generating means for calculating a final cumulative DSV of the recording code string or the transmission code string, determining whether or not the calculated final cumulative DSV is within a predetermined range, and generating checking information on the basis of the determination result, wherein the DSV-control-bit generating means generates the DSV control bit on the basis of the checking information generated by the checking information generating means. 14. The modulation apparatus according to claim 13, wherein, when it is determined that the final cumulative DSV is out of the predetermined range, the checking information generating means resets the final cumulative DSV to zero and generates an error signal serving as the checking information, and wherein the DSV-control-bit generating means internally calculates a cumulative DSV for generating the DSV control bit and, when the error signal is generated by the checking information generating means, resets the cumulative DSV to zero. 15. A modulation method for a modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string, comprising: a DSV-control-bit generating step of generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into the channel bit string on the basis of a conversion rule (d, k; m, n; r). 16. A recording medium providing a computer-readable computer program for controlling a modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string, the program comprising: a DSV-control-bit generating step of generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into the channel bit string on the basis of a conversion rule (d, k; m, n; r). 17. A program for causing a computer that controls a modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string to perform a process comprising: a DSV-control-bit generating step of generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into the channel bit string on the basis of a conversion rule (d, k; m, n; r). 18. A DSV-control-bit generating method for generating a DSV control bit to be inserted into an input bit string, comprising: a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into a predetermined position of the input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string generated from the input bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step. 19. A recording medium providing a computer-readable program for generating a DSV control bit to be inserted into an input bit string, the program comprising: a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into a predetermined position of the input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string generated from the input bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step. 20. A program for generating a DSV control bit to be inserted into an input bit string, the program causing a computer to perform a process comprising: a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into a predetermined position of the input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string generated from the input bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step. |
<SOH> BACKGROUND ART <EOH>In transferring data to a predetermined transmission line or in recording data onto a recording medium, such as a magnetic disk, an optical disk, or a magneto-optical disk, the data is modulated to suit the transfer or recording. One known modulation method is block coding. Block coding is to divide a data stream into blocks of mΓi bits (hereinafter the blocks are referred to as data words), and each data word is converted into a codeword of nΓi bits in accordance with an appropriate coding rule. When i=1, the resultant code is a fixed-length code. When i can be of multiple values, that is, when i is selected from a range of 1 to i max (maximum i) and conversion is performed with the selected i, the resultant code is a variable-length code. The code generated by block coding is defined as a variable-length code (d, k; m, n; r). In the above description, i denotes the constraint length, and i max is r (maximum constraint length); d denotes the minimum number of consecutive β0sβ between consecutive β1sβ, e.g., the minimum run length of β0β; and k denotes the maximum number of consecutive β0sβ between consecutive β1sβ, e.g., the maximum run length of β0β. In recording the code generated as described above onto an optical disk, a magneto-optical disk, or the like, such as a compact disk (CD) or mini disk (MD), the variable-length code is subjected to NRZI (Non Return to Zero Inverted) modulation in which β1β is inverted whereas β0β is not inverted, and recording is done on the basis of the NRZI-modulated variable-length code (hereinafter referred to as the recording code string). There is a system, such as that on a magneto-optical disk in an initial ISO format having a not-so-high recording density, which records a modulated recording bit string that has not been subjected to NRZI modulation. The minimum inversion interval of the recording code string is denoted by Tmin, and the maximum inversion interval of the recording code string is denoted by Tmax. Higher recording density in the direction of linear velocity is realized when the minimum inversion interval Tmin is longer, that is, when the minimum run length d is greater. On the other hand, in terms of clock reading, it is preferable to have a shorter maximum inversion interval Tmax, that is, a smaller maximum run length k. Various modulations methods have been proposed. Specifically, modulation systems proposed or actually used in, for example, optical disks, magnetic disks, magneto-optical disks, and the like are described as follows. For example, RLL codes (Run Length Limited Code) with the minimum run length d=2 include an EFM (Eight to Fourteen Modulation) code (may also be represented as (2, 10: 8, 17; 1)) used in CD, MD, and the like; an 8-16 code (may also be represented as (2, 10: 8, 16; 1)) used in DVD (Digital Video Disk); an RLL (2-7) (may also be represented as (2, 7; 1, 2; r)) used in PD (Phase Change Disk); and the like. RLL codes with the minimum run length d=1 include a fixed-length RLL (1-7) (may also be represented as (1, 7; 2, 3; 1)) used in an ISO-format MO disk (Magnetic-Optical Disk); and a variable-length RLL (1-7) (may also be represented as (1, 7; 2, 3; r)) used in a disk drive for a high-density optical disk, magneto-optical disk, or the like. A conversion table for the variable-length RLL (1-7) is as follows: TABLE 1 RLL (1, 7, 2, 3, 2) Data word Codeword i = 1 11 00x 10 010 01 10x i = 2 0011 000 00x 0010 000 010 0001 100 00x 0000 100 010 The symbol x in the conversion table corresponds to 1 when the subsequent codeword is 0 and corresponds to 0 when the subsequent codeword is 1. The maximum constraint length r is 2. The parameters of the variable-length RLL (1-7) are (1, 7; 2, 3; 2). When the bit interval of the recording code string is denoted by T, the minimum inversion interval Tmin expressed as (d+1) is 2(=1+1)T. When the bit interval of the data stream is denoted by Tdata, the minimum inversion interval Tmin expressed as (m/n)Γ2 is 1.33(=(β
)Γ2)Tdata. In the above description, m/n denotes conversion at the ratio m:n. For example, β
denotes conversion at 2:3 (conversion of a data word of 2Γi bits into a codeword of 3Γi bits). The maximum inversion interval Tmax expressed as (k+1)T is 8(=7+1)T ((=(β
)Γ8Tdata=5.33Tdata). The detection window margin Tw is expressed as (m/n)ΓTdata and is 0.67(=β
)Tdata. In a code string (channel bit string) generated by RLL (1-7) modulation in Table 1, 2T which is Tmin occurs most frequently, which is followed by 3T, 4T, and so forth. The fact that edge information, such as 2T or 3T, occurs many times with a short cycle is advantageous in reading clock. In contrast, when the recording linear density becomes higher, the minimum run length causes a problem. Specifically, when 2T (the minimum run length) occurs consecutively, the recording waveform is easily distorted because the waveform output of 2T is smaller than the others and is influenced more easily by defocus, tangential tilt, or the like. In recording at high linear density, recording with consecutive minimum marks is influenced more easily by disturbance, such as noise, and this may easily lead to a data read error. In such a case in which a data read error occurs, the error often resides in shift of start-edge and end-edge of the consecutive minimum marks. In other words, the generated bit error length becomes longer. In order to solve this problem, the consecutive minimum run lengths are controlled to better suit high linear density. In contrast, in recording onto a recording medium or in data transfer, code modulation based on each medium (transfer) is done. When a modulated code contains a DC component, fluctuation or jitter may be caused in various error signals, such as for a tracking error in servo control of a disk drive. It is preferable for the modulated code not to contain a DC component. In order to solve this problem, DSV (Digital Sum Value) control is proposed. DSV is the sum of bits of an NRZI-modulated (level-coded) bit string (channel bit string) in which β1β corresponds to +1 and β0β corresponds to β1. DSV serves as a reference for a DC component in the code string. Minimizing the absolute value of DSV, that is, performing DSV control, enables suppression of a DC component in the code string. In the code modulated according to the variable-length RLL (1-7) table shown in Table 1, no DSV control is performed. In such a case, DSV control is performed by calculating DSV of the modulated channel bit string at a predetermined interval and inserting predetermined DSV control bits into the code string. Basically, the DSV control bits are redundant bits. In view of the efficiency of code conversion, the fewer the DSV control bits, the better. It is preferable that the minimum run length d and the maximum run length k remain unchanged by the inserted DSV control bits. Changes of (d, k) influence the reading and writing characteristics. In order to satisfy the above requirements, DSV control must be performed in as efficiently as possible. While the actual RLL code must satisfy the minimum run length requirement, the maximum run length requirement need not be satisfied. There is a format that uses a pattern exceeding the maximum run length for a sync signal. For example, although EFM plus for DVD has a maximum run length of 11T, EFM plus for DVD allows for 14T as a matter of convenience of the format. By exceeding the maximum run length, for example, the capability of detecting a sync signal or the like is enhanced greatly. In the RLL (1-7) format with improved conversion efficiency, it is important to βcontrol the consecutive minimum run lengths so as to better suit the high linear densityβ and to βperform DSV control as efficiently as possibleβ in association with an increase in linear density. Accordingly, the assignee of the present invention et al. discloses, in Japanese Patent Application No. 10-150280, a conversion table including, as a conversion code, a basic code where d=1, k=7, m=2, and n=3; a coding rule that the remainder of the number of β1sβ in each element of a data stream divided by two must be one or zero and equal the remainder of the number of β1sβ in a converted channel bit string divided by two; a first replacement code for limiting the consecutive minimum run lengths d to a predetermined number or less; and a second replacement code for satisfying the run length limitation. Specifically, when a disk drive with high linear density reads/writes an RLL code, a pattern with consecutive minimum run lengths often causes a long error. When DSV control is performed on an RLL code, such as the RLL (1-7) code, DSV control bits need to be inserted into a code string (channel bit string) at arbitrary intervals. As described above, since the DSV control bits are redundant bits, it is preferable to have fewer DSV control bits. In order to maintain the minimum run length or the maximum run length, the DSV control bits of at least 2 bits or greater are necessary. The assignee of the present invention et al. discloses, in Japanese Patent Application No. 10-150280, an RLL code with the minimum run length d=1 (d, k; m, n) and a conversion table, which is shown in Table 2, for limiting the number of consecutive minimum run lengths and for performing complete DSV control using efficient control bits while maintaining the minimum run length and the maximum run length (hereinafter referred to as a 1,7PP table; and a code according to 1,7PP table is referred to as a 1,7PP code): TABLE 2 1, 7PP (d, k, m, n, r) = (1, 7, 2, 3, 4) Data word Codeword 11 *0* 10 001 01 010 0011 010 100 0010 010 000 0001 000 100 000011 000 100 100 000010 000 100 000 000001 010 100 100 000000 010 100 000 β110111 001 000 000 (next010) 00001000 000 100 100 100 00000000 010 100 100 100 if xx1 then *0* = 000 xx0 then *0* = 101 Termination table 00 000 0000 010 100 β110111 001 000 000 (next010) When next channel bits are β010β convert β11 01 11β to β001 000 000β after using main table and termination table. As an example of a modulation apparatus using the 1,7PP table, the assignee of the present invention discloses, in Japanese Patent Application No. 10-150280, a modulation apparatus 1 shown in FIG. 1 . The modulation apparatus 1 includes a DSV control bit determination and insertion unit 11 for determining β1β or β0β serving as a DSV control bit and inserting the DSV control bit into an input data stream at arbitrary intervals; a modulator 12 for modulating the data stream containing the DSV control bits; and an NRZI unit 13 for converting the output of the modulator 12 into a recording code string. Although not shown in the diagram, the modulation apparatus 1 includes a timing management unit for generating a timing signal, supplying the timing signal to the above components, and managing timing. In Japanese Patent Application No. 09-342416, the assignee of the present invention et al. discloses a specific example of another modulation apparatus, namely, a modulation apparatus 2 shown in FIG. 2 . The modulation apparatus includes a DSV control bit insertion unit 21 for inserting β1β or β0β serving as a DSV control bit into a data stream at arbitrary intervals. At this time, there is a data stream into which the DSV control bit β 1 β is inserted and another data stream into which the DSV control bit β 0 β is inserted. The modulation apparatus further includes a modulator 22 for modulating the data stream containing the DSV control bits and a DSV controller 23 for NRZI-modulating the modulated code string into level data, calculating DSV of the level data, and consequently outputting a DSV-controlled recording code string. As described above, the 1,7PP code is advantageous in solving the above problems. In contrast, compared with a modulation apparatus using a known method or a technique for performing DSV control on the RLL (1,7) code, the configuration of the known modulation apparatus using the 1,7PP code is complicated, and the circuit size is increased. For example, in the modulation apparatus 2 shown in FIG. 2 , the register configuration in the modulator 22 is shown in FIG. 3 . Specifically, the modulator 22 has a modulation (1,7PP modulation) portion and a delay portion corresponding to a DSV control interval (DSV section) in one integrated unit in order to transfer data corresponding to the DSV control interval to the DSV controller 23 at a subsequent stage. As a result, the modulator 22 needs two registers, that is, an input register 22 a (register 22 a for the data stream) and an output register 22 b (register 22 b for the channel bit string). The number of registers required corresponds to the DSV control interval. Two pairs of registers (registers 22 a and 22 b ) are necessary for the DSV control bit β0β and the DSV control bit β1β. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a block diagram showing an example of the configuration of a known modulation apparatus. FIG. 2 is a block diagram showing an example of the configuration of another known modulation apparatus. FIG. 3 is a diagram showing an example of the register configuration in a modulator of the modulation apparatus shown in FIG. 2 . FIG. 4 is a block diagram showing an example of the configuration of a modulation apparatus according to the present invention. FIG. 5 is a block diagram showing details of the configuration of the modulation apparatus shown in FIG. 4 . FIG. 6 is a flowchart for describing the operation of the modulation apparatus shown in FIG. 4 . FIG. 7 is a diagram for describing the data format at each stage of a data stream modulated by the modulation apparatus shown in FIG. 4 . FIG. 8 is a chart for describing timing of data to be input to the modulation apparatus shown in FIG. 4 . FIG. 9 is a diagram showing an example of the register configuration in a modulator of the modulation apparatus shown in FIG. 4 . FIG. 10 is a block diagram showing an example of the configuration of another modulation apparatus according to the present invention. detailed-description description="Detailed Description" end="lead"? |
Systems and methods for automatic carburetor enrichment during cold start |
An automatic carburetor (20) enrichment system that controls the air-fuel mixture during cold start of an engine (10) having a carburetor including a fuel bowl (22) and an induction passage (23), includes a sensor (50) that provides a signal indicative of an engine temperature at engine start, a fuel line (30) connected between the fuel bowl (22) and the induction passage, a solenoid valve (40) disposed in the fuel line, and a controller (10) that receives the signal and sets a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture at engine start. The automatic carburetor enrichment system reduces cranking time during cold start, eliminates the need for driver input during cold start, prevents engine stalling without assistance from the operator during the warm up phase, provides a simpler, more cost effective and reliable carburetor enrichment, provides self-drowning protection without the use of an electronic idle switch and eliminates the risk of engine drowning when the engine is cranked with the choke ON and the ignition switches OFF. |
1. A carburetor enrichment system that controls an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start, the engine having a carburetor, that is supplied fuel from a fuel reservoir, and includes an induction passage, the carburetor enrichment system comprising: a sensor that provides a signal indicative of an engine temperature; a fuel line connected between the fuel reservoir and the induction passage; a solenoid valve disposed in the fuel line; and a controller that receives the signal and sets a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture. 2. A carburetor enrichment system according to claim 1, wherein the controller sets the duty cycle of the solenoid valve at one of a) an engine start time and b) a boost duration time associated with the engine temperature. 3. A carburetor enrichment system according to claim 2, wherein the engine start time is determined when the engine reaches a predetermined idle threshold speed. 4. A carburetor enrichment system according to claim 2, wherein the controller stores a plurality of engine temperatures and associated boost duration times. 5. A carburetor enrichment system according to claim 1, wherein the controller stores a plurality of engine temperatures and associated duty cycles. 6. A carburetor enrichment system according to claim 2, wherein the controller reduces the duty cycle of the solenoid valve at a predetermined rate after one of a) the engine start time and b) the boost duration time. 7. A carburetor enrichment system according to claim 6, wherein the predetermined rate is constant. 8. A carburetor enrichment system according to claim 2, wherein the duty cycle of the solenoid valve is a maximum duty cycle prior to one of a) the engine start time and b) the boost duration time. 9. A carburetor enrichment system that controls an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start the engine having a carburetor, that is supplied fuel from a fuel reservoir, and includes an induction passage, the carburetor enrichment system comprising: a sensor that provides a signal indicative of an engine temperature; a fuel line connected between the fuel reservoir and the induction passage; a solenoid valve disposed in the fuel line; and a controller that sets a duty cycle of the solenoid valve to a maximum duty cycle, receives the signal, reduces the duty cycle from the maximum duty cycle to a determined duty cycle associated with the engine temperature, and further reduces the duty cycle at a predetermined rate after one of a) engine start and b) a boost duration time associated with the engine temperature. 10. A carburetor enrichment system according to claim 9, wherein the engine start time is determined when the engine reaches a predetermined idle threshold speed. 11. A method of controlling an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start, the engine having a carburetor including an induction passage that is supplied fuel by a fuel line connected between a fuel reservoir and the induction passage, the fuel line having a solenoid valve disposed therein, the method comprising: determining an engine temperature; and setting a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture. 12. A method according to claim 11, wherein setting the duty cycle occurs at one of a) an engine start time and b) a boost duration time associated with the engine temperature. 13. A method according to claim 12, wherein the engine start time is determined when the engine reaches a predetermined idle threshold speed. 14. A method according to claim 12, wherein a plurality of engine temperatures are associated with a plurality of boost duration times. 15. A method according to claim 11, wherein a plurality of engine temperatures ar associated with a plurality of duty cycles. 16. A method according to claim 12, further comprising reducing the duty cycle of the solenoid valve at a predetermined rate after one of a) the engine start time and b) the boost duration time. 17. A method according to claim 16, wherein the predetermined rate is constant. 18. A method according to claim 12, wherein the duty cycle of the solenoid valve is a maximum duty cycle prior to one of a) the engine start time and b) the boost duration time. 19. A method for controlling an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start the engine having a carburetor including an induction passage that is supplied by a fuel line connected between a fuel reservoir and the induction passage, the fuel line having a solenoid valve disposed therein, the method comprising: determining an engine temperature; setting a duty cycle of the solenoid valve to a maximum duty cycle; reducing the duty cycle from the maximum duty cycle to a determined duty cycle associated with the engine temperature; and further reducing the duty cycle at a predetermined rate after one of a) engine start and b) a boost duration time associated with the engine temperature. 20. A method according to claim 19, wherein the engine start time is determined when the engine reached a predetermined idle threshold speed. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates to systems and methods for automatic carburetor enrichment during cold start of an engine. 2. Description of the Related Art In internal combustion engines having carburetor controlled fuel supplies, for example, engines used in vehicles such as snowmobiles, personal watercraft, and all terrain vehicles, the rate of fuel flow in a fixed or variable venturi carburetor is dependent on the pressure differential between the venturi and the fuel bowl, also known as the float bowl or float chamber. In a conventional float bowl carburetor the pressure differential is measured between the pressure in the fluid float chamber, which is normally atmospheric, and the pressure at the discharge orifice of the fuel metering system which is normally located in or adjacent the venturi in the induction passage. For optimum combustion, the relationship between the mass air flow and the mass fuel flow delivered to the engine by the carburetor should be kept to a controllable rate. A fixed or variable venturi may be used to provide a constant relationship between the mass air flow and the mass fuel flow. As the air velocity in the induction passage increases, a pressure reduction, or vacuum, is created in the venturi. The pressure reduction creates a pressure differential between the induction passage and the fuel in the float chamber causing fuel to be drawn into the induction passage at a flow rate that is proportional to the pressure differential. The pressure reduction is mainly a function of the air velocity through the induction passage. However, at a given velocity, the mass air flow rate is dependent on the air density which in turn is dependent on the barometric pressure and temperature. For a given air velocity, the induction passage delivers a reduced mass air flow at higher altitudes due to the reduced barometric pressure and correspondingly reduced air density. Operating the engine at higher altitudes thus causes the engine to be supplied with an over rich air-fuel mixture. Conversely, for a given air velocity, the induction passage delivers an increased mass air flow at lower temperatures due to the increased air density. Operating the engine at lower temperatures thus causes the engine to be supplied with an over lean air-fuel mixture. U.S. Pat. No. 5,021,198 to Bostelmann, the entire contents of which are herein incorporated by reference, discloses a carburetor with a high altitude compensator that includes a pressure splitter connected with the lower pressure of the venturi throat in the area where the fuel delivery line opens out and with the induction pressure in the area of the inlet end of the air flow passage. The pressure splitter includes a pressure line with two chokes that are connected in series. The fuel bowl is connected to the pressure line between the two chokes and one or both of the chokes is controlled as a function of the specific air density. The control system of U.S. Pat. No. 5,021,198 cannot provide an enriched air-fuel mixture during cold start When the engine is cold it is difficult to start because it is difficult to create a sufficient amount of fuel vapor in the combustion chamber because atomization and vaporization are less effective at lower temperatures. It is therefore necessary to increase the amount of fuel in order to compensate for the lack of atomization. It has been known to increase the amount of fuel by using a manual primer or an air pump enrichment system at the carburetor. U.S. application Ser. No. 08/948,064, the entire contents of which are hereby incorporated by reference, discloses an electronic compensation system for an internal combustion engine including a manifold connected to the float chamber and the venturi of each carburetor. A barometric pressure sensor and an engine temperature sensor provide signals to an electronic control unit that controls first and second solenoids connected to the manifold. During a cold start, the first solenoid is controlled to provide pressurized gas, provided by a pressure line in communication with the crankcase interior, from the manifold to the float chambers of the carburetors to increase the fuel flow and enrich the air-fuel mixture. As the engine temperature increases, the electronic control unit responds to signals from the engine temperature sensor to reduce the duty cycle of the first solenoid and thus reduce the supply of pressurized gas to the float chambers until the normal engine operating temperature is reached. The second solenoid is controlled during normal engine operation to apply the underpressure, or vacuum, from the venturis of the carburetors to the manifold to reduce the float chamber pressure to decrease the fuel flow when the air density is reduced, for example, at increased altitudes. The electronic compensation system of U.S. application Ser. No. 08/948,064 requires the use of an air pump when the rotation speed of the engine c corresponds to idling or higher. While completely reliable during operation, condensation of fuel, oil, and water in the air pump decreases the reliability of the air pump. The electronic compensation system of U.S. application Ser. No. 08/948,064 also requires an expensive idle switch to signal the position of the throttle during idling to the electronic control unit and to limit fuel distribution to avoid flooding the engine. In addition, the electronic compensation system of U.S. application Ser. No. 08/948,064 requires the insertion of an impulse line from the interior of the crankcase to the air pump to activate a diaphragm of the air pump. |
<SOH> SUMMARY OF THE INVENTION <EOH>There exists a need for an engine control system that automatically provides an enriched air-fuel mixture during cold start and that does not require a manifold and an air pump for providing pressurized gas to the float chamber and an impulse line for activating the diaphragm of the air pump. There also exists a need for an engine control system that automatically reduces the enriched air-fuel mixture as the engine operating temperature approaches a normal engine operating temperature and that does not require an idle switch for limiting the fuel distribution to the engine when in off-idle mode. A system for automatic carburetor enrichment during cold start according to the present invention controls an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start, the engine having a carburetor and being supplied fuel from a fuel reservoir, for example a fuel bowl, and an induction passage, and includes a sensor that provides a signal indicative of an engine temperature at engine start, a fuel line connected between the fuel bowl and the induction passage, a solenoid valve disposed in the fuel line, and a controller that receives the signal and sets a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture at engine start A method according to the present invention for controlling the air-fuel ratio of an air fuel mixture supplied to a carburetor of an internal combustion engine during cold start, the engine having a carburetor including a fuel bowl, a fuel line between the fuel reservoir and an induction passage of the carburetor, and a solenoid valve disposed in the fuel line, includes determining a temperature of the engine, determining a duty cycle of the solenoid valve associated with the determined temperature, setting the duty cycle of the solenoid valve to the associated duty cycle at an engine start time to increase the air-fuel ratio of the air-fuel mixture at engine start. |
Sequence of the photorhabdus luminescens strain tt01 genome and uses |
The present invention relates to the genomic sequence and to nucleotide sequences encoding polypeptides of Photorhabdus luminescens. The present invention further relates to polypeptides involved in operons involved in the biosynthesis of antibiotics or toxins, as well as polypeptides with activity of the antibiotic or toxin. Uses of the aforementioned polypeptides in pesticides, bactericides, or fungicides is provides. In addition, the present invention provides vectors, cells, or animals containing the sequences of the present invention. |
1-56. (canceled) 57. An isolated nucleotide sequence derived from the Photorhabdus luminescens genome, comprising a sequence selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 41 and SEQ ID No. 5826 to SEQ ID No. 5834. 58. An isolated nucleotide sequence derived from the Photorhabdus luminescens genome, selected from the group consisting of: a) a nucleotide sequence comprising at least 75% identity with a sequence chosen selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 41 and SEQ ID No. 5826 to SEQ ID No. 5834; b) a nucleotide sequence comprising a fragment of a sequence selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 41 and SEQ ID No. 5826 to SEQ ID No. 5834; c) a nucleotide sequence complementary to a nucleotide sequence as defined in a) or b); d) a nucleotide sequence of the RNA corresponding to one of the sequences as defined in a), b) or c); and e) a nucleotide sequence as defined in a), b), c) or d), which has been modified. 59. The nucleotide sequence as claimed in claim 58, wherein the nucleotide sequence is a fragment of a nucleotide sequence selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 41 and SEQ ID No. 5826 to SEQ ID No. 5834, and wherein said fragment encodes a polypeptide selected from the group consisting of SEQ ID No. 42 to SEQ ID No. 3855 or a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No. 5835 to SEQ ID No. 10784. 60. The nucleotide sequence as claimed in claim 59, wherein: a) said nucleotide sequence encodes a polypeptide selected from the group consisting of SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 67, SEQ ID No. 171, SEQ ID No. 221, SEQ ID No. 268, SEQ ID No. 288, SEQ ID No. 380, SEQ ID No. 426, SEQ ID No. 438, SEQ ID No. 448, SEQ ID No. 453, SEQ ID No. 455, SEQ ID No. 456, SEQ ID No. 458, SEQ ID No. 501, SEQ ID No. 516, SEQ ID No. 530, SEQ ID No. 542, SEQ ID No. 551, SEQ ID No. 720, SEQ ID No. 761, SEQ ID No. 762, SEQ ID No. 814, SEQ ID No. 859, SEQ ID No. 860, SEQ ID No. 861, SEQ ID No. 862, SEQ ID No. 869, SEQ ID No. 1079, SEQ ID No. 1168, SEQ ID No. 1174, SEQ ID No. 1176, SEQ ID No. 1413, SEQ ID No. 1414, SEQ ID No. 1415, SEQ ID No. 1416, SEQ ID No. 1417, SEQ ID No. 1457, SEQ ID No. 1651, SEQ ID No. 1856, SEQ ID No. 1869, SEQ ID No. 2021, SEQ ID No. 2080, SEQ ID No. 2152, SEQ ID No. 2162, SEQ ID No. 2173, SEQ ID No. 2251, SEQ ID No. 2295, SEQ ID No. 2306, SEQ ID No. 2317, SEQ ID No. 2328, SEQ ID No. 2340, SEQ ID No. 2342, SEQ ID No. 2351, SEQ ID No. 2500, SEQ ID No. 3228, SEQ ID No. 3230, SEQ ID No. 3311, SEQ ID No. 3317, SEQ ID No. 3318, SEQ ID No. 3319, SEQ ID No. 3320, SEQ ID No. 3322, SEQ ID No. 3323, SEQ ID No. 3326, SEQ ID No. 3327, SEQ ID No. 3328, SEQ ID No. 3375, SEQ ID No. 3376, SEQ ID No. 3377, SEQ ID No. 3378, SEQ ID No. 3422, SEQ ID No. 3489, SEQ ID No. 3503, SEQ ID No. 3609, SEQ ID No. 3623, SEQ ID No. 3624, SEQ ID No. 3772, SEQ ID No. 3783, SEQ ID No. 3788 and SEQ ID No. 3794; or b) said nucleotide sequence is selected from the group consisting of SEQ ID No. 5835 to SEQ ID No. 10784. 61. A nucleotide sequence, comprising a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence as claimed in claim 59; b) a nucleotide sequence comprising at least 75% identity with a nucleotide sequence as claimed in claim 59; c) a complementary or RNA nucleotide sequence corresponding to a sequence as defined in a) or b); d) a nucleotide sequence of a representative fragment of a sequence as defined in a) or c); and e) a sequence as defined in a) or c), which has been modified. 62. A polypeptide encoded by a nucleotide sequence as claimed in claim 58. 63. A polypeptide as claimed in claim 62, wherein: a) said polypeptide is selected from the group consisting of SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 67, SEQ ID No. 171, SEQ ID No. 221, SEQ ID No. 268, SEQ ID No. 288, SEQ ID No. 380, SEQ ID No. 426, SEQ ID No. 438, SEQ ID No. 448, SEQ ID No. 453, SEQ ID No. 455, SEQ ID No. 456, SEQ ID No. 458, SEQ ID No. 501, SEQ ID No. 516, SEQ ID No. 530, SEQ ID No. 542, SEQ ID No. 551, SEQ ID No. 720, SEQ ID No. 761, SEQ ID No. 762, SEQ ID No. 814, SEQ ID No. 859, SEQ ID No. 860, SEQ ID No. 861, SEQ ID No. 862, SEQ ID No. 869, SEQ ID No. 1079, SEQ ID No. 1168, SEQ ID No. 1174, SEQ ID No. 1176, SEQ ID No. 1413, SEQ ID No. 1414, SEQ ID No. 1415, SEQ ID No. 1416, SEQ ID No. 1417, SEQ ID No. 1457, SEQ ID No. 1651, SEQ ID No. 1856, SEQ ID No. 1869, SEQ ID No. 2021, SEQ ID No. 2080, SEQ ID No. 2152, SEQ ID No. 2162, SEQ ID No. 2173, SEQ ID No. 2251, SEQ ID No. 2295, SEQ ID No. 2306, SEQ ID No. 2317, SEQ ID No. 2328, SEQ ID No. 2340, SEQ ID No. 2342, SEQ ID No. 2351, SEQ ID No. 2500, SEQ ID No. 3228, SEQ ID No. 3230, SEQ ID No. 3311, SEQ ID No. 3317, SEQ ID No. 3318, SEQ ID No. 3319, SEQ ID No. 3320, SEQ ID No. 3322, SEQ ID No. 3323, SEQ ID No. 3326, SEQ ID No. 3327, SEQ ID No. 3328, SEQ ID No. 3375, SEQ ID No. 3376, SEQ ID No. 3377, SEQ ID No. 3378, SEQ ID No. 3422, SEQ ID No. 3489, SEQ ID No. 3503, SEQ ID No. 3609, SEQ ID No. 3623, SEQ ID No. 3624, SEQ ID No. 3772, SEQ ID No. 3783, SEQ ID No. 3788 and SEQ ID No. 3794; or b) said polypeptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID No. 5835 to SEQ ID No. 10784. 64. A polypeptide, comprising a polypeptide selected from the group consisting of: a) a polypeptide as claimed in claim 62; b) a polypeptide exhibiting at least 80% identity with a polypeptide of sequence selected from the group consisting of SEQ ID No. 42 to SEQ ID No. 3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No. 5835 to SEQ ID No. 10784; c) a fragment of at least 5 amino acids of a polypeptide of sequence selected from the group consisting of SEQ ID No. 42 to SEQ ID No. 3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No. 5835 to SEQ ID No. 10784; d) a biologically active fragment of a polypeptide of sequence selected from the group consisting of SEQ ID No. 42 to SEQ ID No. 3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No. 5835 to SEQ ID No. 10784; and e) a polypeptide of sequence selected from the group consisting of SEQ ID No. 42 to SEQ ID No. 3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No. 5835 to SEQ ID No. 10784, or as defined in c) or d), which has been modified. 65. A nucleotide sequence encoding a polypeptide as claimed in claim 62. 66. The nucleotide sequence as claimed in claim 58, wherein said nucleotide sequence encodes a polypeptide of P. luminescens having toxin activity or antibiotic activity, or is involved in the synthesis of toxins or antibiotics. 67. The polypeptide as claimed in claim 62, wherein said polypeptide is a polypeptide of P. luminescens having toxin activity or antibiotic activity, or is involved in the synthesis of toxins or antibiotics, or a fragment thereof. 68. A recording medium, comprising one or more nucleotide sequences as claimed in claim 57. 69. The recording medium as claimed in claim 68, wherein said recording medium is selected from the group consisting of a CD-ROM, a computer disk and a computer server. 70. A method of identifying primers or probes for determining genes in strains related to P. luminescens comprising inspecting the sequences recorded on a recording medium as claimed in claim 68 and identifying a primer pair or probe corresponding to a desired nucleotide or to a nucleotide sequence encoding a polypeptide with a desired function. 71. A method of studying the genetic polymorphism of strains related to P. luminescens comprising obtaining a nucleotide sequence from a strain related to P. luminescens sequencing the nucleotide sequence comparing the nucleotide sequence to the nucleotide sequences recorded on a recording medium as claimed in claim 68. 72. A method for the automatic annotation of genes originating from a genome other than P. luminescens comprising obtaining genomic DNA from a strain other than P. luminescens sequencing the genomic DNA comparing the nucleotide sequence of the genomic DNA to the nucleotide sequences recorded on a recording medium as claimed in claim 68 to assign putative function associated therewith. 73. A recording medium, comprising one or more polypeptides sequences as claimed in claim 62. 74. The recording medium as claimed in claim 73, wherein said recording medium is selected from the group consisting of a CD-ROM, a computer disk and a computer server. 75. A method of identifying primers or probes for determining genes in strains related to P. luminescens comprising inspecting the sequences recorded on a recording medium as claimed in claim 73 and identifying a primer pair or probe corresponding to a desired nucleotide or to a nucleotide sequence encoding a polypeptide with a desired function. 76. A method of studying the genetic polymorphism of strains related to P. luminescens comprising obtaining a nucleotide sequence from a strain related to P. luminescens sequencing the nucleotide sequence comparing the nucleotide sequence to the nucleotide sequences encoding the polypeptide sequences recorded on a recording medium as claimed in claim 73. 77. A method for the automatic annotation of genes originating from a genome other than P. luminescens comprising obtaining genomic DNA from a strain other than P. luminescens sequencing the genomic DNA comparing the nucleotide sequence of the genomic DNA to the nucleotide sequences encoding the polypeptide sequences recorded on a recording medium as claimed in claim 73 to assign putative function associated therewith 78. A primer or a probe, comprising one or more sequences as claimed in claim 57. 79. The nucleotide sequence as claimed in claim 78, wherein said primer or probe is labeled with a radioactive compound or with a nonradioactive compound. 80. The nucleotide sequence as claimed in claim 78, wherein said primer or probe is immobilized on a support by a covalent or noncovalent interaction. 81. The nucleotide sequence as claimed in claim 80, wherein said support is a high density filter or a DNA chip. 82. A DNA chip or a filter, comprising one or more nucleotide sequences as claimed in claim 78. 83. The DNA chip or the filter as claimed in claim 82, further comprising one or more nucleotide sequences from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said nucleotide sequences are immobilized on the support of said chip. 84. The DNA chip or the filter as claimed in claim 83, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens. 85. A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a DNA chip or a filter as claimed in claim 82. 86. A cloning and/or expression vector, comprising a nucleotide sequence as claimed in claim 57. 87. The cloning and/or expression vector as claimed in claim 86, comprising a nucleotide sequence selected from the group consisting of SEQ ID No. 3856 to SEQ ID No. 5825 and SEQ ID No. 5835 to SEQ ID No. 10784, or fragments thereof. 88. A host cell, transformed with a vector as claimed in claim 86. 89. A plant or an animal, except a human, comprising a transformed cell as claimed in claim 88. 90. A method for preparing a polypeptide, comprising culturing a cell transformed with a vector as claimed in claim 88 under conditions which allow the expression of said polypeptide, and recovering the resultant recombinant polypeptide. 91. A recombinant polypeptide obtained by the method as claimed in claim 90. 92. A method for preparing a polypeptide as claimed in claim 62, comprising chemical synthesizing said polypeptide and isolating said polypeptide. 93. An antibody selected from the group consisting of a monoclonal antibody, a polyclonal antibody, and a chimeric antibody, or a fragment thereof, wherein said antibody specifically recognizes a polypeptide as claimed in claim 62. 94. The antibody as claimed in claim 93, wherein said antibody is a labeled antibody. 95. A method for detecting and/or identifying bacteria belonging to the species P. luminescens, in a biological sample, comprising a) contacting said biological sample with an antibody as claimed in claim 93; b) detecting the formation of an antigen-antibody complex. 96. A method for detecting the expression of a gene of P. luminescens, comprising contacting a strain of P. luminescens with an antibody as claimed in claim 93, and detecting the formation of an antigen-antibody complex. 97. A kit for performing the method as claimed in claim 95, comprising the following elements: a) said antibody; and at least one of either b) reagents for constituting the medium suitable for an immunoreaction; or c) reagents for detecting the antigen-antibody complexes resulting from the immunoreaction. 98. A kit for performing the method as claimed in claim 96, comprising the following elements: a) said antibody; b) reagents for constituting the medium suitable for an immunoreaction; and c) reagents for detecting the antigen-antibody complexes resulting from the immunoreaction. 99. The polypeptide as claimed in claim 62, wherein said polypeptide is immobilized on a support 100. The polypeptide as claimed in claim 99, wherein said support a protein chip. 101. The polypeptide as claimed in claim 91, wherein said polypeptide is immobilized on a support 102. The polypeptide as claimed in claim 101, wherein said support a protein chip. 103. The antibody as claimed in claim 93, wherein said polypeptide is immobilized on a support 104. The antibody as claimed in claim 103, wherein said support a protein chip. 105. A protein chip comprising one or more polypeptide as claimed in claim 62 immobilized on the support of said chip. 106. The protein chip as claimed in claim 105, further comprising one or more polypeptides from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said polypeptides are immobilized on the support of said chip. 107. The protein chip as claimed in claim 106, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens. 108. A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a protein chip as claimed in claim 105. 109. A protein chip comprising one or more polypeptide as claimed in claim 91 immobilized on the support of said chip. 110. The protein chip as claimed in claim 109, further comprising one or more polypeptides from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said polypeptides are immobilized on the support of said chip. 111. The protein chip as claimed in claim 110, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens. 112. A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a protein chip as claimed in claim 109. 113. A protein chip comprising one or more antibodies as claimed in claim 93 immobilized on the support of said chip. 114. The protein chip as claimed in claim 113, further comprising one or more polypeptides from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said polypeptides are immobilized on the support of said chip. 115. The protein chip as claimed in claim 114, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens. 116. A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a protein chip as claimed in claim 113. 117. A method for detecting and/or identifying bacteria belonging to the species P. luminescens, in a biological sample, comprising: a) isolating the DNA, or cDNA from the RNA, from the biological sample; b) specifically amplifying the DNA of bacteria belonging to the species P. luminescens using at least one primer as claimed in claim 78; c) identifying the amplification products. 118. A kit or set for detecting and/or identifying bacteria belonging to the species P. luminescens, comprising: a) a nucleotide probe and/or primer as claimed in claim 78; and at least one of b) reagents required for carrying out a hybridization reaction; or c) reagents for a DNA amplification reaction. 119. A composition comprising one or more nucleotide sequences as claimed in claim 57. 120. A pharmaceutical composition comprising the composition as claimed in claim 119 and a pharmaceutically acceptable vehicle. 121. A biopesticidal composition comprising the composition as claimed in claim 119. 122. A composition comprising one or more polypeptides as claimed in claim 62. 123. A pharmaceutical composition comprising the composition as claimed in claim 122 and a pharmaceutically acceptable vehicle. 124. A biopesticidal composition comprising the composition as claimed in claim 122. 125. A composition comprising a vector as claimed in claim 86. 126. A pharmaceutical composition comprising the composition as claimed in claim 125 and a pharmaceutically acceptable vehicle. 127. A biopesticidal composition comprising the composition as claimed in claim 125. 128. A composition comprising one or more antibodies as claimed in claim 93. 129. A pharmaceutical composition comprising the composition as claimed in claim 128 and a pharmaceutically acceptable vehicle. 130. A biopesticidal composition comprising the composition as claimed in claim 128. 131. A method of preparing a toxin or an antibiotic comprising culturing a cell as claimed in claim 88, and expressing a polypeptide involved in production of a toxin or an antibiotic. 132. A genomic library of a bacterium of the genus Photorhabdus. 133. The genomic DNA library of a bacterium of the genus Photorhabdus as claimed in claim 132, wherein said DNA library is cloned into a plasmid. 134. The genomic DNA library as claimed in claim 132, wherein said bacterium is P. luminescens or P. luminescens strain TT01. 135. The genomic DNA library as claimed in claim 132, wherein said genomic DNA library is the genomic DNA library deposited with the CNCM on May 12, 2000, under accession No. I-2478. 136. A method for identifying at least one nucleotide sequence of P. luminescens not present in the genome of another species of bacterium or for identifying at least one nucleotide sequence of a genome of a bacterium of a species other than P. luminescens and not present in the P. luminescens genome, comprising: a) aligning the genomic sequences of the other bacterial species with the nucleotide sequences of P. luminescens as claimed in claim 57; and b) analyzing the data obtained by said aligning to identify and isolate said sequences only present in one or the other genome. 137. A method for identifying at least one nucleotide sequence of P. luminescens not present in the genome of another species of bacterium or for identifying at least one nucleotide sequence of a genome of a bacterium of a species other than P. luminescens and not present in the P. luminescens genome, comprising: a) aligning the genomic sequences of the other bacterial species with the genomic DNA library as claimed in claim 134; and b) analyzing the data obtained by said aligning to identify and isolate said sequences only present in one or the other genome. |
Benzo'fisoindole derivatives with affinity to the ep4 receptor |
The present invention relates to a compound of formula (I): corresponding pharmaceutical compositions, preparation processes, and/or methods of using the aforementioned compounds and/or compositions. |
1. A compound of formula (I): wherein: R1 and R3 are the same or different and represent βO, hydrogen, C1-6alkyl, C1-6-dialkyl, βCHC1-C5alkyl, βS, or a 5- or 6-membered aryl; R4 to R9 are the same or different and represent hydrogen, C1-6alkoxy, OCF3, OCH2CF3, O-cyclopropyl, OCH2-cyclopropyl, C1-C6alkyl, S-alkyl, NR210 where R10 is hydrogen or C1-6alkyl, halogen, NO2, OH, CH2OC1-C6alkyl, CH2OH, or CF3; Q1 is hydrogen, C1-6alkyl, C1-6dialkyl, C1-6alkoxy, NHAc, NR210 where R10 is hydrogen or C1-6alkyl, difluoro, fluoro, βO, or OH; Q2, Q3, Q4 and Q5 are the same or different and represent hydrogen, C1-6alkoxy, OCF3, OCH2CF3, O-cyclopropyl, OCH2-cyclopropyl, C1-C6alkyl, S-alkyl, NR210 where R10 is hydrogen or C1-6alkyl, halogen, NO2, OH, CH2OC1-C6alkyl, CH2OH, or a 5- or 6-membered aryl; provided that the compound of formula (I) is not: [4-(1-oxo-1,3-dihydro-2H-benzo [f]isoindol-2-yl)phenyl]-2-propionic acid, and sodium salt and [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; and pharmaceutically acceptable derivatives thereof. 2. A compound according to claim 1 wherein R1 is βO. 3. A compound according to claim 1 wherein R3 is methyl, dimethyl, 2-furanyl, βO or hydrogen. 4. A compound according to claim 1 wherein R4 and R9 are each hydrogen, methoxy, ethoxy, i-propoxy, n-propoxy, n-butyloxy or n-hexyloxy. 5. A compound according to claim 1 wherein R6 and R7 are each hydrogen, methyl, methoxy, chlorine, bromine, iodine, NO2, or CF3. 6. A compound according to claim 1 wherein Q1 is hydrogen, methyl, ethyl, NHAc, NH2 or methoxy. 7. A compound according to claim 1 wherein Q2, Q3, Q4 and Q5 are each hydrogen, methyl, methoxy, chlorine, bromine, iodine, 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, or phenyl. 8. A compound of formula (I) wherein said compound is selected from the group consisting of: [4-(4,9-dimethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid and [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid and [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo [f]isoindol-2-yl)phenyl]acetic acid; [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid and [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; and [4-(4-butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid. 9. A process for preparing a compound of formula (I) and pharmaceutically acceptable derivatives thereof according to claim 1 which comprises: (A) where R1 and R3 are both βO and R4 and R9 are the same or different and represent C1-6alkoxy, by reacting a compound of formula (II): with a 4-aminophenylacetic acid of formula (III): in glacial acetic acid at elevated temperature; (B) where one of R1 and R3 is βO and the other is hydrogen and R4 and R9 are the same or different and represent C1-6alkoxy, by reducing a compound of formula (IV): with a suitable reducing agent, followed by separation of isomers and deprotection; (C) where one of R1 and R3 is βO and the other is hydrogen and R4 and R9 are the same or different and represent C1-6alkoxy, by reacting a compound of formula (V): with a 4-aminophenylacetic acid of formula (III) as defined above in presence of triethylamine and dimethylformamide, followed by deprotection; (D) where one of R1 and R3 is βO and the other is hydrogen and one of R4 and R9 is C1-6alkoxy, by reacting a compound of formula (VI): where RD is in the β4 or -9 position and is C1-6 alkoxy, with a 4-aminophenylacetate of formula (VII): in presence of sodium triacetoxyborohydride in a suitable solvent, followed by deprotection; (E) where R1 and R3 are both hydrogen and R4 and R9 are the same or different and represent C1-6 alkoxy, by reacting a compound of formula (VIII): with a 4-aminophenylacetate of formula (VII) as described above in a suitable solvent, at elevated temperature followed by deprotection; (F) where R3 is βCHC1-5alkyl, by reacting a compound of formula (IV) as defined above with a Grignard reagent C1-C6alkyl-MgBr under conventional conditions, followed by separation of isomers and deprotection; (G) where R3 is C1-6dialkyl, by reacting a compound of formula (IX): with a 4-aminophenylacetate of formula (VII) as defined above in presence of aluminium trichloride, followed by deprotection; or (H) converting compounds of formula (I), prepared according to processes (A) to (G), into other compounds of formula (I) using conventional procedures; and optionally converting compounds of formula (I) prepared by any one of processes (A) to (H) into pharmaceutically acceptable derivatives thereof. 10. A compound of formula (I) or a pharmaceutically acceptable derivative thereof as defined in claim 1 for use in human or veterinary medicine. 11. (Cancelled) 12. A method of treating a human or animal subject suffering from a condition which is mediated by the action of PGE2 at EP4 receptors which comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof as defined in claim 1. 13. A pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable derivative thereof, as defined in claim 1 in admixture with one or more physiologically acceptable carriers or excipients. |
Protein-coupled receptor |
The present application provides a gene encoding a G protein-coupled receptor termed nGPCR-14; constructs and recombinant host cells incorporating the genes; the nGPCR-14 polypeptides encoded by the gene; antibodies to the nGPCR-x polypeptides; and methods of making and using all of the foregoing. |
1. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to a sequence of SEQ ID NO: 192, and fragments thereof; said nucleic acid molecule encoding at least a portion of nGPCR-14, wherein the isolated nucleic acid comprises at least one or more nucleotides from one or more of the following regions of SEQ ID NO: 191: nucleotides 1 to 193 of SEQ ID NO: 191; nucleotides 612 to 644 of SEQ ID NO:191; nucleotides 697 to 706 of SEQ ID NO:191; nucleotides 1011 to 1049 of SEQ ID NO: 191; nucleotides 1051 to 1057 of SEQ ID NO:191; nucleotides 1090 to 1096 of SEQ ID NO:191; or nucleotides 1141 to 1642 of SEQ ID NO:191. 2. The isolated nucleic acid molecule of claim 1 comprising a sequence that encodes a polypeptide comprising a sequences of SEQ ID NO: 192, and fragments thereof. 3. The isolated nucleic acid molecule of claim 1 comprising a sequence homologous to a sequence of SEQ ID NO: 191, and fragments thereof. 4. The isolated nucleic acid molecule of claim 1 comprising a sequence of SEQ ID NO:191, and fragments thereof. 5. The isolated nucleic acid molecule of claim 1 wherein said nucleic acid molecule is DNA. 6. The isolated nucleic acid molecule of claim 1 wherein said nucleic acid molecule is RNA. 7. An expression vector comprising a nucleic acid molecule of any one of claims 1 to 5. 8. The expression vector of claim 7 wherein said nucleic acid molecule comprises a sequence of SEQ ID NO:191. 9. The expression vector of claim 7 wherein said vector is a plasmid. 10. The expression vector of claim 7 wherein said vector is a viral particle. 11. The expression vector of claim 10 wherein said vector is selected from the group consisting of adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, and retroviruses. 12. The expression vector of claim 7 wherein said nucleic acid molecule is operably connected to a promoter selected from the group consisting of simian virus 40, mouse mammary tumor virus, long terminal repeat of human immunodeficiency virus, maloney virus, cytomegalovirus immediate early promoter, Epstein Barr virus, rous sarcoma virus, human actin, human myosin, human hemoglobin, human muscle creatine, and human metalothionein. 13. A host cell transformed with an expression vector of claim 7. 14. The transformed host cell of claim 13 wherein said cell is a bacterial cell. 15. The transformed host cell of claim 14 wherein said bacterial cell is E. coli. 16. The transformed host cell of claim 13 wherein said cell is yeast. 17. The transformed host cell of claim 16 wherein said yeast is S. cerevisiae. 18. The transformed host cell of claim 13 wherein said cell is an insect cell. 19. The transformed host cell of claim 18 wherein said insect cell is S. frugiperda. 20. The transformed host cell of claim 13 wherein said cell is a mammalian cell. 21. The transformed host cell of claim 20 wherein mammalian cell is selected from the group consisting of chinese hamster ovary cells, HeLa cells, African green monkey kidney cells, human 293 cells, and murine 3T3 fibroblasts. 22. An isolated nucleic acid molecule comprising a nucleotide sequence complementary to at least a portion of a sequence of SEQ ID NO:191, said portion comprising at least 10 nucleotides, wherein said isolated nucleic acid comprises at least one or more nucleotides from one or more of the following regions of SEQ ID NO: 191: nucleotides 1 to 193 of SEQ ID NO: 191; nucleotides 612 to 644 of SEQ ID NO:191; nucleotides 697 to 706 of SEQ ID NO: 191; nucleotides 1011 to 1049 of SEQ ID NO: 191; nucleotides 1051 to 1057 of SEQ ID NO:191; nucleotides 1090 to 1096 of SEQ ID NO:191; or nucleotides 1141 to 1642 of SEQ ID NO:191. 23. The nucleic acid molecule of claim 22 wherein said molecule is an antisense oligonucleotide directed to a region of a sequence of SEQ ID NO: 191. 24. The nucleic acid molecule of claim 23 wherein said oligonucleotide is directed to a regulatory region of a sequence of SEQ ID NO: 191. 25. A composition comprising a nucleic acid molecule of any one of claims 1 to 5 or 22 and an acceptable carrier or diluent. 26. A composition comprising a recombinant expression vector of claim 7 and an acceptable carrier or diluent. 27. A method of producing a polypeptide that comprises a sequence of SEQ ID NO: 192, and homologs and fragments thereof, said method comprising the steps of: a) introducing a recombinant expression vector of claim 7 into a compatible host cell; b) growing said host cell under conditions for expression of said polypeptide; and c) recovering said polypeptide, wherein the polypeptide comprising a sequence of SEQ ID NO:192 comprises at least one or more amino acid residues from one or more of the following regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQ ID NO:192; amino acid residues 68 to 77 of SEQ ID NO:192; amino acid residues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513 of SEQ ID NO: 192. 28. The method of claim 27 wherein said host cell is lysed and said polypeptide is recovered from the lysate of said host cell. 29. The method of claim 27 wherein said polypeptide is recovered by purifying the culture medium without lysing said host cell. 30. An isolated polypeptide encoded by a nucleic acid molecule of claim 1, wherein the polynucleotide comprises at least one or more amino acid residues from one or more of the following regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQ ID NO:192; amino acid residues 68 to 77 of SEQ ID NO:192; amino acid residues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513 of SEQ ID NO: 192. 31. The polypeptide of claim 30 wherein said polypeptide comprises a fragment of SEQ ID NO:192. 32. The polypeptide of claim 30 wherein said polypeptide comprises an amino acid sequence homologous to a sequence of SEQ ID NO: 192. 33. The polypeptide of claim 30 wherein said sequence homologous to a sequence of SEQ ID NO:192 comprises at least one conservative amino acid substitution compared to the sequence of SEQ ID NO: 192. 34. The polypeptide of claim 30 wherein said polypeptide comprises a fragment of a polypeptide with a sequence of SEQ ID NO:192. 35. A composition comprising a polypeptide of claim 30 and an acceptable carrier or diluent. 36. An isolated antibody which binds to an epitope on a polypeptide of claim 30. 37. The antibody of claim 36 wherein said antibody is a monoclonal antibody. 38. A composition comprising an antibody of claim 36 and an acceptable carrier or diluent. 39. A method of inducing an immune response in a mammal against a polypeptide of claim 30 comprising administering to said mammal an amount of said polypeptide sufficient to induce said immune response. 40. A method for identifying a compound which binds nGPCR-14 comprising the steps of: a) contacting nGPCR-14 with a compound; and b) determining whether said compound binds nGPCR-14. 41. The method of claim 40 wherein the nGPCR-14 comprises an amino acid sequence of SEQ ID NO: 192. 42. The method of claim 40 wherein binding of said compound to nGPCR-14 is determined by a protein binding assay. 43. The method of claim 40 wherein said protein binding assay is selected from the group consisting of a gel-shift assay, Western blot, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross linking, interaction trap/two-hybrid analysis, southwestern analysis, and ELISA. 44. A compound identified by the method of claim 40. 45. A method for identifying a compound which binds a nucleic acid molecule encoding nGPCR-14 comprising the steps of: a) contacting said nucleic acid molecule encoding nGPCR-14 with a compound; and b) determining whether said compound binds said nucleic acid molecule. 46. The method of claim 45 wherein binding is determined by a gel-shift assay. 47. A compound identified by the method of claim 45. 48. A method for identifying a compound which modulates the activity of nGPCR-14 comprising the steps of: a) contacting nGPCR-14 with a compound; and b) determining whether nGPCR-14 activity has been modulated. 49. The method of claim 48 wherein the nGPCR-14 comprises an amino acid sequence of SEQ ID NO: 192. 50. The method of claim 48 wherein said activity is neuropeptide binding. 51. The method of claim 48 wherein said activity is neuropeptide signaling. 52. A compound identified by the method of claim 48. 53. A method of identifying an animal homolog of nGPCR-14 comprising the steps: a) comparing the nucleic acid sequences of the animal with a sequence of SEQ ID NO: 191, and portions thereof, said portions being at least 10 nucleotides; and b) identifying nucleic acid sequences of the animal that are homologous to said sequence of SEQ ID NO: 191, and portions thereof. 54. The method of claim 53 wherein comparing the nucleic acid sequences of the animal with a sequence selected of SEQ ID NO: 191, and portions thereof, said portions being at least 10 nucleotides, is performed by DNA hybridization. 55. The method of claim 53 wherein comparing the nucleic acid sequences of the animal with a sequence selected of SEQ ID NO: 191, and portions thereof, said portions being at least 10 nucleotides, is performed by computer homology search. 56. A method of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor, comprising the steps of: (a) assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering an amino acid sequence, expression, or biological activity of at least one nGPCR that is expressed in the brain, wherein the nGPCR comprises an amino acid sequence of SEQ ID NO:192, and allelic variants thereof, and wherein the nucleic acid corresponds to a gene encoding the nGPCR; and (b) diagnosing the disorder or predisposition from the presence or absence of said mutation, wherein the presence of a mutation altering the amino acid sequence, expression, or biological activity of the nGPCR in the nucleic acid correlates with an increased risk of developing the disorder. 57. A method according to claim 56, wherein the nGPCR is nGPCR-14 comprising an amino acid sequence set forth in SEQ ID NO:192 or an allelic variant thereof. 58. A method according to claim 56, wherein the assaying step comprises at least one procedure selected from the group consisting of: a) comparing nucleotide sequences from the human subject and reference sequences and determining a difference of at least a nucleotide of at least one codon between is the nucleotide sequences from the human subject that encodes an nGPCR-14 allele and an nGPCR-14 reference sequence; (b) performing a hybridization assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; (c) performing a polynucleotide migration assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; and (d) performing a restriction endonuclease digestion to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences. 59. A method according to claim 58 wherein the assaying step comprises: performing a polymerase chain reaction assay to amplify nucleic acid comprising nGPCR-14 coding sequence, and determining nucleotide sequence of the amplified nucleic acid. 60. A method of screening for an nGPCR-14 mental disorder genotype in a human patient, comprising the steps of: (a) providing a biological sample comprising nucleic acid from said patient, said nucleic acid including sequences corresponding to allelles of nGPCR-14; and (b) detecting the presence of one or more mutations in the nGPCR-14 allelle; wherein the presence of a mutation in an nGPCR-40 allelle or nGPCR-54 allele is indicative of a mental disorder genotype. 61. The method according to claim 59 wherein said biological sample is a cell sample. 62. The method according to claim 59 wherein said detecting the presence of a mutation comprises sequencing at least a portion of said nucleic acid, said portion comprising at least one codon of said nGPCR-14 alleles. 63. The method according to claim 59 wherein said nucleic acid is DNA. 64. The method according to claim 59 wherein said nucleic acid is RNA. 65. A kit for screening a human subject to diagnose a mental disorder or a genetic predisposition therefor, comprising, in association: (a) an oligonucleotide useful as a probe for identifying polymorphisms in a human nGPCR-14 gene, the oligonucleotide comprising 6-50 nucleotides in a sequence that is identical or complementary to a sequence of a wild type human nGPCR-14 gene sequence or nGPCR-14 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution; and (b) a media packaged with the oligonucleotide, said media containing information for identifying polymorphisms that correlate with schizophrenia or a genetic predisposition therefor, the polymorphisms being identifiable using the oligonucleotide as a probe. 66. A method of identifying a nGPCR allelic variant that correlates with a mental disorder, comprising steps of: (a) providing a biological sample comprising nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny; (b) detecting in the nucleic acid the presence of one or more mutations in an nGPCR that is expressed in the brain, wherein the nGPCR comprises an amino acid sequence of SEQ ID NO: 192, and allelic variants thereof, and wherein the nucleic acid includes sequence corresponding to the gene or genes encoding nGPCR; wherein the one or more mutations detected indicates an allelic variant that correlates with a mental disorder. 67. A method according to claim 66 wherein the at least one nGPCR is nGPCR-14, or an allelic variant thereof. 68. A purified and isolated polynucleotide comprising a nucleotide sequence encoding a nGPCR-14 allelic variant identified according to claim 67. 69. A host cell transformed or transfected with a polynucleotide according to claim 68 or with a vector comprising the polynucleotide. 70. A purified polynucleotide comprising a nucleotide sequence encoding nGPCR-14 of a human with a mental disorder; wherein said polynucleotide hybridizes to the complement of SEQ ID NO:191 under the following hybridization conditions: (a) hybridization for 16 hours at 42Β° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60Β° C. in a wash solution comprising 0.1ΓSSC and 1% SDS; and wherein the polynucleotide that encodes the nGPCR-14 amino acid sequence of the human differs from SEQ ID NO:192 by at least one residue and comprises at least one or more amino acid residues from one or more of the following regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQ ID NO: 192; amino acid residues 68 to 77 of SEQ ID NO: 192; amino acid residues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513 of SEQ ID NO: 192. 71. A vector comprising a polynucleotide according to claim 70. 72. A host cell that has been transformed or transected with a polynucleotide according to claim 70 and that expresses the nGPCR-14 protein encoded by the polynucleotide. 73. A host cell according to claim 72 that has been co-transfected with a polynucleotide encoding the nGPCR-14 amino acid sequence set forth in SEQ ID NO:192 and that expresses the nGPCR-14 protein having the amino acid sequence set forth in SEQ ID NO:192. 74. A method for identifying a modulator of biological activity of nGPCR-14 comprising the steps of: a) contacting a cell according to claim 72 in the presence and in the absence of a putative modulator compound; b) measuring nGPCR-14 biological activity in the cell; wherein decreased or increased nGPCR-14 biological activity in the presence versus absence of the putative modulator is indicative of a modulator of biological activity. 75. A method to identify compounds useful for the treatment of a mental disorder, said method comprising steps of: (a) contacting a composition comprising nGPCR-14 with a compound suspected of binding nGPCR-14; (b) detecting binding between nGPCR-14 and the compound suspected of binding nGPCR-14; wherein compounds identified as binding nGPCR-14 are candidate compounds useful for the treatment of a mental disorder. 76. A method for identifying a compound useful as a modulator of binding between nGPCR-14 and a binding partner of nGPCR-14 comprising the steps of: (a) contacting the binding partner and a composition comprising nGPCR-14 in the presence and in the absence of a putative modulator compound; (b) detecting binding between the binding partner and nGPCR-14; wherein decreased or increased binding between the binding partner and nGPCR-14 in the presence of the putative modulator, as compared to binding in the absence of the putative modulator is indicative a modulator compound useful for the treatment of schizophrenia. 77. A method according to claim 75 or 76 wherein the composition comprises a cell expressing nGPCR-14 on its surface. 78. A method according to claim 77 wherein the composition comprises a cell transformed or transfected with a polynucleotide that encodes nGPCR-14. 79. A method of purifying a G protein from a sample containing said G protein comprising the steps of: a) contacting said sample with a polypeptide of claim 1 for a time sufficient to allow said G protein to form a complex with said polypeptide; b) isolating said complex from remaining components of said sample; c) maintaining said complex under conditions which result in dissociation of said G protein from said polypeptide; and d) isolating said G protein from said polypeptide. 80. The method of claim 79 wherein said sample comprises an amino acid sequence of SEQ ID NO: 192. 81. The method of claim 79 wherein said polypeptide comprises an amino acid sequence homologous to a sequence of SEQ ID NO:192. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The G protein-coupled receptors (GPCRs) form a vast superfamily of cell surface receptors which are characterized by an amino-terminal extracellular domain, a carboxyl terminal intracellular domain, and a serpentine structure that passes through the cell membrane seven times. Hence, such receptors are sometimes also referred to as seven transmembrane (7TM) receptors. These seven transmembrane domains define three extracellular loops and three intracellular loops, in addition to the amino- and carboxy-terminal domains. The extracellular portions of the receptor have a role in recognizing and binding one or more extracellular binding partners (e.g., ligands), whereas the intracellular portions have a role in recognizing and communicating with downstream molecules in the signal transduction cascade. The G protein-coupled receptors bind a variety of ligands including calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and even photons, and are important in the normal (and sometimes the aberrant) function of many cell types. [See generally Strosberg, Eur. J. Biochem. 196:1-10 (1991) and Bohm et al., Biochem J. 322:1-18 (1997).] When a specific ligand binds to its corresponding receptor, the ligand typically stimulates the receptor to activate a specific heterotrimeric guanine-nucleotide-binding regulatory protein (G-protein) that is coupled to the intracellular portion of the receptor. The G protein in turn transmits a signal to an effector molecule within the cell, by either stimulating or inhibiting the activity of that effector molecule. These effector molecules include adenylate cyclase, phospholipases and ion channels. Adenylate cyclase and phospholipases are enzymes that are involved in the production of the second messenger molecules cAMP, inositol triphosphate and diacyglycerol. It is through this sequence of events that an extracellular ligand stimuli exerts intracellular changes through a G protein-coupled receptor. Each such receptor has its own characteristic primary structure, expression pattern, ligand-binding profile, and intracellular effector system. Because of the vital role of G protein-coupled receptors in the communication between cells and their environment, such receptors are attractive targets for therapeutic intervention, for example by activating or antagonizing such receptors. For receptors having a known ligand, the identification of agonists or antagonists may be sought specifically to enhance or inhibit the action of the ligand. Some G protein-coupled receptors have roles in disease pathogenesis (e.g., certain chemokine receptors that act as HIV co-receptors may have a role in AIDS pathogenesis), and are attractive targets for therapeutic intervention even in the absence of knowledge of the natural ligand of the receptor. Other receptors are attractive targets for therapeutic intervention by virtue of their expression pattern in tissues or cell types that are themselves attractive targets for therapeutic intervention. Examples of this latter category of receptors include receptors expressed in immune cells, which can be targeted to either inhibit autoimmune responses or to enhance immune responses to fight pathogens or cancer; and receptors expressed in the brain or other neural organs and tissues, which are likely targets in the treatment of schizophrenia, depression, bipolar disease, or other neurological disorders. This latter category of receptor is also useful as a marker for identifying and/or purifying (e.g., via fluorescence-activated cell sorting) cellular subtypes that express the receptor. Unfortunately, only a limited number of G protein receptors from the central nervous system (CNS) are known. Thus, a need exists for G protein-coupled receptors that have been identified and show promise as targets for therapeutic intervention in a variety of animals, including humans. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to an isolated nucleic acid molecule that comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a fragment thereof. The nucleic acid molecule encodes at least a portion of nGPCR-x. In some embodiments, the nucleic acid molecule comprises a sequence that encodes a polypeptide comprising even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a fragment thereof. In some embodiments, the nucleic acid molecule comprises a sequence homologous to odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a fragment thereof. In some embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, and fragments thereof. According to some embodiments, the present invention provides vectors which comprise the nucleic acid molecule of the invention. In some embodiments, the vector is an expression vector. According to some embodiments, the present invention provides host cells which comprise the vectors of the invention. In some embodiments, the host cells comprise expression vectors. The present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence complementary to at least a portion of a sequence from an odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, said portion comprising at least 10 nucleotides. The present invention provides a method of producing a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof. The method comprising the steps of introducing a recombinant expression vector that includes a nucleotide sequence that encodes the polypeptide into a compatible host cell, growing the host cell under conditions for expression of the polypeptide and recovering the polypeptide. The present invention provides an isolated antibody which binds to an epitope on a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof. The present invention provides an method of inducing an immune response in a mammal against a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof. The method comprises administering to a mammal an amount of the polypeptide sufficient to induce said immune response. The present invention provides a method for identifying a compound which binds nGPCR-x. The method comprises the steps of: contacting nGPCR-x with a compound and determining whether the compound binds nGPCR-x. The present invention provides a method for identifying a compound which binds a nucleic acid molecule encoding nGPCR-x. The method comprises the steps of contacting said nucleic acid molecule encoding nGPCR-x with a compound and determining whether said compound binds said nucleic acid molecule. The present invention provides a method for identifying a compound which modulates the activity of nGPCR-x. The method comprises the steps of contacting nGPCR-x with a compound and determining whether nGPCR-x activity has been modulated. The present invention provides a method of identifying an animal homolog of nGPCR-x. The method comprises the steps screening a nucleic acid database of the animal with an odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof and determining whether a portion of said library or database is homologous to said odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or portion thereof. The present invention provides a method of identifying an animal homolog of nGPCR-x. The methods comprises the steps screening a nucleic acid library of the animal with a nucleic acid molecule having an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof; and determining whether a portion of said library or database is homologous to said odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof. Another aspect of the present invention relates to methods of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor. The methods comprise the steps of assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering an amino acid sequence, expression, or biological activity of at least one nGPCR that is expressed in the brain. The nGPCR comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, and SEQ ID NO:94, and allelic variants thereof. A diagnosis of the disorder or predisposition is made from the presence or absence of the mutation. The presence of a mutation altering the amino acid sequence, expression, or biological activity of the nGPCR in the nucleic acid correlates with an increased risk of developing the disorder. The present invention further relates to methods of screening for a nGPCR-40 or nGPCR-54 hereditary schizophrenia genotype in a human patient. The methods comprise the steps of providing a biological sample comprising nucleic acid from the patient, in which the nucleic acid includes sequences corresponding to alleles of nGPCR-40 or nGPCR-54. The presence of one or more mutations in the nGPCR-40 allele or the nGPCR-54 allele is detected indicative of a hereditary schizophrenia genotype. The present invention provides kits for screening a human subject to diagnose schizophrenia or a genetic predisposition therefor. The kits include an oligonucleotide useful as a probe for identifying polymorphisms in a human nGPCR-40 gene or a human nGPCR-54 gene. The oligonucleotide comprises 6-50 nucleotides in a sequence that is identical or complementary to a sequence of a wild type human nGPCR-40 or nGPCR-54 gene sequence or nGPCR-40 or nGPCR-54 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution. The kit also includes a media packaged with the oligonucleotide. The media contains information for identifying polymorphisms that correlate with schizophrenia or a genetic predisposition therefor, the polymorphisms being identifiable using the oligonucleotide as a probe. The present invention further relates to methods of identifying nGPCR allelic variants that correlates with mental disorders. The methods comprise the steps of providing biological samples that comprise nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny, and detecting in the nucleic acid the presence of one or more mutations in an nGPCR that is expressed in the brain. The nGPCR comprises an amino acid sequence selected from the group consisting of SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, and SEQ ID NO:94, and allelic variants thereof. The nucleic acid includes sequences corresponding to the gene or genes encoding nGPCR. The one or more mutations detected indicate an allelic variant that correlates with a mental disorder. The present invention further relates to purified polynucleotides comprising nucleotide sequences encoding alleles of nGPCR-40 or nGPCR-54 from a human with schizophrenia. The polynucleotide hybridizes to the complement of SEQ ID NO:83 or of SEQ ID NO:85 under the following hybridization conditions: (a) hybridization for 16 hours at 42Β° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60Β° C. in a wash solution comprising 0.1ΓSSC and 1% SDS. The polynucleotide that encodes nGPCR-40 or nGPCR-54 amino acid sequence of the human differs from SEQ ID NO:84 or SEQ ID NO:86 by at least one residue. The present invention also provides methods for identifying a modulator of biological activity of nGPCR-40 or nGPCR-54 comprising the steps of contacting a cell that expresses nGPCR-40 or nGPCR-54 in the presence and in the absence of a putative modulator compound and measuring nGPCR-40 or nGPCR-54 biological activity in the cell. The decreased or increased nGPCR-40 or nGPCR-54 biological activity in the presence versus absence of the putative modulator is indicative of a modulator of biological activity. The present invention further provides methods to identify compounds useful for the treatment of schizophrenia. The methods comprise the steps of contacting a composition comprising nGPCR-40 with a compound suspected of binding nGPCR-40 or contacting a composition comprising nGPCR-54 with a compound suspected of binding nGPCR-54. The binding between nGPCR-40 and the compound suspected of binding nGPCR-40 or between nGPCR-54 and the compound suspected of binding nGPCR-54 is detected. Compounds identified as binding nGPCR-40 or nGPCR-54 are candidate compounds useful for the treatment of schizophrenia. The present invention further provides methods for identifying a compound useful as a modulator of binding between nGPCR-40 and a binding partner of nGPCR-40 or between nGPCR-54 and a binding partner of nGPCR-54. The methods comprise the steps of contacting the binding partner and a composition comprising nGPCR-40 or nGPCR-54 in the presence and in the absence of a putative modulator compound and detecting binding between the binding partner and nGPCR-40 or nGPCR-54. Decreased or increased binding between the binding partner and nGPCR-40 or nGPCR-54 in the presence of the putative modulator, as compared to binding in the absence of the putative modulator is indicative a modulator compound useful for the treatment of schizophrenia. Another aspect of the present invention relates to methods of purifying a G protein from a sample containing a G protein. The methods comprise the steps of contacting the sample with an nGPCR for a time sufficient to allow the G protein to form a complex with the nGPCR; isolating the complex from remaining components of the sample; maintaining the complex under conditions which result in dissociation of the G protein from the nGPCR; and isolating said G protein from the nGPCR. detailed-description description="Detailed Description" end="lead"? |
Pharmaceutical formulation |
The present invention provides a pharmaceutical formulation which comprises a core comprising a first active ingredient, a coating comprising a second active ingredient which is incompatible with the first active ingredient and a barrier between the core and the coating which prevents physical contact between the core and the coating, characterised in that the barrier is formed on the core by film-coating and the coating is formed on the barrier by press-coating. |
1. A pharmaceutical formulation which comprises a core comprising a first active ingredient, a coating comprising a second active ingredient which is incompatible with the first active ingredient and a barrier between the core and the coating which prevents physical contact between the core and the coating, characterised in that the barrier is formed on the core by film-coating and the coating is formed on the barrier by press-coating. 2. A pharmaceutical formulation as claimed in claim 1 wherein the core further comprises one or more pharmaceutical excipients, disintegrants, lubricants, anti-adhesion agents, flow agents or diluents. 3. A pharmaceutical formulation as claimed in claim 1 wherein the coating further comprises one or more pharmaceutical excipients, disintegrants, lubricants, anti-adhesion agents, flow agents or diluents. 4. A pharmaceutical formulation as claimed in claim 1 wherein the core comprises ranitidine or a physiologically acceptable salt thereof and the coating comprises acetylsalicylic acid or a physiologically acceptable salt thereof. 5. A pharmaceutical formulation as claimed in claim 4 wherein the ranitidine is present in the core in an amount of from 10 to 200 mg or a physiologically acceptable salt of ranitidine is present in the core in an amount which is equivalent to from 10 to 200 mg of ranitidine. 6. A pharmaceutical formulation as claimed in claim 4 wherein the ranitidine is present in the form of ranitidine hydrochloride. 7. A pharmaceutical formulation as claimed in claim 4 wherein acetylsalicylic acid is present in the coating in an amount of from 50 to 1000 mg or a physiologically acceptable salt of acetylsalicylic acid is present in the core in an amount which is equivalent to from 50 to 1000 mg of acetylsalicylic acid. 8. A pharmaceutical formulation as claimed in claim 4 wherein the acetylsalicylic acid or the physiologically acceptable salt of acetylsalicylic acid is microencapsulated with ethyl cellulose. 9. A pharmaceutical formulation as claimed in claim 1 wherein the barrier also reduces the transmission of moisture between the core and the coating. 10. A pharmaceutical formulation as claimed in claim 1 wherein the barrier is present in an amount of from 1 to 20% by weight based on the total weight of the core. |
Method of education and scholastic management for cyber education system utilizing internet |
This invention relates to the method of cyber education through internet capable to check the true attendance of a cyber class by a fingerprint and calculate the accumulative learning time of each registered lectures by the host computer for the scholastic management of the cyber education system. In addition, utilizing this invention, it provides a pre-school system by using a game and animation created for pre-school curriculums capable to evaluate the education development of each children with a method for calculating the accumulative learning time of each subject by individual ID, and a software system for studying subjects at a stage of character formation for kids and evaluating the mental development of human being by means of playing game. |
1. The method for checking the attendance of the cyber class through internet, comprising the step of, (a) registering the fingerprint of registrant for completing a course of study and being issued ID number. (b) confirming the attendance of the cyber class by inputting the registrant's fingerprint through internet; (c) confirming the time to depart from the class by inputting the fingerprint when the registrant finishes attending a lecture; (d) verifying the true attendance of class by the fingerprint data inputted from the registrant by comparing the fingerprint data with the fingerprint data memorized in the host computer in accordance with ID data of each registrant. 2. The method of claim 1, further comprising the step of alarming with a sound signal every set of time to the student for inputting the fingerprint, and verifying the true attendance of the cyber class by checking the fingerprint for a certain interval of the time. 3. The method of claim 1 or claim 2, further comprising the step of giving credits for the completion of the course by calculating the accumulated time of each student's log in time checked by means of fingerprint. 4. The method of claim 1 and 2, further comprising the step of creating database for each subject and lessons, and divide total running time of the program to small quantity, and when student logs-in to database with fingerprint and only views a few parts of the lesson then host computer records partial time spent and accumulates and giving credits when partial lessons are accumulating to complete lesson time which host computer recognizes as attendance. 5. The method of claim 1, wherein checking the attendance of the class through the verification of the iris of the eye, the voice signal or a passwords of the students instead of fingerprint. 6. The method of the scholastic management for granting academic degree at cyber school comprising of; (a) calculating by the host computer the starting time and ending time of each lesson by student's fingerprint data inputted and accumulate the total logging time in the memory device of the host computer; (b) granting credits when the host computer recognizes individual's accumulative time spent of each lesson is qualified according to the school's guide line for completion of the course; (c) giving a qualification to the student to submit a thesis through on-line or off-line when student finishes academic requirements according to the records of credits by the host computer; (d) granting a degree to the student when student passes all examinations concerning a thesis and complete academic cyber courses. 7. The method of claim 6, further comprising the step of giving an examination by the host computer and receiving the answer through internet to earn enough credits to graduate. 8. The method of claim 6, wherein further comprising the step of examining the thesis by attending the student on the thesis examination committee of the institute and giving the degree after the said examination. 9. The method of the scholastic management for operating the universalized university such as a cyber university or a cyber graduate school for worldwide, comprising the step of; (a) constructing the database of the lectures in different languages comprising at least one or more different languages of the each lecture of the each professor; (b) registering the fingerprint of the student for completing a course of study and being issued ID code; (c) logging into the system with registered ID and selecting the lecture by the language and the professor of the database; (d) attending the cyber class by confirming the starting time and the ending time of each lecture by inputting the student's fingerprint to check the true attendance of the cyber class; (e) granting credits when the host computer recognizes the student's accumulative time spent of each lesson is qualified according to the school's guide line for completion of the course; (f) giving a qualification to the student to submit a thesis through on-line or off-line when student finishes academic requirements according to the records of credits calculated by the host computer; (g) granting a degree to the student when student passes all examinations concerning a thesis and complete academic cyber courses. 10. The educational system for operating the cyber pre-school system utilizing internet comprising the step of; (a) a database comprising animation and/or game of each subject suitable for constructing the pre-school curriculums for the kids for studying by playing the game and/or animation; (b) a host computer for, (i) registering the cyber pre-school for completing a course of study by the parents and being issued ID code of the kids, (ii) calculating the time of log-in and log-out of each subject by the ID code for recording the accumulated playing time of each subject, (iii) qualifying the kids for completion of the program when individual's accumulative time spent of the each subject according to school's guide line, and (iv) recognizing the kids as completion of the academic programs when the kids complete all the courses, according to the records of the host computer. 11. The educational system of claim 10, further comprising a means for registering kid's fingerprint to indicate starting time and ending time for accessing DB to check an accurate attendance. 12. The educational system of claim 10, further comprising a database of the educational curriculums such as a language course, a music course or mathematics course for issuing a certificate for completion of the said educational curriculums by recognizing the kids as completion of the course when kid's accumulative time spent of the course is qualified by the host computer. 13. The educational system of claim 10, further comprising a database of internet on-line games for providing internet on-line game service to kids for interchanging among other kids. 14. The educational system of claim 10, further comprising a database for providing internet on-line game service and a means for a visual chatting service for kids to interchange among other kids. 15. The educational system of claim 10, further registering the kids of off-line pre-school to cyber pre-school for logging into the database through internet and recognizing the completion of courses with data calculated by the host computer. 16. The business method for operating the cyber pre-school utilizing internet comprising the step of; (a) creating a DB (database) for studying the each subject divided into session according to the pre-school curriculums; (b) issuing ID to log in to the DB by paying the tuition; (c) calculating the accumulated time of studying by the kids according to the time of log-in and log-out of each subject with the host computer; (d) advising the accumulated studying time of each subject through internet to the parents to select the subject for their kids to study more according to the said accumulated learning time data of each subject; (e) qualifying the kids for completion of the pre-school when individual's accumulative time spent of the each subject is qualified according to the guide line of the school by the host computer. 17. The business method of claim 16, further comprising a step for evaluating the development of study by calculating the signal from the kids for selecting answer among the questionnaires comprising animations or games with explanation voice. 18. The business method of claim 17, wherein the step for calculating further comprising a step for calculating the accumulated time of student's study by the subject and the number of the complete study of the subject. 19. The business method of claim 17, wherein the database is comprising educational game created to learn the subject of the pre-school curriculums for studying through playing the games. 20. The business method of claim 17, wherein the database is comprising educational animation and/or animated game created to study according to the curriculums for studying through playing the game and/or viewing animation. 21. The business method of claim 20, further comprising a cyber class created to study the subject according to the curriculums for studying through the explanation of the cyber teacher and the class activities of the characters in the cyber class. 22. The method for evaluating the mental development of a human being through internet comprising the step of, (a) making a DB (database) of games created to evaluate the mental development such as a character, a disposition and/or a morality of a human being in a category of each checking subject; (b) registering ID to log in the database with/without the fingerprint of the player; (c) accessing the database with said ID with/without the fingerprint of the player and playing the games through internet; (d) calculating the each action of the cyber characters played by the player and giving different points according to the favorable action and the non-favorable action based on the mental development evaluating standard in a category of each subject by the host computer of the DB system; (e) advising the result of the test in a category of each checking subject, calculated by the host computer for evaluating the mental developments of the player. 23. The method of claim 22, wherein the database comprising games to check the mental development and/or knowledge of pre-school kids and evaluating the mental development and/or knowledge of the kids according to the action of the cyber characters played by kids. 24. The method of claim 22 or claim 23, wherein the DB contents comprising animated questionnaires with/without a sound explanation to be selected by the player for evaluating the mental development of the player. 25. The software system for evaluating the mental development of human being with the PC comprising, (a) an animated game created to evaluate the player's intention according to the action of the game character in the categories of each situation through playing the game by the player; (b) a calculating means for calculating the each action of the game characters played by the player by giving different points according to the favorable action and the non-favorable action based on the mental development evaluation standard in a category of each subject to check; (c) a displaying means for displaying the result of the calculation by the point in a category of each subject to evaluate the mental development of the player. 26. The software system of claim 25, wherein the calculating means calculating the accumulated points in category of each subject and the number of the completion of the game to display according to the player. 27. The software system for studying the educational subjects with PC comprising, an animated game created to learn the subject through playing the game by the kids; a means for calculating the playing time of the each subject of the software by the each subject; and a means for displaying the accumulated playing time according to the subject to check the studying intensive of the kids. 28. The software system of claim 27 further comprising a cyber class created to study the subject to learn through the activities of the kids and the guidance of the teacher in the cyber class. 29. The software system of claim 27 or claim 28, further comprising a animated questionnaires for the kids to select the answer for calculating points to display on the monitor of the PC to evaluate the kid's understanding. |
<SOH> FIELD OF INVENTION <EOH>The present invention relates to the method of cyber education and scholastic management system related to the cyber education through Internet capable to check the true attendance of the cyber class by a fingerprint and calculate the accumulative learning time of each registered lectures by the host computer for determining automatically the completion of program for cyber education's scholastic management. In addition, utilizing the system that calculates accumulative learning time of each registered lectures by the host computer, a children's educational software program can be created for the pre-school children at the stage of character formation where they start learning language, morality and human nature by imitation on the model of parents and home environment, the system can accumulate how much they absorb knowledge on each subject and how many subjects are covered according to the accumulated learning time data of the each subject by the child's individual ID, to provide the data for evaluating each child's progress in data by the category of each subject, so that the parents may know what kind of the subjects to be covered more for their kids. |
<SOH> BRIEF DESCRIPTION OF THE DRAWING <EOH>The figures in the drawings are briefly described as follows: FIG. 1 is a block diagram of the registering fingerprint data into education management system. FIG. 2 is a block diagram of the system that calculates logging time on educational institution's database detailed-description description="Detailed Description" end="lead"? |
Fuel cell arrangement and method for operating a fuel cell arrangement |
A fuel cell arrangement (10) and its operation are described. This fuel cell arrangement (10) comprises one or more fuel cells (1) to which a combustible gas or a cathode gas is fed and which supply an electric power Pext to an external consuming device (2). One or more internal electrical consuming devices (3) can optionally be switched on and off by means of a control device (5) as a function of the operating condition of the fuel cell arrangement (10). Excessive heat is removed from the fuel cell arrangement by means of a cooling device (4). According to the invention, a portion of the electric power PBZ generated by the fuel cells (1) is converted at the internal electrical consuming devices (3) during the operation of the fuel cell arrangement (10). When the electric power Pext supplied to the external electrical consuming device (2) increases or decreases, the electric power Pint converted at the internal electrical consuming devices (3) is reduced or increased inversely in the sense of making the electric power PBZ generated by the fuel cells (1) uniform. This is carried out by the control device (5) of the fuel cell arrangement (10). |
1-40. (Cancelled) 41. A method of operating a fuel cell arrangement having at least one fuel cell comprising: feeding a combustible gas and a cathode gas to said fuel cell arrangement; supplying an electric power Pext to an external consuming device; optionally switching on and off at least one internal electrical consuming device; and removing excessive heat from said fuel cell arrangement by a cooling device; wherein an electric power PBZ is generated by said at least one fuel cell, and an electric power Pint is consumed by said at least one internal electrical consuming device, said power Pint being a portion of said power PBZ. 42. The method of claim 41, wherein an increase or decrease of said power Pext causes a decrease or increase in said power Pint, respectively. 43. The method of claim 41, wherein said power Pext and said power Pint are inversely related. 44. The method of claim 42, wherein said power PBZ is uniform. 45. The method of claim 43, wherein, for a load-following operation, an increase of said power Pext supplied to said external electrical consuming device is compensated by a reduction of said power Pint, and a reduction of said power Pext is compensated by an increase of said power Pint. 46. The method of claim 45, wherein, for a standby operation, a switching-off of said external electrical consuming device is compensated by an increase in said power Pint. 47. The method of claim 46, wherein a change of said power Pint takes place essentially immediately with a change of the power Pext. 48. The method of claim 47, wherein said power PBZ is kept essentially constant. 49. The method of claim 48, wherein said power Pint maximally amounts to approximately one fourth to approximately one half of a nominal electric power of said at least one fuel cell. 50. The method of claim 48, wherein said power Pint is removed from said fuel cell arrangement by an increased use of said cooling device. 51. A fuel cell arrangement having at least one fuel cell comprising: at least one internal electrical consuming device, said at least one internal electrical consuming device being optionally switched on and off; a cooling device for removing excess heat from said fuel cell arrangement; and a control device, said control device controlling said at least one internal electrical consuming device as a function of an operating condition of said fuel cell arrangement; wherein an electric power PBZ is generated by said fuel cell arrangement, an electric power Pint is consumed by said at least one internal electrical consuming device, said power Pint being a portion of said power PBZ, and an electric power Pext is consumed by an external consuming device. 52. The fuel cell arrangement of claim 51, wherein an increase or decrease of said power Pext causes a decrease or increase in said power Pint, respectively. 53. The fuel cell arrangement of claim 51, wherein said power Pext and said power Pint are inversely related. 54. The fuel cell arrangement of claim 52, wherein said power PBZ is uniform. 55. The fuel cell arrangement of claim 53, wherein, for a load-following operation, said control device controlling said at least one internal consuming device such that an increase of said power Pext is compensated by a reduction of said power Pint, and a reduction of said power Pext is compensated by an increase of said power Pint. 56. The fuel cell arrangement of claim 55, wherein, for a standby operation, said control device controlling said at least one internal consuming device such that a switching-off of said external electrical consuming device is compensated by an increase in said power Pint. 57. The fuel cell arrangement of claim 56, wherein said control device controlling said at least one internal consuming device such that a change of said power Pint takes place essentially immediately with a change of the power Pext. 58. The fuel cell arrangement of claim 57, wherein said control device controlling said at least one internal consuming device such that said power PBZ is kept essentially constant. 59. The fuel cell arrangement of claim 58, wherein said control device controlling said at least one internal consuming device such that said power Pint maximally amounts to approximately one fourth to approximately one half of a nominal electric power of said fuel cell arrangement. 60. The fuel cell arrangement of claim 58, wherein said cooling device is constructed such that it removes said power Pint from said fuel cell arrangement. 61. The fuel cell arrangement of claim 60, said at least one internal electrical consuming device further comprising at least one of a start heating device, a ventilator device, and another aggregate. 62. The fuel cell arrangement of claim 61, further comprising an electrical inverter for converting a direct voltage supplied by said at least one fuel cell into an alternating voltage, said alternating voltage being supplied to said external consuming device. 63. The fuel cell arrangement of claim 62, wherein said at least one internal electrical consuming device being connected, via said control device, to said external electrical consuming device in parallel with an output of said inverter. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Fuel cell arrangements are known which comprise one or more fuel cells to which a combustible gas and a cathode gas are fed and which supply electric power to an external consuming device. One or more internal electrical consuming devices of the fuel cell arrangement can optionally be switched on and off. A cooling device is provided for eliminating excessive heat from the fuel cell arrangement. One difficulty during the operation of fuel cell arrangements of the above-mentioned type is the fact that, with respect to their performance, they cannot arbitrarily rapidly follow a change of the electric load of the connected external consuming device. The reason is based less on the dynamics of the electrochemical events taking place in the fuel cells than in the slowness of supplying the fuel cells with their reaction gases: the combustible gas and the cathode gas. The load-following operation therefore requires electric power at the expense of the efficiency in order to be able to immediately react in the event of a load demand. In the case of a power supply system failure, i.e., in the event of a sudden failure of the load demand of the connected external consuming device, the fuel cell arrangement, if possible, should not switch off but change to a standby operation, in which the current requirement for the internal consuming devices of the fuel cell arrangement is covered. The intrinsic consumption of the fuel cell arrangement is minimized for economical reasons, so that the system generates little electric power. In the load-following operation, the amount of the current production of a fuel cell arrangement is defined by the external consuming devices connected to it. Such a condition, which is also called an βisland operationβ, particularly in the case of small system sizes, results in load demands, which fluctuate considerably with respect to their percentages as a result of the connecting or disconnecting of individual consumption points. Rapid load changes occur during failures of the external consuming device, for example, in the event of a power supply system failure. The fuel cell arrangement will then supply no electric power to the external consuming device. In this case, the fuel cell arrangement is to change to a standby operation in which it generates just enough electric power in order to satisfy the intrinsic consumption. |
<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the invention to provide a method of operating a fuel cell arrangement of the initially mentioned type by means of which the fuel cell arrangement can be operated in the sense of a fast response to changing load requirements. Furthermore, by means of the invention, a fuel cell arrangement may be created which is capable of rapidly responding to changing load requirements. With respect to the method, the object is achieved by means of the method of operating a fuel cell arrangement indicated herein. With respect to the device, the object is achieved by means of the fuel cell arrangement indicated herein. The invention creates a method of operating a fuel cell arrangement having one or more fuel cells, to which a combustible gas and a cathode gas are fed and which supply electric power to an external consuming device, and having one or more internal electrical consuming devices which can optionally be switched on and off, as well as having a cooling device by which excessive heat is eliminated from the fuel cell arrangement. According to the invention, it is provided that, in the operation of the fuel cell arrangement, a portion of the electric power generated by the fuel cells is converted at the internal electrical consuming devices, and that, in the case of an increase or decrease of the electric power supplied to the external electrical consuming device, the electric power converted at the internal electrical consuming devices is reduced or increased inversely in the sense of making the electric power generated by the fuel cells uniform. It is an advantage of the method according to the invention that, while the generating of the electric power is uniform, the fuel cells can rapidly respond to a change of the load demand by the external electrical consuming device. According to a preferred embodiment of the invention, it is provided that, for a load-following operation, an increase of the power supplied to the external electrical consuming device is compensated by a reduction of the electric power converted at the internal electrical consuming devices, and a reduction of the power supplied to the external electrical consuming device is compensated by an increase of the power converted at the internal electrical consuming devices. According to another aspect of the invention, it is provided that, for a standby operation, a switching-off of the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices. It is preferably provided that the change of the electric power inverted at the internal electrical consuming devices takes place essentially immediately with the change of the power supplied to the external electrical consuming device. Here, it is particularly advantageous for the electric power generated by the fuel cells to be kept essentially constant. According to an advantageous embodiment of the invention, it is provided that maximally an electric power is converted at the internal electrical consuming devices which amounts to approximately one fourth to approximately half of the nominal electric power of the fuel cells. Preferably, the electric power converted at the internal electrical consuming devices is removed from the fuel cell arrangement by an increased use of the cooling device. Furthermore, by means of the invention, a fuel cell arrangement is created which has one or more fuel cells to which a combustible gas and a cathode gas are fed and which supply an electric power to an external consuming device, and which has one or more internal electrical consuming devices which can optionally be switched on and off, and which has a cooling device by means of which the excessive heat is removed from the fuel cell arrangement, and which has a control device for controlling the operation of the internal consuming devices as a function of the operating condition of the fuel cell arrangement. According to the invention, it is provided that the control device controls the internal electrical consuming devices such that a portion of the electric power generated by the fuel cells is converted at the internal electrical consuming devices, and that, in the case of an increase or decrease of the electric power supplied to the external electrical consuming device, the electric power converted at the internal electrical consuming devices is reduced or increased inversely in the sense of making the electric power generated by the fuel cells uniform. It is an advantage of the fuel cell arrangement according to the invention that, while the generating of the electric power is essentially uniform, this fuel cell arrangement can rapidly respond to a change of the load demand of the connected electrical consuming device. According to an aspect of the invention, it is provided that the control device controls the internal electrical consuming devices such that, for a load-following operation, an increase of the power supplied to the external electrical consuming device is compensated by a reduction of the electric power converted at the internal electrical consuming devices, and a reduction of the power supplied to the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices. According to another aspect of the invention, it is provided that the control device controls the internal electrical consuming devices such that, for a standby operation, a switching-off of the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices. Preferably, it is provided that the control device controls the internal electrical consuming devices such that the change of the electric power converted at the internal electrical consuming devices takes place essentially immediately with the change of the power supplied to the external electrical consuming device. Furthermore, it is an advantage for the control device to control the internal electrical consuming devices such that the electric power generated by the fuel cells is essentially kept constant. According to an advantageous embodiment of the invention, it is provided that maximally an electric power is converted at the internal electrical consuming devices which amounts to approximately one fourth to approximately half of the nominal electric power of the fuel cells. Furthermore, it is advantageous for the cooling device to be constructed such that it removes the electric power converted at the internal electrical consuming devices from the fuel cell arrangement. Advantageously, the internal electrical consuming devices comprise a start heating device and/or a ventilating device and/or other aggregates of the fuel cell arrangement. According to a preferred embodiment of the invention, it is provided that the fuel cell arrangement comprises an electrical inverter for converting the direct voltage supplied by the fuel cells into an alternating voltage which is fed to the external consuming device. Finally, according to an advantageous embodiment of the invention, it is provided that the internal electrical consuming devices can be connected by means of the control device optionally parallel to the external electric consuming device with the output of the inverter. |
Recombinant proteinase k |
The invention concerns recombinant proteinase K. Furthermore a method for producing recombinant proteinase K is disclosed, which is characterized in that a) a host cell is transformed with a recombinant nucleic acid which codes for the zymogenic precursor of proteinase K, b) the host cell is cultured in such a manner that the zymogenic precursor of proteinase K is formed in the form of inclusion bodies in the host cell, c) the inclusion bodies are isolated and natured under conditions which result in the formation of the protease part of the zymogenic precursor in its natural conformation, d) the natured proteinase K is activated and purified. |
1-22. (canceled) 23. A method for the naturation of denatured zymogenic proteinase K comprising transferring the denatured zymogenic proteinase K to a folding buffer, the buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH of 7.5 to 10.5 and the method being carried out at a temperature between 0Β° C. and 37Β° C. 24. The method of claim 23 wherein the redox shuffling system comprises mixed disulfides or thiosulfonates. 25. The method of claim 23 wherein the pH range is 8 to 9. 26. The method of claim 23 wherein the temperature is between 0Β° C. and 25Β° C. 27. The method of claim 23 wherein the buffer further comprises denaturing agents at a concentration of less than 50 mM. 28. The method of claim 23 wherein the low molecular weight substances are selected from the group consisting of L-arginine at a concentration of 0.5 to 2.0 M, Tris at a concentration of 0.5 M to 2.0 M, triethanolamine at a concentration of 0.5 M to 2.0 M, and Ξ±-cyclodextrin at a concentration of 60 mM to 120 mM. 29. The method of claim 23 wherein the Ca2+ ion concentration is 1 to 20 mM. 30. The method of claim 23 wherein the denatured zymogenic proteinase K is transferred to the folding buffer while reducing the concentration of denaturing agents that may be present. 31. A folding buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH value in the range of 7.5 to 10.5. 32. The buffer of claim 31 wherein the pH value is 8 to 9 and the redox shuffling system comprises mixed disulfides or thiosulfonates. 33. A method for activating a natured zymogenic precursor of active proteinase K comprising adding a detergent to an inactive complex comprising a native proteinase K and an inhibitory propeptide of the active proteinase K, thereby releasing the active proteinase K from the inactive complex. 34. The method of claim 33 wherein the detergent is SDS at a concentration of 0.1 to 2% (w/v). 35. A method for producing active recombinant proteinase K comprising (a) producing an inactive zymogenic proform of proteinase K in an inclusion body, (b) naturing in vitro the zymogenic proform of proteinase K, and (c) activating the zymogenic proform by autocatalytic cleavage, thereby converting it to the active proteinase K. 36. The method of claim 35 wherein the naturing step comprises transferring the denatured zymogenic proteinase K to a folding buffer, the buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH of 7.5 to 10.5 and the naturing step being carried out at a temperature between 0Β° C. and 37Β° C. 37. The method of claim 35 wherein the inclusion body is solubilized by a denaturing agent and a reducing agent. 38. The method of claim 37 wherein the denaturing agent is guanidinium hydrochloride at a concentration of 6-8 M or urea at a concentration of 8-10 M and the reducing agent is DTT or DTE at a concentration of 50-200 mM. 39. A method for producing active recombinant proteinase K comprising (a) transforming a host cell with a vector containing a DNA sequence coding for a zymogenic precursor of proteinase K (b) expressing the zymogenic precursor in inclusion bodies, (c) isolating the inclusion bodies and solubilizing the zymogenic precursor, (d) naturing the zymogenic precursor with a folding buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH of 7.5 to 10.5 and the naturing step being carried out at a temperature between 0Β° C. and 37Β° C., and (e) activating the zymogenic precursor by autocatalytic cleavage, thereby converting it to the active proteinase K 40. The method of claim 39 wherein the host cell is a prokaryotic cell. 41. The method of claim 39 wherein the host cell is Escherichia coli. 42. A codon-optimized recombinant nucleic acid comprising DNA coding for a recombinant zymogenic proteinase K which has been optimized for expression in Escherichia coli. 43. A vector containing the recombinant nucleic acid of claim 42. 44. A host cell transformed with the vector of claim 43. |
Antibody complexes and methods for immunolabeling |
The present invention provides labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents. The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins whereby the labeling proteins have affinity for a specific region of the target-binding antibody and are covalently attached to a label. Typically, the labeling reagent is an anti-Fc Fab or Fabβ² fragment that was generated by immunizing a goat or rabbit with the Fc fragment of an antibody. The present invention provides for discrete subsets of labeling reagent and immuno-labeled complexes that facilitate the simultaneous detection of multiple targets in a sample wherein the immuno-labeled complexes are distinguished by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody. This is particularly useful for fluorophore labels that can be attached to labeling reagents and subsequently immuno-labeled complexes in ratios for the detection of multiple targets. |
1. A method of forming an immuno-labeled complex, wherein said method comprises the steps of: b) contacting a solution of target-binding antibodies with a discreet labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent whereby a discreet immuno-labeled complex subset is formed; c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof; and, c) optionally repeating said steps a), b), and c) to form a panel of immuno-labeled complex subsets wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody. 2. The method according to claim 1, wherein said target binding antibody is a murine monoclonal antibody, a rabbit polyclonal antibody or a goat polyclonal antibody. 3. The method according to claim 2, wherein said target-binding antibodies are in a solution comprising serum proteins or ascites proteins. 4. The method according to claim 1, wherein said labeling reagent is a Fab or Fabβ²fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof. 5. The method according to claim 4, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope. 6. The method according to claim 5, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof. 7. The method according to claim 5, wherein said fluorescent protein is a phycobiliprotein. 8. The method according to claim 5, wherein said tandem dye is selected from the group consisting of a cyanine-phycobiliprotein derivative and xanthene-phycobiliprotein derivative. 9. The method according to claim 5, wherein said enzyme is selected from the group consisting of a peroxidase, a phosphatase, a glycosidase, and a luciferase. 10. A method for detecting a target in a sample, wherein said method comprises the steps of: a) contacting said sample with an immuno-labeled complex comprising a target-binding antibody and a labeling reagent; b) incubating said sample of step a) for a time sufficient to permit said immuno-labeled complex to selectively bind to said target; and, c) illuminating said immuno-labeled complex whereby said target is detected. 11. The method according to claim 11, wherein said sample comprises a population of cells, cellular extract, subcellular component, proteins, peptides, tissue culture, tissue, a bodily fluid, or a portion or combination thereof. 12. The method according to claim 11, wherein said sample is immobilized on a solid or semi-solid matrix. 13. The method according to claim 12, wherein said matrix is a gel, a membrane, an array, a glass surface or a microparticle. 14. The method according to claim 10, wherein said labeling reagent is a Fab or Fabβ²fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof. 15. The method according to claim 14, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope. 16. The method according to claim 15, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof. 17. The method according to claim 15, wherein said fluorescent protein is a phycobiliprotein. 18. The method according to claim 15, wherein said tandem dye is selected from the group consisting of a cyanine-phycobiliprotein derivative and xanthene-phycobiliprotein derivative. 19. The method according to claim 15, wherein said enzyme selected from the group consisting of a peroxidase, a phosphatase, a glycosidase, and a luciferase. 20. The method according to claim 19, wherein said method further comprises adding a colorimetric, fluorescent or chemiluminescent enzyme substrate. 21. The method according to claim 10, wherein said immuno-labeled complex comprises a labeling reagent that is a Fab or Fabβ² anti-Fc fragment wherein said fragment is independently covalently bonded to one or more labels that are selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a phycobiliprotein, a borapolyazaindacene, a peroxidase, a phosphatase, a tandem dye and derivatives thereof. 22. A method for detecting multiple targets in a sample, said method comprising: a) contacting said sample with a solution comprising A) a pooled subset of immuno-labeled complexes wherein each said complex comprises a target-binding antibody and a labeling reagent, wherein each said subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody or B) an individual immuno-labeled complex subset wherein multiple individual subsets are added to said sample; b) incubating said sample for a time sufficient to allow said immuno-labeled complex to selectively bind to said target; and, c) illuminating said immuno-labeled complex whereby said multiple targets are detected. 23. The method according to claim 22, wherein said sample comprises a population of cells, cellular extract, subcellular component, tissue culture, tissue, a bodily fluid, or a portion or combination thereof. 24. The method according to claim 23, wherein said sample is immobilized on a solid or semi-solid matrix. 25. The method according to claim 24, wherein said matrix is a gel, a membrane, an array, a glass surface or a microparticle. 26. The method according to claim 23, wherein said labeling reagent is a Fab or Fabβ²fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof. 27. The method according to claim 26, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an electron transfer agent, a hapten, an enzyme and a radioisotope. 28. The method according to claim 27, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof. 29. The method according to claim 27, wherein said fluorescent protein is a phycobiliprotein. 30. The method according to claim 27, wherein said tandem dye is selected from the group consisting of a cyanine-phycobiliprotein derivative and xanthene-phycobiliprotein derivative. 31. The method according to claim 27, wherein said enzyme is selected from the group consisting of a peroxidase, a phosphatase, a glycosidase, and a luciferase. 32. The method according to claim 31, wherein said method further comprises adding a calorimetric, fluorescent or chemiluminescent enzyme substrate. 33. A method for identifying and quantitating multiple targets in a sample, wherein said method comprises the steps of: a) contacting a population of cells or fragments thereof in a sample with A) a solution comprising a pooled subset of immuno-labeled complexes wherein said complex comprises a target-binding antibody and a labeling reagent, wherein each subset is. distinguished from another subset i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody or B) an individual subset of immuno-labeled complexes wherein multiple individual subsets are added to said sample; b) incubating said cells or fragments thereof for a time period sufficient to allow said immuno-labeled complex to bind said targets; c) passing said incubated population of cells or fragments thereof through an examination zone; and, d) collecting data from said cells or fragments thereof passed through said examination zone whereby said identification and quantitation of multiple targets is determined. 34. The method according to claim 33, wherein said labeling reagent is a Fab or Fabβ²fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof. 35. The method according to claim 34, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a radioisotope, and a hapten. 36. The method according to claim 35, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof. 37. The method according to claim 35, wherein said fluorescent protein is a phycobiliprotein. 38. The method according to claim 35, wherein said tandem dye is selected from the group consisting of cyanine-phycobiliprotein and xanthene-phycobiliprotein. 39. A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of: a) cleaving an intact anti-region antibody with an enzyme to generate Fab fragments; b) isolating said anti-region Fab fragments of step a); c) contacting a matrix comprising intact immunoglobulin proteins or fragments that selectively bind anti-region Fab fragments with a solution comprising said anti-region fragments of step b) wherein said anti-region Fab fragments are immobilized on said matrix; d) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group; e) washing said matrix of step d) to remove unbound label, and; f) eluting said labeling reagent from said matrix whereby said isolated labeling reagent is manufactured. 40. The method according to claim 39, wherein said anti-region Fab fragment are selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment. 41. The method according to claim 40, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof. 42. A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of: a) contacting a matrix comprising intact immunoglobulin proteins or fragments thereof that selectively bind non-antibody proteins with a solution comprising said non-antibody proteins wherein said non-antibody proteins are immobilized on said matrix; b) contacting said matrix of step a) with a solution comprising a fluorophore label that contains a reactive group; c) washing said matrix to remove unbound label, and; d) eluting said labeling reagent from said matrix whereby said isolated labeling reagent is manufactured that comprises a fluorophore label. 43. The method according to claim 42, wherein said non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof. 44. The method according to claim 43, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof. 45. A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of: a) cleaving an intact anti-region antibody with an enzyme resulting in Fab or Fabβ²fragments; a) contacting said anti-region Fab or Fabβ² fragments of step a) with a solution comprising a label that contains a reactive group; and, c) isolating labeled anti-region Fab or Fabβ² fragments by size exclusion or affinity chromatography whereby an isolated labeling reagent is manufactured. 46. The method according to claim 45, wherein said anti-region Fab or Fabβ² fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment. 47. The method according to claim 46, wherein said label is a fluorescent protein or a tandem dye. 48. The method according to claim 47, wherein said fluorescent protein is a phycobiliprotein. 49. The method according to claim 47, wherein said tandem dye is selected from the group consisting of cyanine-phycobiliprotein and xanthene-phycobiliprotein. 50. An isolated labeling reagent made by a process comprising: a) cleaving an intact anti-region antibody with an enzyme resulting in Fab or Fabβ²fragments; b) contacting said Fab or Fabβ² fragments of step a) with a solution comprising a label that contains a reactive group; and, c) isolating labeled anti-region Fab or Fabβ² fragments by size exclusion or affinity chromatography. 51. The labeling reagent according to claim 50, wherein said anti-region Fab or Fabβ²fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment. 52. The labeling reagent according to claim 51, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a radioisotope, and a hapten. 53. The labeling reagent according to claim 52, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof. 54. An isolated labeling reagent made by a process comprising: a) cleaving an intact anti-region antibody with an enzyme to generate F(abβ²)2 fragments; b) contacting said anti-region F(abβ²)2 with a reducing agent to produce anti-region Fabβ² fragments containing a thiol group; c) contacting said Fabβ² fragments with a solution comprising a label that contains a reactive group; and, d) isolating said Fabβ² fragments of step d) that are covalently attached to a label by size exclusion or affinity chromatography. 55. The labeling reagent according to claim 54, wherein said anti-region Fabβ² fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment. 56. The method according to claim 55, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a radioisotope, and a hapten. 57. An isolated labeling reagent made by a process comprising: a) cleaving an intact anti-region antibody with an enzyme to generate Fab fragments; b) isolating said anti-region Fab fragments of step a); c) contacting a matrix comprising intact immunoglobulin proteins or fragments thereof that specifically bind anti-region Fab fragments with a solution comprising said anti-region Fab fragments of step b) wherein said anti-region Fab fragments are immobilized; d) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group; e) washing said matrix to remove unbound label, and; f) eluting said labeling reagent from said matrix whereby said labeling reagent is manufactured comprising a label and being isolated from other proteins and fragments thereof. 58. The labeling reagent according to claim 57, wherein said anti-region Fab fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment. 59. The labeling reagent according to claim 58, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof. 60. An isolated labeling reagent made by a process comprising: a) contacting a matrix comprising intact immunoglobulin proteins or fragments that selectively bind non-antibody proteins with a solution comprising said non-antibody proteins wherein said non-antibody proteins are immobilized; b) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group; c) washing said matrix to remove unbound label, and; d) eluting said labeling reagent from said matrix whereby said labeling reagent is manufactured comprising a label. 61. The labeling reagent according to claim 60, wherein said non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof. 62. The labeling reagent according to claim 61, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof. 63. An immuno-labeled complex generated by a process comprising: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subset is distinguished from another labeling reagent subset by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent; and, c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof whereby an immuno-labeled complex is produced. 64. The immuno-labeled complex according to claim 63, wherein said process further comprises repeating said steps a), b), and c) to form subsets of immuno-labeled complexes wherein each subset is distinguished from another subset by i) ratio of label to labeling reagent or ii) a physical properties of said label, or iii) a ratio of labeling reagent to said target-binding antibody or iv) by said target-binding antibody. 65. The immuno-labeled complex according to claim 63, wherein said target-binding antibody is a murine monoclonal antibody, a rabbit polyclonal antibody or a goat polyclonal antibody. 66. The immuno-labeled complex according to claim 65, wherein said labeling reagent is a Fab or Fabβ² fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof. 67. The immuno-labeled complex according to claim 66, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope. 68. The immuno-labeled complex according to claim 67, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof. 69. A labeling solution comprising a buffer and either an individual labeling reagent subset or a pooled subset of labeling reagents, wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) by a physical property of said label. 70. The solution according to claim 69, wherein said labeling reagent is a Fab or Fabβ²fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof. 71. The solution according to claim 70, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an enzyme, a hapten and a radioisotope. 72. The solution according to claim 71, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof. 73. A kit for preparing an immuno-labeling complex, said kit comprising: a) a labeling solution comprising a labeling reagent that is independently covalently bonded to one or more labels; or b) a panel of labeling solutions wherein each labeling solution comprises a subset of a labeling reagent and each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) by a physical property of said label; and, c) a solution comprising a capture reagent. 74. The kit according to claim 73, wherein said labeling reagent is a Fab or Fabβ²fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof. 75. The kit according to claim 74, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an enzyme, a hapten and a radioisotope. 76. The kit according to claim 75, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof. 77. The kit according to claim 76, wherein the kit further comprises an addition component selected from the group consisting of (a) stains for characterization of cellular organelles, cell viability, or cell proliferation state, (b) enzyme substrates and (c) enzyme conjugates. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Immunolabeling is a method for qualitative or quantitative determination of the presence of a target in a sample, wherein antibodies are utilized for their specific binding capacity. The antibodies form a complex with the target (antigen), wherein a detectable label is present on the antibody or on a secondary antibody. The detectable label is a key feature of immunolabeling, which can be detected directly or indirectly. The label provides a measurable signal by which the binding reaction is monitored providing a qualitative and/or quantitative measure of the degree of binding. The relative quantity and location of signal generated by the labeled antibodies can serve to indicate the location and/or concentration of the target. The label can also be used to select and isolate labeled targets, such as by flow sorting or using magnetic separation media. Examples of labels include but are not limited to radioactive nucleotides ( 125 I, 3 H, 14 C, 32 P), chemiluminescent, fluorescent, or phosphorescent compounds (e.g., dioxetanes, xanthene, or carbocyanine dyes, lanthanide chelates), particles (e.g., gold clusters, colloidal gold, microspheres, quantum dots), and enzymes (e.g., peroxidases, glycosidases, phosphatases, kinases). Ideally, the label is attached to the antibody in a manner that does not perturb the antibody's binding characteristics but enables the label to be measured by an appropriate detection technology. The choice of labels is influenced by factors such as ease and sensitivity of detection, equipment availability, background in the sample (including other labels) and the degree to which such labels are readily attached to the particular antibody. Both direct and indirect labeling of antibodies is utilized for immunolabeling. Direct labeling utilizes only a primary antibody, i.e. the antibody specific for the target, bound to the label. In contrast, indirect labeling utilizes a secondary antibody bound to the label, which is specific for the primary antibody, e.g. a goat anti-rabbit antibody. The principal differences in immunolabeling methods and materials reside in the way that the label is attached to the antibody-antigen complex, the type of label that is used, and the means by which the antibody-antigen complex is detected. Limitations for direct labeling primary antibodies include the need for buffers free of primary amines, or carrier proteins such as bovine serum albumin (BSA), and other compounds such as tris-(hydroxymethyl)aminomethane (TRIS), glycine, and ammonium ions. These materials are, however, common components in antibody buffers and purification methods, and it may not be possible or feasible to remove them prior to the coupling reaction. In particular, many monoclonal antibodies are available only as ascites fluid or in hybridoma culture supernatants, or diluted with carrier proteins, such as albumins. Thus, direct labeling of antibodies in ascites fluid or other medias containing interfering compounds is not attainable. The indirect immunolabeling method typically involves a multi-step process in which an unlabeled first antibody (typically a primary antibody) is directly added to the sample to form a complex with the antigen in the sample. Subsequently, a labeled secondary antibody, specific for the primary antibody, is added to the sample, where it attaches noncovalently to the primary antibody-antigen complex. Alternatively, a detectable label is covalently attached to an immunoglobulin-binding protein such as protein A and protein G to detect the antibody-antigen complex that has previously been formed with the target in the sample. Using ligands, such as streptavidin, that are meant to amplify the detectable signal also expands this cascade binding. Indirect immunolabeling often results in false positives and high background. This is due to the fact that secondary antibodies, even when purified by adsorption against related species, nevertheless can exhibit significant residual cross-reactivity when used in the same sample. For example, when mouse tissue is probed with a mouse monoclonal antibody, the secondary antibody must necessarily be a labeled anti-mouse antibody. This anti-mouse antibody will detect the antibody of interest but will inevitably and additionally detect irrelevant, endogenous mouse immunoglobulins inherent in mouse tissue. This causes a significant background problem, especially in diseased tissues, which reduces the usefulness and sensitivity of the assay. Thus, the simultaneous detection of more than one primary antibody in a sample without this significant background interference depends on the availability of secondary antibodies that 1) do not cross-react with proteins intrinsic to the sample being examined, 2) recognize only one of the primary antibodies, and 3) do not recognize each other (Brelje, et al., METHODS IN CELL BIOLOGY 38, 97-181, especially 111-118 (1993)). To address the background problem in indirect labeling, a number of strategies have been developed to block access of the anti-mouse secondary antibodies to the endogenous mouse immunoglobulins. One such strategy for blocking involves complexing the primary antibody with a selected biotinylated secondary antibody to produce a complex of the primary and secondary antibodies, which is then mixed with diluted normal murine serum (Trojanowski et al., U.S. Pat No. 5,281,521 (1994)). This method is limited by the necessity to utilize an appropriate ratio of primary-secondary complex. Too low a ratio of primary-secondary complex will cause a decrease in specific staining and increased background levels due to the uncomplexed secondary anti-mouse antibody binding to endogenous mouse antibodies. However, the ability of a whole IgG antibody (as was used in the referenced method) to simultaneously bind and cross-link two antigens results in too high a ratio, causing the complex to precipitate or form complexes that are too large to penetrate into the cell or tissue. Another strategy for blocking access to endogenous immunoglobulins in the sample involves pre-incubating the sample with a monovalent antibody, such as Fabβ² fragments, from an irrelevant species that recognize endogenous immunoglobulins. This approach requires large quantities of expensive Fabβ² fragments and gives mixed results and adds at least two steps (block and wash) to the overall staining procedure. The addition of a cross-linking reagent has resulted in improved reduction of background levels (Tsao, et al., U.S. Pat. No. 5,869,274 (1997)) but this is problematic when used with fluorophore-labeled antibodies. The cross-linking causes an increase in the levels of autofluorescence and thus the background (J. Neurosci. Meth. 83, 97 (1998); Mosiman et al., Methods 77, 191 (1997); Commun. Clin. Cytometry 30,151 (1997); Beisker et al., Cytometry 8, 235 (1987)). In addition, pre-incubation with a cross-linking reagent often masks or prevents the antibody from binding to its antigen (J. Histochem. Cytochem. 45, 327 (1997); J. Histochem. Cytochem. 39, 741 (1991); J. Histochem. Cytochem. 43,193 (1995); Appl. Immunohistochem. Molecul. Morphol. 9,176 (2001)). In a variation of this blocking strategy, a multi-step sequential-labeling procedure is used to overcome the problems of cross-reactivity. The sample is incubated with a first antibody to form a complex with the first antigen, followed by incubation of the sample with a fluorophore-labeled goat Fab anti-mouse IgG to label the first antibody and block it from subsequently complexing when the second antibody is added. In the third step, a second mouse antibody forms a complex with the second antigen. Because the second antibody is blocked from cross-reacting with the first antibody, the second mouse antibody is detected with a standard indirect-labeling method using a goat anti-mouse antibody conjugated to a different fluorescent dye (J. Histochem. Cytochem. 34, 703 (1986)). This process requires multiple incubation steps and washing steps and it still cannot be used with mouse antibodies to probe mouse tissue. Another blocking method is disclosed in the animal research kit (ARK) developed by DAKO. In this kit, a primary antibody is complexed with biotin-labeled goat Fab anti-mouse IgG and excess free Fab is blocked with normal mouse serum. However, since the Fab used in this process is generated from the intact IgG (rather than a selected region) there is a potential for the formation of anti-paratope or anti-idiotype antibodies that will block the antigen-binding site and prevent immunolabeling. The biotinylated antibody also requires subsequent addition of a labeled avidin or streptavidin conjugate for its subsequent visualization. The present invention is advantageous over previously described methods and compositions in that it provides the benefits of indirect labeling with the easy and flexibility of direct labeling for determination of a desired target in a biological sample. The present invention provides labeled monovalent proteins specific for a target-binding antibody, which are complexed prior to addition with a biological sample. Because these monovalent proteins are not bivalent antibodies, precipitation and cross-linking are not a problem. Therefore the compositions of the present invention can be used with immunologically similar monoclonal or polyclonal antibodies of either an identical isotype or different isotypes. The monovalent labeling reagents are specific for the Fc region of target-binding antibodies, these reagents will not interfere with the binding region of the primary antibody. In addition, the monovalent labeling proteins are not negatively affected by the presence of primary amines like BSA, gelatin, hybridoma culture supernatants or ascites fluid, thus primary antibodies present in these media can be effectively labeled with the labeling reagents of the present invention. Thus, the present invention provides numerous advantages over the conventional methods of immunolabeling. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents. The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins whereby the labeling proteins have affinity for a specific region of the target-binding antibody and are covalently attached to a label. Typically, the labeling reagent is an anti-Fc Fab or Fabβ² fragment that was generated by immunizing a goat or rabbit with the Fc fragment of an antibody. The methods for labeling a target-binding antibody with a labeling reagent comprise a) contacting a solution of target-binding antibodies with a labeling reagent, b) incubating said target-binding antibodies and said labeling reagent wherein a region of said target binding antibody is selectively bound by labeling reagent, and c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof that are optionally immobilized on a matrix. The labeling of the target-binding antibody can be performed irrespective of the solution that the antibody is present in and includes proteins that are normally present in serum or ascites. This feature of the labeling process of the target-binding antibody eliminates the need to purify and concentrate the target-binding antibody. The time required for the labeling reagent to selectively bind to the target-binding antibody is typically very short, often less than 10 minutes. Often the labeling reagent binds the target-binding antibody in the amount of time it takes to add and mix the labeling reagent with the target-binding antibody. This formation of an immuno-labeled complexβa target-binding antibody and a labeling reagentβresults in the formation of an target detection solution that is used to detect a target in a sample. The labeling steps of the target-binding antibody are optionally repeated to form a panel of subsets, these immuno-labeled complex subsets may be used individually or pooled wherein each subset is distinguished from another subset by i) the target-binding antibody, or ii) a ratio of label to labeling reagent, or iii) a ratio of labeling reagent to the target-binding antibody or iv) by a physical property of the label. Thus, it is appreciated that a wide range of subsets can be formed wherein the subsets can be used individually to detect a target in a sample or pooled to simultaneously detect multiple targets in a sample. The simultaneous detection of multiple targets in a sample is especially useful in methods that utilize flow cytometry or methods that immobilize a population of cells or tissue on a surface. The methods for determining a target in a sample using immuno-labeled subsets comprises forming a subset of immuno-labeled complexes, as described above, contacting a sample with said immuno-labeled complexes, incubating the sample for a time sufficient to allow the immuno-labeled complex to selectively bind to a desired target, and illuminating the immuno-labeled complex whereby the target is detected. The sample is any material that may contain a target and typically comprises a population of cells, cellular extract, subcellular component, proteins, peptides, tissue culture, tissue, a bodily fluid, or a portion or combination thereof. When multiple targets are detected a pooled subset of immuno-labeled complexes are formed and incubated with the sample or individual subsets are add sequentially to a sample. For methods using flow cytometry the population of cells is illuminated when they pass through an optical examination zone and the data collected about the label determines the identity and quantity of the targets. |
Gssp4 polynucleotides and polypeptides and uses thereof |
The present invention relates to the field of metabolic research. Metabolic disorders, such as obesity, are a public health problem that is serious and widespread. GSSP4 polypeptides have been identified that are believed to be beneficial in the treatment of metabolic disorders. These compounds should be effective for reducing cholesterol levels, body mass, body fat, and for treating metabolic-related diseases and disorders. The metabolic-related diseases or disorders envisioned to be treated by the methods of the invention include, but are not limited to: obesity, hyperlipidemia, hypercholesterolemia, atherosclerosis, diabetes, glucose intolerance, insulin resistance and hypertension. |
1. A method of reducing circulating free fatty acid levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass. 2. A method of reducing circulating glucose levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass. 3. A method of reducing circulating triglyceride levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass. 4. A method of reducing circulating cholesterol levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass. 5. An isolated polypeptide comprising: a) an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; b) a polypeptide fragment of at least six consecutive amino acids of SEQ ID NO: 3; c) the polypeptide of SEQ ID NO: 3; d) homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; e) a heterologous polypeptide fused to the polypeptide of a), b), c), or d); or f) a polypeptide according to a), b), c), d), or e) that has been differentially modified. 6. A composition comprising an isolated polypeptide according to claim 3 and a pharmaceutically or physiologically acceptable carrier. 7. An isolated or purified polynucleotide: a) encoding a polypeptide comprising an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; b) encoding a polypeptide comprising a fragment of at least six consecutive amino acids of SEQ ID NO: 3; c) encoding a polypeptide comprising SEQ ID NO: 3; d) encoding homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; e) encoding a polypeptide comprising a heterologous polypeptide fused to the polypeptide of a), b), c), or d); f) encoding a polypeptide according to a), b), c), d), or e) that has been differentially modified; g) comprising a polynucleotide according to a), b), c), d), e), or f) operably linked to a promoter, other regulatory element, or selectable marker; or h) comprising a vector comprising a polynucleotide according to a), b), c), d), e), f), or g). 8. A recombinant cell comprising a polynucleotide according to claim 7. 9. An antibody that specifically binds to a polypeptide according to claim 5. 10. A method of producing a polypeptide comprising culturing a recombinant cell according to claim 8 under conditions that allow for the expression of said polypeptide. 11. The method according to claim 10, further comprising the isolation, recovery, or purification of said polypeptide. 12. A method of treating a obesity, a metabolic disease or a metabolic disorder comprising the administration of a therapeutically effective amount of a composition comprising: a) an isolated polypeptide comprising: 1) an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; 2) a polypeptide fragment of at least six consecutive amino acids of SEQ ID NO: 3; 3) the polypeptide of SEQ ID NO: 3; 4) homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; 5) a heterologous polypeptide fused to the polypeptide of a(1), a(2), a(3), or a(4); or 6) a polypeptide according to a(1), a(2), a(3), a(4), or a(5) that has been differentially modified; b) an isolated or purified polynucleotide: 1) encoding a polypeptide comprising an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; 2) encoding a polypeptide comprising a fragment of at least six consecutive amino acids of SEQ ID NO: 3; 3) encoding a polypeptide comprising SEQ ID NO: 3; 4) encoding homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; 5) encoding a polypeptide comprising a heterologous polypeptide fused to the polypeptide of b(1), b(2), b(3), or b(4); 6) encoding a polypeptide according to b(1), b(2), b(3), b(4), or b(5) that has been differentially modified; 7) comprising a polynucleotide according to b(1), b(2), b(3), b(4), b(5), or b(6) operably linked to a promoter, other regulatory element, or selectable marker; or 8) comprising a vector comprising a polynucleotide according to b(1), b(2), b(3), b(4), b(5), b(6), or b(7); or c) a recombinant cell comprising: 1) a polynucleotide encoding a polypeptide comprising an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; 2) a polynucleotide encoding a polypeptide comprising a fragment of at least six consecutive amino acids of SEQ ID NO: 3; 3) a polynucleotide encoding a polypeptide comprising SEQ ID NO: 3; 4) a polynucleotide encoding homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; 5) a polynucleotide encoding a polypeptide comprising a heterologous polypeptide fused to the polypeptide of c(1), c(2), c(3), or c(4); 6) a polynucleotide encoding a polypeptide according to c(1), c(2), c(3), c(4), or c(5) that has been differentially modified; 7) a polynucleotide according to c(1), c(2), c(3), c(4), c(5), or c(6) operably linked to a promoter, other regulatory element, or selectable marker; or 8) comprising a vector comprising a polynucleotide according to c(1), c(2), c(3), c(4), c(5), c(6), or c(7). 13. A transgenic animal comprising a polynucleotide according to claim 7. |
<SOH> BACKGROUND OF TH INVENTION <EOH>The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention. Obesity is a public health problem that is serious, widespread, and increasing. In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634). Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643). Even modest weight loss ameliorates these associated conditions. While still acknowledging that lifestyle factors including environment, diet, age and exercise play a role in obesity, twin studies, analyses of familial aggregation, and adoption studies all indicate that obesity is largely the result of genetic factors (Barsh et al (2000) Nature 404:644-651). In agreement with these studies, is the fact that an increasing number of obesity-related genes are being identified. Some of the more extensively studied genes include those encoding leptin (ob) and its receptor (db), pro-opiomelanocortin (Pomc), melanocortin-4-receptor (Mc4r), agouti protein (A y ), carboxypeptidase E (fat), 5-hydroxytryptamine receptor 2C (Htr2c), nescient basic helix-loop-helix 2 (Nhlh2), prohormone convertase 1 IPCSK1), and tubby protein (tubby) (rev'd in Barsh et al (2000) Nature 404:644-651). |
<SOH> SUMMARY OF THE INVENTION <EOH>The instant invention is based on the discovery that GSSP4 polypeptides have unexpected effects in vitro and in vivo, including utility for weight reduction, prevention of weight gain, reduction of cholesterol levels, and control of blood glucose levels in humans and other mammals. These unexpected effects of administration of GSSP4 polypeptides in mammals also include reduction of elevated free fatty acid levels caused by administration of epinephrine, i.v. injection of βintralipidβ, or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, reduction of circulating cholesterol levels, modulation of blood glucose and weight reduction in mammals, particularly those consuming a high fat/high carbohydrate diet. These effects are unexpected and surprising given that proteins of similar structure or homology (such as colipase and mamba intestinal toxin 1) have not been shown to have utility for weight reduction, prevention of weight gain, reduction of cholesterol levels, and control of blood glucose levels. However, the GSSP4 polypeptides of the invention are effective and can be provided at levels that are feasible for treatments in humans. Thus, the invention is drawn to GSSP4 polypeptides, polynucleotides encoding said polypeptides, vectors comprising said GSSP4 polynucleotides, and cells recombinant for said GSSP4 polynucleotides, as well as to pharmaceutical and physiologically acceptable compositions comprising said GSSP4 polypeptide and methods of administering said GSSP4 polypeptides or polynucleotides in a pharmaceutical and physiologically acceptable compositions in order to reduce body weight, cholesterol levels or glucose levels, or to treat metabolic-related diseases and disorders. Assays for identifying agonists and antagonists of metabolic-related activity are also part of the invention. In a first aspect, the invention features a purified, isolated, or recombinant GSSP4 polypeptides. In preferred embodiments, said polypeptides comprise, consist essentially of, or consist of, those having significant activity wherein the said activity is selected from the group consisiting of cholesterol reduction, cholesterol regulation, lipid partitioning, lipid metabolism, glucose control, and insulin-like activity. In preferred embodiments, said polypeptides comprise, consist essentially of, or consist of, the full length polypeptide of SEQ ID NO:3 or a fragment of consecutive amino acids of the full length polypeptide sequence of SEQ ID NO:3. In other preferred embodiments, said polypeptides comprise an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of the polypeptide sequences identified in SEQ ID NO:3. In a further preferred embodiment, the GSSP4 polypeptide is able to lower circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides. Further preferred GSSP4 polypeptides are those that significantly stimulate muscle lipid or free fatty acid oxidation. Further preferred GSSP4 polypeptides are those that cause C2C12 cells differentiated in the presence of said polypeptides to undergo at least 10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared to untreated cells. Further preferred GSSP4 polypeptides are those that are at least 30% more efficient than untreated cells at increasing leptin uptake in a liver cell line (preferably BPRCL mouse liver cells (ATCC CRL-2217)). Further preferred GSSP4 polypeptides are those that significantly reduce the postprandial increase in plasma free fatty acids, particularly following a high fat meal. Further preferred GSSP4 polypeptides are those that significantly reduce or eliminate ketone body production, particularly following a high fat meal. Further preferred GSSP4 polypeptides are those that increase glucose uptake in skeletal muscle cells. Further preferred GSSP4 polypeptides are those that increase glucose uptake in adipose cells. Further preferred GSSP4 polypeptides are those that increase glucose uptake in neuronal cells. Further preferred GSSP4 polypeptides are those that increase glucose uptake in red blood cells. Further preferred GSSP4 polypeptides are those that increase glucose uptake in the brain. Further preferred GSSP4 polypeptides are those that significantly reduce the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal. Further preferred GSSP4 polypeptides are those that significantly prevent the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal. Further preferred GSSP4 polypeptides are those that improve insulin sensitivity. Further preferred GSSP4 polypeptides are those that modulate food intake or food selection. Further preferred GSSP4 polypeptides are those that modulate satiety. Further preferred GSSP4 polypeptides are those that modulate fatty acid metabolism. Further preferred GSSP4 polypeptides are those that modulate cholesterol metabolism, particularly in steroidogenic tissues. Therefore, said polypeptides have a potential role in effecting, either directly or indirectly or both, levels of reproductive hormones (eg. estradiol, progesterone, testosterone). Further preferred GSSP4 polypeptides are those that modulate cortisol levels. Further preferred GSSP4 polypeptides are those that modulate aldosterone levels. Therefore, said polypeptides have a potential role in effecting, either directly or indirectly or both, levels of sodium and potassium. Further preferred GSSP4 polypeptides are those that modulate blood pressure preferably to normalize blood pressure within a normal range. Further preferred GSSP4 polypeptides are those that form multimers (e.g., heteromultimers or homomultimers) in vitro and/or in vivo. Preferred multimers are homodimers or homotrimers. Other preferred multimers are homomultimers comprising at least 4, 6, 8, 9, 10 or 12 GSSP4 polypeptides. Other preferred mulimers are hetero multimers comprising GSSP4 polypeptides of the invention. Further preferred embodiments include heterologous polypeptides comprising a GSSP4 polypeptide of the invention. In a second aspect, the invention features purified, isolated, or recombinant polynucleotides encoding said GSSP4 polypeptides described in the first aspect, or the complement thereof. A further preferred embodiment of the invention is a recombinant, purified or isolated polynucleotide comprising, or consisting of a mammalian genomic sequence, gene, cDNA, or fragments thereof. In one aspect the sequence is derived from a human, mouse or other mammal. In a preferred aspect, the genomic sequence includes isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 22, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 2000, 5000, 6000 or 7500 nucleotides of any one of the polynucleotide sequences described in SEQ ID NO:1, 2, or the complements thereof, wherein said contiguous span comprises a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding nucleotide sequence in SEQ ID NO: 1, 2, or 3. In further embodiments the polynucleotides are DNA, RNA, DNA/RNA hybrids, single-stranded, and double-stranded. Further preferred are GSSP4 polynucleotides and polypeptides that have cholesterol regulating activies. Further preferred are GSSP4 polynucleotides and polypeptides that have body weight regulating activies. Further preferred are GSSP4 polynucleotides and polypeptides that have body fat regulating activies. Further preferred are GSSP4 polynucleotides and polypeptides that have glucose regulating activies. Further preferred are GSSP4 polynucleotides and polypeptides that have lipid regulating activies. In a third aspect, the invention features a recombinant vector comprising, consisting essentially of, or consisting of, said polynucleotide described in the second aspect. In a fourth aspect, the invention features a recombinant cell comprising, consisting essentially of, or consisting of, said recombinant vector described in the third aspect. A further embodiment includes a host cell recombinant for a polynucleotide of the invention. In a fifth aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said GSSP4 polypeptides described in the first aspect and, a pharmaceutical or physiologically acceptable diluent. In a sixth aspect, the invention features a method of controlling cholesterol levels comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect. In further preferred embodiments, the invention features a method of lowering body weight comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect. In further preferred embodiments, the invention features a method of lowering body fat comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect. In further preferred embodiments, the invention features a method of lowering controlling blood glucose comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect. In a seventh aspect, the invention features a method of preventing or treating a metabolic-related disease or disorder comprising, providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect. Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. In preferred embodiments, said individual is a mammal, preferably a human. In a further preferred embodiment, a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect suggest that a compound may have utility in alleviating insulin resistance in individuals, particularly those that are obese or overweight. In a further preferred embodiment, the present invention may be used in complementary therapy in individuals to improve their cholesterol, weight or glucose level, comprising a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect in combination with known agents. The present invention further provides a method of improving the cholesterol levels, body weight or glucose control in individuals comprising the administration of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect alone, without known agents. In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with known agents for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the known agent). Accordingly, the present invention further provides a product containing a composition a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect and a known agent as a combined preparation for simultaneous, separate or sequential use for the improvement of cholesterol levels, body weight or glucose control in individuals, particularly those who are obese or overweight. The ratio of the present composition to known agent is such that the quantity of each active ingredient employed will be such as to provide a therapeutically effective level, but will not be larger than the quantity recommended as safe for administration. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect can be used as a method to improve insulin sensitivity in some persons, particularly those with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) or noninsulin dependent diabetics (Type II) in combination with insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect can be used as a method to improve insulin sensitivity in some persons, particularly those with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) or noninsulin dependent diabetes mellitus (NIDDM, Type II) in combination with alternate therapies. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used as a method in prophylaxis of long-term detrimental effects caused by prolonged high dosage of insulin in humans having IDDM or NIDDM. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing hypersecretion of insulin and disorders or conditions resulting therefrom. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing obesity and consequences or complications thereof. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing hypercholesterolemia and consequences or complications thereof. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing NIDDM or IDDM and consequences or complications thereof. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing impaired glucose tolerance (IGT). Further preferred embodiment thus provides therapeutics and methods for normalizing insulin resistance. Further preferred embodiment thus provides therapeutics and methods for reducing, slowing or preventing the progression to NIDDM. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing the appearance of insulin-resistance syndrome. In further preferred embodiments, other conditions, particularly obesity, associated with insulin resistance are treated or prevented according to the methods of the invention. Thus, by preventing or treating obesity, the methods of the invention will allow an individual to have a more comfortable life and avoid the onset of various diseases triggered by obesity. In further preferred embodiments, the target of the methods according to the present invention includes individuals with normal glucose tolerance (NGT) who are obese or who have fasting hyperinsulinemia, or who have both. In an eighth aspect, the invention features a method of controlling blood free fatty acid (FFA) levels and lipid metabolism comprising, providing, or administering to individuals in need of increasing mobilization and utilization of fat stores and decreasing total fat stores with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect. In a further preferred embodiment, the identification of said individuals in need of increasing mobilization and utilization of fat stores and decreasing total fat stores to be treated with said pharmaceutical or physiologically acceptable composition comprises a person who is involved in physical activity which increases metabolic demand. Furthermore, increasing mobilization and utilization of fat stores and decreasing total fat stores would provide a means to decrease body weight, preventing weight gain, decrease body fat in overweight and obese individuals. Reduction in weight and obesity will thus decrease the risk of chronic disease associated with obesity such as but not limited to the onset of various lipid metabolism disorders, hypertension, Type II diabetes, atherosclerosis, cardiovascular disease and stroke. In related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising a GSSP4 polypeptide, wherein said biological response is selected from the group consisting of: (a) lowering circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids; (b) lowering circulating (either blood, serum or plasma) levels (concentration) of glucose; (c) lowering circulating (either blood, serum or plasma) levels (concentration) of triglycerides; (d) stimulating muscle lipid or free fatty acid oxidation; (e) increasing leptin uptake in the liver or liver cells; (f) reducing the postprandial increase in plasma free fatty acids, particularly following a high fat meal; and, (g) reducing or eliminating ketone body production, particularly following a high fat meal; (h) increasing tissue sensitivity to insulin, particularly muscle, adipose, liver or brain, (i) reducing cholesterol levels, particularly in those with elevated cholesterol (ie. greater than 200 mg/dl); (j) modulating circulating (either blood, serum or plasma) levels (concentration) of glucose within physiological range, preferably maintaining glucose between 60-190 mg/dl; (k) modulating circulating (either blood, serum or plasma) levels (concentration) of FFA within physiological range preferably maintaining FFA between 190-420 mg/dl; (l) modulating ketone body production as the result of a high fat meal, wherein said modulating is preferably reducing or eliminating; (m) reducing body weight particularly in individuals with a BMI of greater than 27. In a ninth aspect, the invention features a method of making the GSSP4 polypeptide described in the first aspect, wherein said method is selected from the group consisting of: proteolytic cleavage, recombinant methodology and artificial synthesis. In a tenth aspect, the present invention provides a method of making a recombinant GSSP4 polypeptides, the method comprising providing a transgenic, non-human mammal whose milk contains said recombinant GSSP4 polypeptides, and purifying said recombinant GSSP4 polypeptides from the milk of said non-human mammal. In one embodiment, said non-human mammal is a cow, goat, sheep, rabbit, or mouse. In another embodiment, the method comprises purifying a recombinant GSSP4 polypeptides from said milk, and further comprises cleaving said protein in vitro to obtain a desired GSSP4 polypeptides. In an eleventh aspect, the invention features a purified or isolated antibody capable of specifically binding to a protein comprising the sequence of one of the polypeptides of the present invention. In one aspect of this embodiment, the antibody is capable of binding to a polypeptide comprising at least 6 consecutive amino acids, at least 8 consecutive amino acids, or at least 10 consecutive amino acids of the sequence of one of the polypeptides of the present invention. In a twelfth aspect, the invention features a use of polypeptides described in the first aspect or polynucleotides described in the second aspect for treatment of metabolic-related diseases and disorders or reducing or increasing body mass. Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. In preferred embodiments, said individual is a mammal, preferably a human. In a thirteenth aspect, the invention features a use of polypeptides described in the first aspect or polynucleotides described in the second aspect for the preparation of a medicament for the treatment of metabolic-related diseases and disorders or for reducing body mass. Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. In preferred embodiments, said individual is a mammal, preferably a human. In a fourteenth aspect, the invention provides polypeptides of the first aspect of the invention or a composition of the fifth aspect for use in a method of treatment of the human or animal body. In a fifteenth aspect, the invention provides polynucleotides described in the second aspect or an acceptable composition thereof, for use in a method of treatment of the human or animal body. In a sixteenth aspect, the invention features methods of reducing body weight for cosmetic purposes comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect, or polypeptides described in the first aspect. Preferably, for said reducing body weight said individual has a BMI of at least 20, 25, 30, 35, or 40. In a seventeenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect or a polypeptide described in the first aspect for reducing body mass in said individuals with a BMI of at least 30, 35, 40, or 45 or for treatment or prevention of metabolic-related diseases or disorders. Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. In preferred embodiments, said individual is a mammal, preferably a human. In preferred embodiments, the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GSSP4 single nucleotide polymorphisms (SNPs) or measuring GSSP4 polypeptide or mRNA levels in clinical samples from said individuals. Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva. In an eighteenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect for reducing body weight for cosmetic reasons. In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect for reducing glucose levels. In a nineteenth aspect, the invention features methods of treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect, or a polypeptide described in the first aspect. |
Method, module, device and server for voice recognition |
The invention concerns a voice recognition method implemented in at least a terminal, the voice recognition method using a language model, comprising the following steps: detecting at least a non-recognised expression in one of the terminals; recording in the terminal data representing the non-recognised expression; transmission by the terminal of the recorded data to a remote server; analysis, at the remote server, of said data and generation of data for correcting said language model taking into account at least part of the non-recognised expressions; and transmitting from the server to at least a terminal correction data, so as to enable subsequent recognition of at least some of the non-recognised expressions. The invention also concerns corresponding modules, devices and remote server. |
1. Voice recognition process implemented in at least one terminal, the said voice recognition process using a language model, wherein it comprises the following steps: detection of at least one unrecognized expression in one of the said terminals; recording in the said terminal of data representative of the said unrecognized expression; transmission by the said terminal of the said recorded data to a remote server, via a first transmission channel; analysis, at the level of the said remote server, of the said data and generation of information for correcting the said language model taking account of at least one part of the said unrecognized expressions; and transmission via a second transmission channel from the said server to at least one terminal of the said correcting information, so as to allow future recognition of at least certain of the said unrecognized expressions. 2. Process according to claim 1, wherein the said data representative of the said unrecognized expressions comprise a compressed voice recording representative of parameters descriptive of the acoustic signal. 3. Process according to claim 1, wherein during the said step of transmission by the said terminal, the latter furthermore transmits to the said server at least one of the items of information forming part of the group comprising: information of context of use of the said voice recognition process when an expression has not been recognized; and information relating to the speaker who has uttered an unrecognized expression. 4. Process according to claim 1, wherein it implements an encryption and/or a scrambling of the said recorded data and/or of the said correcting information. 5. Voice recognition module using a language model, wherein it comprises: an analyser detecting unrecognized expressions; a recorder of data representative of at least one unrecognized expression; a transmitter transmitting the said recorded data to a remote server; and a receiver of correcting information allowing the correcting of the said language model transmitted to the said module allowing future recognition of at least certain of the said unrecognized expressions by the said module, the correcting information having been transmitted by the said remote server after analysis at the level of the said remote server of the said data, and after generation of information for correcting the said language model taking account of at least one part of the unrecognized expressions. 6. Voice recognition device using a language model, wherein it comprises: an analyser detecting unrecognized expressions; a recorder of data representative of at least one unrecognized expression; a transmitter transmitting the said recorded data to a remote server; and a receiver of correcting information allowing the correcting of the said language model transmitted to the said device allowing future recognition of at least certain of the said unrecognized expressions by the said device, the correcting information having been transmitted by the said remote server after analysis at the level of the said remote server of the said data, and after generation of information for correcting the said language model taking account of at least one part of the unrecognized expressions. 7. Voice recognition server, the said recognition being implemented in a set of at least one remote terminal, using a language model, wherein it comprises the following means: a receiver of data representative of at least one expression unrecognized by at least one terminal forming part of the said set and having detected the said unrecognized expression during a voice recognition operation; and a sender sending to the said set of at least one remote terminal correcting information obtained on the basis of an analysis of the said data received at the level of the said server, the said correcting information allowing the correcting by each of the terminals of the said set, of the said language model allowing future recognition of at least one part of the unrecognized expressions. |
Tissue transglutaminase |
The present invention relates to tissue transglutaminase (tTG), and more particularly to DNA coding for and polypeptides containing, the primary structural conformaion of one or more epitopes of tTG and to the use thereof in the detection of autoantibodies and/or lymphocytes produced in response to tTG. |
1. A DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes,: which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq. Ids 1, 3, 5, 7, 9, 11, 13, 15, 17, 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq. Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq. Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes. 2. A DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq. Ids 1, 3, 5, 7 and 9; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq. Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq. Ids 2 4, 6, 8 and 10, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes. 3. A DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq. Ids 11, 13, 15, 17 or 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq. Ids 12, 14, 16 and 18, or one or more active fragments of amino acids as depicted in one or more of Seq. Ids 12, 14, 16 and 18, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes. 4. A DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq. Ids 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq. Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq. Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies. 5. A DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq. Ids 1, 3, 5, 7 or 9; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq. Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq. Ids 2, 4, 6, 8 and 10, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies. 6. A DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq. Ids 11, 13, 15, 17 or 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq. Ids 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of FIGS. 18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies. 7. A DNA sequence as depicted in any of Seq. Ids 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19, or a DNA sequence differing therefrom due to the degeneracy of the genetic code. 8. A biologically functional plasmid or DNA vector including a DNA sequence as defined in claim 1. 9. A host cell which is transformed or transfected with a DNA sequence as defined in claim 1. 10. A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG. 11. A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG. 12. A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes; with the exception of human, guinea pig, mouse, bovine or rat full length tTG. 13. A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; with the exception of human, guinea pig, mouse, bovine or rat full length tTG. 14. A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG. 15. A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; with the exception of human, guinea pig, mouse, bovine or rat full length tTG. 16. A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact,under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq. Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq. Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG. 17. A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq. Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq. Ids 2, 4, 6, 8 and 10, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG. 18. A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq. Ids 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq. Ids 12, 14, 16, 18 and 20, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG. 19. A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq. Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq. Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG. 20. A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq. Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq. Ids 2, 4, 6, 8 and 10, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG. 21. A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq. Ids 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq. Ids 12, 14, 16, 18 and 20, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG. 22. A polypeptide as depicted in any of Seq. Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. 23. A process of preparing a polypeptide as defined in claim 10, which process comprises: (i) providing a host cell which is transformed or transfected with a DNA sequence or a biologically functional plasmid or DNA vector; (ii) growing the host cell; and (iii) recovering a polypeptide as defined in claim 10. 24. A method of screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing either (i) said sample of body fluid from said subject or (ii) lymphocytes isolated from said sample; (b) contacting said sample or isolated lymphocytes with a polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies or lymphocytes produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to tTG, and present in, or isolated from, said sample; and (c) monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to tTG and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or said lymphocytes, in said sample, or isolated from said sample. 25. A method according to claim 24, for screening for autoantibodies produced in response to tTG and present in said sample. 26. A method of screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing said sample of body fluid from said subject; (b) contacting said sample with a polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies produced in response to tTG and present in said sample; and (c) monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to tTG and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample. 27. A method according to claim 24, which comprises directly monitoring interaction of (i) autoantibodies to tTG present in the sample of body fluid from the subject and (ii) a polypeptide as defined in any of claims 10 to 22. 28. A method according to claim 24, which further comprises providing at least one competitor, whereby in step (b) a polypeptide as defined in any of claims 10 to 22, can interact with either the competitor or autoantibodies to tTG and present in said sample. 29. A method of screening for autoantibodies to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing said sample of body fluid from said subject; (b) contacting said sample with (i) full length tTG, and (ii) at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies to tTG, so as to permit said full length tTG to interact with either autoantibodies to tTG present in said sample, or said competitor; and (c) monitoring the interaction of said full length tTG with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to tTG in said sample. 30. A kit for screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) a polypeptide as defined in claim 10; (b) means for contacting either (i) a sample of body fluid obtained from said subject, or (ii) lymphocytes isolated from a sample of body fluid obtained from said subject, with said polypeptide as defined in claims 10, under conditions that allow interaction of tTG with autoantibodies or lymphocytes produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to tTG, and present in, or isolated from, said sample; and (c) means for monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to tTG and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or lymphocytes, in said sample, or isolated from said sample. 31. A kit according to claim 30, for screening for autoantibodies produced in response to tTG in said sample of body fluid. 32. A kit for screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) a polypeptide as defined in claim 10; (b) means for contacting a sample of body fluid obtained from said subject with said polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies produced in response to tTG and present in said sample; and (c) means for monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to tTG and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample. 33. A kit according to claim 30, which comprises means for directly monitoring interaction of (i) autoantibodies to tTG present in the sample of body fluid from the subject and (ii) a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG. 34. A kit according to of claim 30 to 32, which further comprises at least one competitor, whereby a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG, can interact with either the competitor or autoantibodies to tTG present in said sample of body fluid being screened. 35. A kit for screening for autoantibodies to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) full length tTG; (b) at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide as defined in claim 10; (c) means for contacting said sample of body fluid from said subject, said full length tTG and said competitor, under conditions that allow interaction of tTG with autoantibodies to tTG, so as to permit said full length tTG to interact with either autoantibodies to tTG present in said sample, or said competitor; and (d) means for monitoring the interaction of said full length tTG with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to tTG in said sample. 36. An antibody produced in response to one or more epitope regions of tTG, which epitope region comprises part or all of the primary structural conformation of amino acids 1 to 89 of tTG, or the primary structural conformation of amino acids 401 to 494 or 401 to 491 of tTG, or one or more fragments of an antibody produced in response to one or more epitope regions of tTG, which epitope region comprises part or all of the primary structural conformation of amino acids 1 to 89 of tTG, or the primary structural conformation of amino acids 401 to 494 or 401 to 491 of tTG. 37. An antibody according to claim 36, which is obtained by the Examples. 38. An antibody according to claim 36, which is monoclonal, recombinant or polyclonal. 39. An antibody according to claim 38, which is monoclonal. 40. A host cell containing an antibody as defined in claims 36. 41. A hybridoma capable of secreting a monoclonal antibody as defined in claim 36. 42. For use in diagnosis, an antibody as defined in claim 36. 43. Use according to claim 42, in the diagnosis of autoimmune disease associated with an immune reaction to tTG. 44. For use in therapy, an antibody as defined in claim 36. 45. Use according to claim 44, in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG. 46. Use according to claim 43, wherein the autoimmune disease is coeliac disease. 47. An antibody according to claim 36, in the manufacture of a medicament for the treatment of coeliac disease. 48. For use in diagnosis, a polypeptide as defined in claim 10. 49. Use according to claim 48, in the diagnosis of autoimmune disease associated with an immune reaction to tTG. 50. For use in therapy, a polypeptide as defined in claim 10. 51. Use according to claim 50, in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG. 52. Use according to claim 49, wherein the autoimmune disease is coeliac disease. 53. A pharmaceutical composition comprising a polypeptide as defined in claim 10, together with a pharmaceutically acceptable carrier, diluent or excipient therefor, wherein said polypeptide can interact with autoantibodies and/or lymphocytes produced in response to tTG. 54. A polypeptide as defined in claim 10, for use in the manufacture of a medicament for the treatment of coeliac disease. 55. A method of diagnosing the likely onset or presence of autoimmune disease associated with an immune reaction to tTG in a subject suspected of suffering from, susceptible to, having or recovering from, autoimmune disease associated with an immune reaction to tTG, the method comprising detecting autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid from the subject according to a method as defined in claim 24, and whereby the detected autoantibodies and/or lymphocytes can provide a diagnosis of the likely onset or presence of autoimmune disease associated with an immune reaction to tTG in the subject 56. A method of delaying or preventing the onset of autoimmune disease associated with an immune reaction to tTG in an animal subject suspected of suffering from, susceptible to or recovering from autoimmune disease associated with an immune reaction to tTG, which method comprises initially detecting autoantibodies or lymphocytes indicative of the onset or presence of autoimmune disease associated with an immune reaction to tTG in a sample of body fluid obtained from the subject according to a method as defined in claim 24, thereby providing a diagnosis of the likely onset of autoimmune disease associated with an immune reaction to tTG in the subject, and thereafter therapeutically treating the subject so as to delay the onset and/or prevent autoimmune disease associated with an immune reaction to tTG. 57. A method of monitoring dietary compliance by a subject having, suspected of suffering from, susceptible to or recovering from gluten sensitive enteropathy, which method comprises screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from the subject according to a method as defined in claim 24, and whereby the detection of such autoantibodies and/or lymphocytes produced in response to tTG in the sample of body fluid provides an indication as to the dietary compliance of the subject to a gluten-free or substantially gluten free diet. 58. A method of treating autoimmune disease associated with an immune reaction to tTG in a subject, which method comprises initially detecting autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from the subject according to a method as defined in claim 24, thereby providing a diagnosis of autoimmune disease in the subject, and administering to the subject a therapeutically effective amount of at least one therapeutic agent effective in the treatment of such autoimmune disease. 59. A method of treating autoimmune disease associated with an immune reaction to tTG in a subject, which method comprises administering to the subject a therapeutically effective amount of a therapeutic agent identified as providing a therapeutic effect by interaction with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG, or amino acids 401 to 492 of tTG. 60. A therapeutic agent identified as providing a therapeutic effect by interaction with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG, or amino acids 401 to 491 of tTG. 61. A therapeutic agent according to claim 60, for use in the therapeutic treatment of an autoimmune disease associated with an immune reaction to tTG. 62. A method of cloning lymphocytes produced in response to tTG, which method comprises: providing a source of lymphocytes; contacting the lymphocytes with a polypeptide as defined in claim 10, so as to effect proliferation of said lymphocytes; and isolating and cloning the proliferating lymphocytes. 63. Use of lymphocytes prepared according to claim 62, in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG. 64. A pharmaceutical composition comprising lymphocytes prepared according to claim 62, together with a pharmaceutically acceptable carrier, diluent or excipient therefor. 65. Use of lymphocytes prepared according to claim 62, in the manufacture of a medicament for the treatment of autoimmune disease associated with an immune reaction to Ttg. 66. Use of lymphocytes prepared according to claim 62, in the manufacture of a medicament for the treatment of coeliac disease. 67. A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 2. 68. A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 3 69. A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 4. 70. A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 5. 71. A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 6. 72. A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 7. 73. A host cell which is transformed or transfected with a plasmid or vector as defined in claim 67. 74. A host cell which is transformed or transfected with a plasmid or vector as defined in claim 68. 75. A host cell which is transformed or transfected with a plasmid or vector as defined in claim 69. 76. A host cell which is transformed or transfected with a plasmid or vector as defined in claim 70. 77. A host cell which is transformed or transfected with a plasmid or vector as defined in claim 71. 78. A host cell which is transformed or transfected with a plasmid or vector as defined in claim 72. |
Information processing apparatus and method |
When reproducing content, a client reads attribute information defined in the header of content data corresponding to a content ID specified by a user. When the read attribute information meets an attribute condition defined in a license stored in a storage unit defining the attribute condition, the encrypted content data is decrypted and output. After the content is distributed, a collected edition or a best edition can be newly released without creating new content by issuing the license having an attribute condition that limits, for example, a release data and an artist. Defining a license with a specific subscription ID as the attribute condition allows the user with the license to use new release content with the subscription ID without additional purchase of a license. |
1. An information processing apparatus comprising: content receiving means for receiving content including encrypted content data and attribute information; content storage means for storing the content; license receiving means for receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; license storage means for storing the license; determining means for determining whether the attribute information on the piece of the content meets the attribute condition of the license stored in the license storage unit; decrypting means for decrypting the encrypted content data of the piece of the content based on the determination of the determining means that the attribute information on the piece of the content meets the attribute condition of the license; and outputting means for outputting the content data decrypted by the decrypting means. 2. An information processing apparatus according to claim 1, wherein the content further includes a content key for decrypting the content data. 3. An information processing apparatus according to claim 1, wherein the attribute information includes a combination of an attribute item and an attribute value. 4. An information processing apparatus according to claim 1, wherein the attribute item includes information on a record company, an artist, a release date, a content provider, a genre, a subscription, and a label. 5. An information processing apparatus according to claim 1, wherein the attribute condition includes a combination of an attribute item, an attribute value, and an operator. 6. An information processing apparatus comprising: receiving means for receiving a license request including a license ID for uniquely identifying the license including the attribute condition defining the condition regarding the attribute information included in the content; storage means for storing the license along with the license ID; obtaining means for obtaining the license corresponding to the license ID included in the license request; signature means for adding a digital signature to the license; and sending means for sending the license with the signature added thereto by the signature means. 7. An information processing apparatus according to claim 6, further comprising license processing means for attaching a terminal ID to the license obtained by the obtaining means. 8. An information processing apparatus comprising: storing means for storing content including encrypted content data and attribute information; receiving means for receiving a content request including a content ID for uniquely identifying the content; and sending means for sending a piece of content corresponding to the content ID included in the content request, wherein: the attribute information included in the piece of the content is information used for determining whether an attribute condition of the license is met when the piece of the content is used; and the attribute condition of the license is information defining a condition regarding the attribute information on the piece of the content that can be used. 9. An information processing method comprising: a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step. 10. A program for causing a computer to execute: a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step. 11. A program storage medium containing a program to cause a computer to execute: a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step. |
<SOH> BACKGROUND ART <EOH>Recently, systems have been realized for exchanging music data for free among a plurality of users in which a user provides his/her own music data to other users via the Internet and receives other users' music data that the user does not have. Theoretically, only with the presence of one piece of music data and another piece of content, such systems allow the other users to take advantage of them. Because of this, many users do not purchase the content, causing a copyright holder to lose their chance to obtain royalties that the copyright holder deserves. Accordingly, there are systems in which the content to be distributed is encrypted and the license for using the content is separately provided, so that the encrypted content cannot be decrypted and reproduced without the license corresponding to the encrypted content. This enables the content to be freely distributed while the copyright of the copyright holder is protected. However, the above system has difficulties in flexibly establishing a correspondence between the license and the content, and difficulties in newly distributing the content to which the already-distributed license can be applied. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a block diagram showing a construction of a content providing system to which the present invention is applied. FIG. 2 is a block diagram showing a construction of a client in FIG. 1 . FIG. 3 is a flowchart illustrating content download processing by the client in FIG. 1 . FIG. 4 is a flowchart illustrating content providing processing by a content server in FIG. 1 . FIG. 5 is a diagram showing an example data format. FIG. 6 is a diagram illustrating types of attribute items. FIG. 7 is a diagram showing a license structure. FIG. 8 is a flowchart illustrating reproduction processing by the client. FIG. 9 is a flowchart illustrating license acquisition processing. FIG. 10 is a flowchart illustrating license acquisition processing. FIG. 11 is a flowchart illustrating license acquisition processing. FIG. 12 is a flowchart illustrating details of the license acquisition processing. FIG. 13 is a flowchart illustrating content data obtaining processing. FIG. 14 is a diagram illustrating a key structure. FIG. 15 is a diagram illustrating a relationship between the key structure and a license. FIG. 16 is a diagram illustrating license granting processing by a license server. detailed-description description="Detailed Description" end="lead"? |
Gonococcal proteins and nucleic acids |
The invention provides proteins from gonococcus (Neisseria gonorrhoeae), including amino acid sequences, the corresponding nucleotide sequences, expression data, and serological data. The proteins are useful antigens for vaccines, immunogenic compositions, and/or diagnostics. They are also useful for distinguishing between gonococcus and meningococcus and, in particular, between gonococcus and serogroup B meningococcus. |
1. A protein comprising an amino acid sequence selected from the group consisting of SEQ IDs 26, 72, 230, 984, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314 and 316: 2. A protein having 50% or greater sequence identity to a protein according to claim 1. 3. A protein comprising a fragment of an amino acid sequence selected from the group consisting of 26, 72, 230, 984, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206,208, 210, 212,214, 216, 218,220,222, 224, 226, 228, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314 and 316. 4. An antibody which specifically binds to a protein according to any one of claims 1 to 3. 5. A nucleic acid molecule which encodes a protein according to any one of claims 1 to 3. 6. A nucleic acid molecule according to claim 5, comprising a nucleotide sequence selected from the group consisting of SEQ IDs 25, 71, 229, 983, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313 and 315. 7. A nucleic acid molecule comprising a fragment of a nucleotide sequence selected from the group consisting of SEQ IDs 25, 71, 229, 983, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313 and 315. 8. A nucleic acid molecule comprising a nucleotide sequence complementary to a nucleic acid molecule according to any one of claims 5 to 7. 9. A nucleic acid molecule comprising a nucleotide sequences having 50% or greater sequence identity to a nucleic acid molecule according to any one of claims 5 to 8. 10. A nucleic acid molecule which can hybridise to a nucleic acid molecule according to any one of claims 5 to 9 under high stringency conditions. 11. A composition comprising a protein, a nucleic acid molecule, or an antibody according to any preceding claim. 12. A composition according to claim 11 being a vaccine composition or a diagnostic composition. 13. A composition according to claim 11 or claim 12 for use as a pharmaceutical. 14. The use of a composition according to claim 13 in the manufacture of a medicament for the treatment or prevention of infection due to streptococcus bacteria, particularly N.gonorrhoeae. 15. A process for distinguishing N.gonorrhoeae from N.meningitidis, comprising the steps of: (a) contacting a protein, a nucleic acid molecule, or an antibody according to any one of claims 1 to 10 with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes. |
<SOH> BACKGROUND ART <EOH>Neisseria gonorrhoeae is a bacterial pathogen. There is currently no effective vaccine against N.gonorrhoeae infection. It is an object of the invention to provide proteins and nucleic acid useful in vaccine study and/or manufacture. N.gonorrhoeae is related to N.meningitidis. Sequence data are now available for serogroup B of meningococcus [e.g. WO99/24578; WO99/36544; WO99/57280; WO00/22430; WO00/66791; Tettelin et al. (2000) Science 287:1809-1815] and also for serogroup A [Parkhill et al. (2000) Nature 404:502-506]. It is a further object of the invention to provide proteins and nucleic acid useful in distinguishing between gonococcus and meningococcus and, in particular, between gonococcus and serogroup B meningococcus. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>There are no drawings. detailed-description description="Detailed Description" end="lead"? |
Extracts derived from chenopodium plants and uses thereof |
The present invention relates to pesticides. More particularly, the present invention relates to botanical pesticides. In particular, the present invention relates to compositions and methods for controlling plant-infesting pests with plant extracts and notably with compositions comprising oil extracts derived from Chenopodium sp. plant material. The invention further relates to compositions comprising such extracts as pesticidal compositions and providing the advantages of minimal development of resistance thereto, minimal toxicity to mammals, minimal residual activity and environmental compatibility. The pesticidal compositions of the present invention comprises Ξ±-terpinene, Ο-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone. |
1. An essential oil extract derived from a Chenopodium species, comprising: from at least 30% to at least 65% Ξ±-terpinene, from at least 8% to at least 26% Ο-cymene, from at least 5% to at least 24% limonene, from at least 0.04% to at least 1% carvacrol, from at least 0.1% to at least 2% carveol, from at least 0.1% to at least 2.0% nerol, from at least 0.04% to at least 2% thymol, and from at least 0.04% to at least 1% carvone, and wherein said extract is active against one or more pests chosen from insects, acari, and fungi. 2. (Canceled) 3. (Canceled) 4. (Canceled) 5. The essential oil extract according to claim 1, wherein said species is Chenopodium ambrosioides. 6. A pesticidal composition, comprising an effective amount of the essential oil extract of claim 1 and a suitable emulsifier, spreader or sticking agent, and carrier. 7. The pesticidal composition according to claim 6, comprising between 0.125% to 10% (by volume) essential oil extract. 8. The pesticidal composition according to claim 7, comprising between 0.25% to 5% (by volume) of said essential oil extract. 9. The pesticidal composition according to claim 6, wherein said composition comprises between 5% to 50% (by volume) of said essential oil extract. 10. The pesticidal composition according to claim 9, comprising between 10% to 25% (by volume) of said essential oil extract. 11. The pesticidal composition according to claim 10, comprising between 1% to 15% (by volume) of a suitable emulsifier, and between 50% to 70% (by volume) of a suitable carrier or solvent. 12. The pesticidal composition according to claim 11, comprising between 2% to 20% (by volume) of a suitable spreader or sticking agent. 13. A pesticidal composition, comprising between 30% to 50% (by volume) of the essential oil extract of claim 1, between 0.5% to 25% (by volume) of a suitable emulsifier, and between 10% to 50% (by volume) water. 14. A method for controlling one or more pests chosen from phytophagous acari, phytophagous insects, and phytophagous fungi, which comprises applying to a locus where control is desired a pesticidally-effective amount of the pesticidal composition of claim 6. 15. (Canceled) 16. (Canceled) 17. The method according to claim 14, wherein said locus is soil. 18. The method according to claim 14, wherein said locus is a plant. 19. The essential oil extract according to claim 1, further comprising ascaridole at a proportion of 9.86% or less. 20. The essential oil extract according to claim 1, further comprising ascaridole at a proportion of 1% or less. 21. The essential oil extract according to claim 20, wherein said extract has trace or undetectable amounts of ascaridole. 22. A formulation for indoor or outdoor fumigation, comprising: between 1% to 15% (by volume) of the essential oil extract of claim 1. 23. A formulation for topical application, comprising: between 1% to 10% (by volume) of the essential oil extract of claim 1. 24. The formulation of claim 23, wherein the formulation further comprises between 2% to 5% (by volume) of an emollient, between 0.1% to 1% (by volume) of a stabilizer and between 0.1% to 0.5% (by volume) of a preservative. 25. The formulation of claim 23, wherein the formulation further comprises between 1% to 10% (by volume) of an emulsifier, between 2% to 5% (by volume) of a coat or skin conditioner, between 0. 1% to 1% (by volume) of a stabilizer, between 0. 1% to 1% (by volume) of a preservative and between 0. 1% to 0.5% (by volume) of an antioxidant. 26. A microemulsion formulation, comprising: between 30% to 50% (by volume) of the essential oil extract of claim 1, between 0.5% to 25% (by volume) of a suitable emulsifier, and between 10% to 50% (by volume) water. 27. The formulation of claim 26, wherein said formulation is for soil delivery. 28. A method for controlling phytophagous pests, which comprises applying to a locus where control is desired a pesticidally-effective amount of the formulation according to any one of claims 22, 23, or 26. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Plant feeding mites are among the most voracious phytophagous pests of crops. To combat these pests, synthetic pesticides have been developed. These synthetic chemical pesticides, however, often have detrimental environmental effects that are harmful to humans and other animals and therefore do not meet the guidelines developed by most Integrated Pest Management programs. Moreover, resistance to these products has been found to develop with many of the new products put on the market (Georghiou, 1990; Nauen et al., 2001). Although resistance follows a highly complex genetic and biochemical process, it can generally develop rapidly with synthetic products because their active ingredients rely on one or more molecules of the same class. The organism can therefore respond to the toxin by developing physiological, behavioral or morphological defense mechanisms to neutralize the effect of the molecule (Roush and MacKenzie, 1987). Acari, such as spider mites, in particular, are extremely difficult to control with pesticides. Tetranychus urticae (the two-spotted spider mite), for example, has accumulated a considerable number of genes conferring resistance to all major classes of acaricides. Resistance to many registered acaricides have been reported, for example, resistance has been reported to hexythiazox, abamectin, and clofentezine. Furthermore, many of these pesticides have been found to exacerbate pest infestation by destroying the natural predators of mites (U.S. Pat. No. 5,839,224). Additionally, many synthetic insecticides have been found to stimulate mite reproduction. For example, it was found that mites reproduce many times faster when exposed to carbaryl, methyl parathion, or dimethoate in the laboratory than untreated populations (Flint, 1990). As a result, there are very few pesticides remaining that are effective against spider mites (Georghiou, 1990). In the Farm Chemical Handbook (Meister, 1999), for example, only 48 products out of a total of 2,050 listed acaricides and insecticides (or 2.4%), were identified as acaricides and only 69 of these products (or 3.4%) were identified as both acaricides and insecticides. The control of insect infestation has also proven to be difficult. For example, European chafer Rhizotrogus majalis Razoumowsky (Coleoptera: Scarabaeidae) insects are an exotic species to Canada and the United States and are commonly known as Scarab beetles. Throughout the northeast, where the beetle has become established, R. majalis causes major damage to turfgrass, consuming the roots of home lawns as well as urban recreational areas and golf courses. The larvae of this species are extremely difficult and expensive to control using conventional insecticides. In several areas of the U.S. insecticide resistance has already developed within populations. The control of plant fungi is another source for growing concern. Phytopathogenic fungi are of great economic importance since fungal growth on plants or on parts of plants inhibits production of root, stem, foliage, fruit or seed, and the overall quality of a cultivated crop. About 25 percent of all fungal diseases in agriculture and horticulture are caused by powdery mildew phytopathogens (U.S. Pat. Nos. 5,882,689 and 5,496,568). In cucumber crops, for example, powdery mildew caused by the plant pathogens, Erysiphe cichoracearum and Sphaerotheca fuliginea , is one of the most problematic disease. The fungus responsible for Gray Mold, Botrytis cinerea, can attack more than 200 species of cultivated plants and especially those growing in a greenhouse environment. It is a saprophyte that attacks dead or senescent plant tissue. B. cinerea is especially damaging to stems (causing stem rot), when it enters scars left by pruning of lower leaves. Following attack, the plant dies, causing heavy economic losses to the grower. Control of stem rot is attempted by treating leaf scars after leaf pruning. Although benomyl is presently used, it will likely be removed from the market shortly. Moreover, iprodione (another pesticide), is less effective because of developing fungal resistance. In spite of their strong fungicidal effect, agricultural fungicides currently in use have been a source of problems, because the required amount is ever increasing as tolerance of target plant pathogens increases. Moreover, many fungicides are synthetic agents, which when used, pose possible drawbacks for humans, animals and the environment. Thus, effective commercial products (to combat fungal attacks on crops), which reduce the dose of synthetic chemicals spread into the environment, are in need; keeping in mind a regard for environmental and human safety issues. As an alternative to synthetic agents, botanical pesticides offer the advantage of being naturally derived compounds that are safe to both humans and the environment. Specifically, botanical pesticides offer such advantages as being inherently less toxic than conventional pesticides, generally affecting only the target pest and closely related organisms, and are often effective in very small quantities. In addition, botanical pesticides often decompose quickly and, therefore, are ideal for use as a component of Integrated Pest Management (IPM) programs. There are few published reports of the acaricidal properties of botanical pesticides. For example, U.S. Pat. No. 4,933,371 describes the use of saponins extracted from various plants (i.e., yucca, quillaja, agave, tobacco and licorice) as acaricides. This patent also describes the use of linalool extracted from the oil of various plants such as Ceylon's cinnamon, sassafras, orange flower, bergamot, Artemisia balchanorum, ylang ylang, rosewood and other oil extracts as acaricides. These methods, however, require the extraction of one active substance from the plant which often does not meet desired levels of toxicity towards acari. Plant essential oils are a complex mixture of compounds of which many can be biologically active against insect and mite pests, the compounds acting individually or in synergy with each other, to either repel or kill the pests by contact. These components are plant secondary metabolites or allelochemicals produced by plants as a defense mechanism against plant feeding pests (Ceske and Kaufman, 1999). Because of the complexity of the mixture, it has been observed that pests do not easily develop resistance to these products as they can to synthetic pesticides or botanical pesticides comprising a single active compound. In this respect, Feng and Isman (1995) demonstrated that repeated treatments of pure azadirachtin, a major active constituent of neem oil, against the green peach aphid led to a 9-fold resistance after 40 generations. However, repeated exposure during 40 generations to crude neem extracts did not lead to resistance. There remains a need to provide new and effective pesticidal products which overcome the shortcomings of products known in the art. For example, there remains a need for broad spectrum compositions which are less likely to enable pests to develop resistance thereto. There also remains a need to provide a method to combat pests at any given locus, using a composition which is not toxic to animals, especially to mammals, nor to any beneficial predator/parasitoid insects. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides essential oil extracts derived from Chenopodium sp. comprising, Ξ±-terpinene, Ο-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone, wherein these extracts have one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities. One or more of these extracts can be formulated into compositions that enable their application into different environments such as plants, soil, animals and buildings. If accordance with another aspect of the invention there is provided composition comprising one or more Chenopodium sp. -derived essential oil extracts, wherein said extract comprises Ξ±-terpinene, Ο-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone, in combination with an emulsifier, carrier, spreader and/or sticking agent, to enable application of the composition to a specific environment, wherein the composition has one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities. |
Dynamic management of access rights lists in a portable electronic object |
Access rights lists, such as capacity or access control lists, are dynamically managed in a data processing element such as a smart card from an administrator server. To access an access rights list from the server, the list is signed in the server so that a signature can be transmitted to the card. The card compares the signature received from the server to signatures determined according to the access rights lists contained in the card and keys associated with those lists. Server access to an identified list is only authorized when it corresponds to a signature which is found among the determined signatures in the card and which is identical to the received signature. |
1. A method for managing lists of rights of access between subjects and objects, stored in a data processing means from an external administrator entity, comprising the following steps: initially associating keys of administrator entities with access rights lists and storing a security algorithm in the data processing means, and subsequently accessing an access rights list from the entity by: signing the access rights list in the entity by applying determined data from the list and an associated key to the security algorithm in order to produce a signature, transmitting the signature from the entity to the data processing means, comparing the signature received in the data processing means with determined signatures according to applications of determined data in lists of access rights contained in the data processing means and of keys respectively associated with these lists to the security algorithm, and allowing access of the entity to an access rights list found only if its determined signature is identical to the received signature. 2. A method according to claim 1, wherein the association of the keys with the access rights lists is performed in advance in the processing means prior to bringing the data processing means into service. 3. A method according to claim 1, wherein the association of the keys with an access rights list to be added to the data processing means is performed by transmitting the list with the keys from the entity to the data processing means, and determination of signatures of the list received in the data processing means is performed by applying the determined data in the list received and the keys received to the algorithm, and validation of the recording of the list received in the data processing means occurs when one of the signatures determined is identical with the signature received. 4. A method according to claim 1, wherein the signing step is replaced by the reception in the entity of a signature transmitted by another entity. 5. A method according to claim 1, wherein delegation information is transmitted with the signature, and access of the entity to the list found is not authorised when the delegation information is detected in association with the list found in the data processing means. 6. A method according to claim 1, wherein the signatures to be compared with the signature received are determined in the data processing means prior to the transmitting step. 7. A method according to claim 1, further including the step before authorising access of the entity to the control list, of updating a duration of life parameter of the access rights list found in order to erase the access rights list found when the updated duration of life parameter exceeds a maximum limit and in order to authorise access to the access rights list found to the entity when the updated duration of life parameter is less than the maximum limit. 8. A method according to claim 7, wherein the duration of life parameter is a cumulative duration of sessions of use of the data processing means or a cumulative duration of absolute time. 9. A method according to claim 7, wherein the duration of life parameter is a number of sessions of use of the data processing means, or a number of sessions using the access rights list found. 10. A method according to claim 7, wherein the duration of life parameter is a number of commands received by the data processing means. 11. A method according to claim 7, wherein the duration of life parameter is a synchronisation value changed periodically in external administrator entities and transmitted with the signature of the access rights list. 12. A method according to claim 1, wherein the signatures of the access list associated with the keys of a list are again determined in the data processing means if the access rights list found has been modified by the entity. 13. A method according to claim 1, wherein the determined data of the list which are applied to the security algorithm depend on characteristics of at least one subject and/or at least one subject group and/or at least one object and/or at least one right of access of a subject to an object relating to the list. 14. A data processing means storing access rights lists managed from at least one external administrator entity comprising: a means for storing keys of administrator entities in association with access rights lists, a means for implementing a security algorithm, a means for determining signatures according to applications of determined data of the lists of access rights and keys respectively associated with said lists to the algorithm, a means for comparing a received signature of an access rights list which results, in the entity, from the application of determined data of the list and of the key of the entity to the algorithm and which is transmitted by the entity, to said determined signatures, and a means for authorising access of the entity to an access rights list found only in correspondence with a signature found amongst the determined signatures and identical to the received signature. 15. A data processing means according to claim 14, further including a means for storing a duration of life parameter and a maximum duration limit for each access rights list, and a means for updating the duration parameter of the list found in order to erase the access rights list found when the duration of life parameter updated exceeds the maximum limit and in order to authorise access to the access list found to the entity when the duration of life parameter updated is less than the maximum limit. |
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