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<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides diagnostic, drug screening, and therapeutic methods that are based on the observation that a mutation in a zebrafish gene, designated nil per os (npo), which is Latin for “nothing by mouth,” leads to abnormal digestive organ growth and development. In a first aspect, the invention provides a method of determining whether a test subject (e.g., a mammal, such as a human) has or is at risk of developing a disease or condition related to an npo protein (e.g., a disease or condition of a digestive organ (e.g., the intestine, liver, bile duct, pancreas, stomach, gall bladder, or esophagus), or cancer). This method involves analyzing a nucleic acid molecule of a sample from the test subject to determine whether the test subject has a mutation (e.g., the npo mutation; see below) in a gene encoding the protein. The presence of a mutation indicates that the test subject has or is at risk of developing a disease related to npo. This method can also involve the step of using nucleic acid molecule primers specific for a gene encoding an npo protein for nucleic acid molecule amplification of the gene by the polymerase chain reaction. It can further involve sequencing a nucleic acid molecule encoding an npo protein from a test subject. In a second aspect, the invention provides a method for identifying a compound that can be used to treat or to prevent a disease or condition of the digestive system or cancer. This method involves contacting an organism (e.g., a zebrafish) having a mutation (e.g., the nil per os mutation) in a gene encoding a nil per os protein and having a phenotype characteristic of such a disease or condition with the compound, and determining the effect of the compound on the phenotype. Detection of an improvement in the phenotype indicates the identification of a compound that can be used to treat or to prevent the disease or condition. In a third aspect, the invention provides a method of treating or preventing a disease or condition of the digestive system or cancer in a patient (e.g., a patient having a mutation (e.g., the nil per os mutation) in a gene encoding a nil per os protein), involving administering to the patient a compound identified using the method described above. Also included in the invention is the use of such compounds in the treatment or prevention of such diseases or conditions, as well as the use of these compounds in the preparation of a medicament for such treatment or prevention. In a fourth aspect, the invention provides a method of treating or preventing a disease or condition of the digestive system or cancer in a patient. This method involves administering to the patient a functional nil per os protein or a nucleic acid molecule (in, e.g., an expression vector) encoding the protein. Also included in the invention is the use of such proteins or nucleic acid molecules in the treatment or prevention of such diseases or conditions, as well as the use of these proteins or nucleic acid molecules in the preparation of a medicament for such treatment or prevention. In a fifth aspect, the invention includes a substantially pure nil per os polypeptide (e.g., a zebrafish or a human npo polypeptide) or a fragment thereof This polypeptide can include or consist essentially of, for example, an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NOs:3 or 5. The encoded polypeptide can include RNA recognition motifs (RRMs) and bind RNA, as is discussed further below. In a sixth aspect, the invention provides a substantially pure nucleic acid molecule (e.g., a DNA molecule) including a sequence encoding a nil per os polypeptide (e.g., a zebrafish or a human npo polypeptide) or a fragment thereof. This nucleic acid molecule can encode a polypeptide including or consisting essentially of an amino sequence that is substantially identical to the amino acid sequence of SEQ ID NOs:3 or 5. The encoded polypeptide can include RNA recognition motifs (RRMs) and bind RNA, as is discussed further below. In a seventh aspect, the invention provides a vector including the nucleic acid molecule described above. In an eighth aspect, the invention includes a cell including the vector described above. In a ninth aspect, the invention provides a non-human transgenic animal (e.g., a zebrafish or a mouse) including the nucleic acid molecule described above. In a tenth aspect, the invention provides a non-human animal having a knockout mutation in one or both alleles encoding a nil per os polypeptide. In an eleventh aspect, the invention includes a cell from the non-human knockout animal described above. In a twelfth aspect, the invention includes a non-human transgenic animal (e.g., a zebrafish) including a nucleic acid molecule encoding a mutant nil per os polypeptide, e.g., a polypeptide having the nil per os mutation. In a thirteenth aspect, the invention provides an antibody that specifically binds to a nil per os polypeptide. In a fourteenth aspect, the invention provides a method of modulating nil per os protein activity by administration of an RNA that stimulates or inhibits this activity. Also included in the invention is the use of such an RNA molecule to stimulate or to inhibit this activity, as well as the use of this RNA molecule in the preparation of a medicament for such stimulation or inhibition. In a fifteenth aspect, the invention provides a method of identifying a stem cell of the gastrointestinal tract, which involves analyzing a pool of candidate cells for expression of nil per os. Cells that express nil per os can then, optionally, be removed from the original pool of candidate cells. By “polypeptide” or “polypeptide fragment” is meant a chain of two or more amino acids, regardless of any post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally or non-naturally occurring polypeptide. By “post-translational modification” is meant any change to a polypeptide or polypeptide fragment during or after synthesis. Post-translational modifications can be produced naturally (such as during synthesis within a cell) or generated artificially (such as by recombinant or chemical means). A “protein” can be made up of one or more polypeptides. By “nil per os protein,” “npo protein,” “nil per os polypeptide,” or “npo polypeptide” is meant a polypeptide that has at least 45%, preferably at least 60%, more preferably at least 75%, 80%, or 85%, and most preferably at least 90% or 95% amino acid sequence identity to the sequence of a human (SEQ ID NO:5) or a zebrafish (SEQ ID NO:3) nil per os polypeptide. Polypeptide products from splice variants of nil per os gene sequences and nil per os genes containing mutations are also included in this definition. A nil per os polypeptide as defined herein plays a role in digestive organ development, modeling, and function. It can be used as a marker of diseases and conditions of the digestive system, digestive organs, or cancer. By a “nil per os nucleic acid molecule” or “npo nucleic acid molecule” is meant a nucleic acid molecule, such as a genomic DNA, cDNA, or RNA (e.g., mRNA) molecule, that encodes a nil per os protein (e.g., a human (encoded by SEQ ID NO:4) or a zebrafish (encoded by SEQ ID NOs:1 or 2) nil per os protein), a nil per os polypeptide, or a portion thereof, as defined above. A mutation in a nil per os nucleic acid molecule can be characterized, for example, by a tyrosine codon to stop codon change (TAT to TAA) in the codon for amino acid 221. In addition to this zebrafish nil per os mutation (hereinafter referred to as “the nil per os mutation”), the invention includes any mutation that results in aberrant nil per os protein production or function, including, only as examples, null mutations and additional mutations causing truncations. The term “identity” is used herein to describe the relationship of the sequence of a particular nucleic acid molecule or polypeptide to the sequence of a reference molecule of the same type. For example, if a polypeptide or a nucleic acid molecule has the same amino acid or nucleotide residue at a given position, compared to a reference molecule to which it is aligned, there is said to be “identity” at that position. The level of sequence identity of a nucleic acid molecule or a polypeptide to a reference molecule is typically measured using sequence analysis software with the default parameters specified therein, such as the introduction of gaps to achieve an optimal alignment (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705, BLAST, or PILEUP/PRETTYBOX programs). These software programs match identical or similar sequences by assigning degrees of identity to various substitutions, deletions, or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, and leucine; aspartic acid, glutamic acid, asparagine, and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. A nucleic acid molecule or polypeptide is said to be “substantially identical” to a reference molecule if it exhibits, over its entire length, at least 51%, preferably at least 55%, 60%, or 65%, and most preferably 75%, 85%, 90%, or 95% identity to the sequence of the reference molecule. For polypeptides, the length of comparison sequences is at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids. For nucleic acid molecules, the length of comparison sequences is at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 110 nucleotides. Of course, the length of comparison can be any length up to and including full length. A nil per os nucleic acid molecule or a nil per os polypeptide is “analyzed” or subject to “analysis” if a test procedure is carried out on it that allows the determination of its biological activity or whether it is wild type or mutated. For example, one can analyze the nil per os genes of an animal (e.g., a human or a zebrafish) by amplifying genomic DNA of the animal using the polyrnerase chain reaction, and then determining whether the amplified DNA contains a mutation, for example, the nil per os mutation, by, e.g., nucleotide sequence or restriction fragment analysis. By “probe” or “primer” is meant a single-stranded DNA or RNA molecule of defined sequence that can base pair to a second DNA or RNA molecule that contains a complementary sequence (a “target”). The stability of the resulting hybrid depends upon the extent of the base pairing that occurs. This stability is affected by parameters such as the degree of complementarity between the probe and target molecule, and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is affected by parameters such as the temperature, salt concentration, and concentration of organic molecules, such as formamide, and is determined by methods that are well known to those skilled in the art. Probes or primers specific for nil per os nucleic acid molecules, preferably, have greater than 45% sequence identity, more preferably at least 55-75% sequence identity, still more preferably at least 75-85% sequence identity, yet more preferably at least 85-99% sequence identity, and most preferably 100% sequence identity to the sequences of human (SEQ ID NO:4) or zebrafish (SEQ ID NOs:1 and 2) nil per os genes. Probes can be detectably labeled, either radioactively or non-radioactively, by methods that are well known to those skilled in the art. Probes can be used for methods involving nucleic acid hybridization, such as nucleic acid sequencing, nucleic acid amplification by the polymerase chain reaction, single stranded conformational polymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP) analysis, Southern hybridization, northern hybridization, in situ hybridization, electrophoretic mobility shift assay (EMSA), and other methods that are well known to those skilled in the art. A molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, a cDNA molecule, a polypeptide, or an antibody, can be said to be “detectably-labeled” if it is marked in such a way that its presence can be directly identified in a sample. Methods for detectably labeling molecules are well known in the art and include, without limitation, radioactive labeling (e.g., with an isotope, such as 32 P or 35 S) and nonradioactive labeling (e.g., with a fluorescent label, such as fluorescein). By “substantially pure” is meant a polypeptide or polynucleotide (or a fragment thereof) that has been separated from the proteins and organic molecules that naturally accompany it. Typically, a polypeptide or polynucleotide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the polypeptide or polynucleotide is a nil per os polypeptide or polynucleotide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure nil per os polypeptide can be obtained, for example, by extraction from a natural source (e.g., an isolated digestive organ), by expression of a recombinant nucleic acid molecule encoding a nil per os polypeptide, or by chemical synthesis. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. The polynucleotide can also be “isolated,” which means that it is separated from flanking nucleotide sequences that naturally accompany it in the genome. An isolated polynucleotide sequence can include coding sequences only or, alternatively, can also include promoter and other regulatory sequences associated with the coding sequences. A polypeptide is substantially free of naturally associated components when it is separated from those proteins and organic molecules that accompany it in its natural state. Thus, a protein that is chemically synthesized or produced in a cellular system that is different from the cell in which it is naturally produced is substantially free from its naturally associated components. Accordingly, substantially pure polypeptides not only include those that are derived from eukaryotic organisms, but also those synthesized in E. coli, other prokaryotes, or in other such systems. An antibody is said to “specifically bind” to a polypeptide if it recognizes and binds to the polypeptide (e.g., a nil per os polypeptide), but does not substantially recognize and bind to other molecules (e.g., non-nil per os related polypeptides) in a sample, e.g., a biological sample, which naturally includes the polypeptide. By “high stringency conditions” is meant conditions that allow hybridization comparable with the hybridization that occurs using a DNA probe of at least 100, e.g., 200, 350, or 500, nucleotides in length, in a buffer containing 0.5 M NaHPO 4 , pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (fraction V), at a temperature of 65° C., or a buffer containing 48% formamide, 4.8×SSC, 0.2 M Tris-Cl, pH 7.6, 1× Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42° C. (These are typical conditions for high stringency northern or Southern hybridizations.) High stringency hybridization is also relied upon for the success of numerous techniques routinely performed by molecular biologists, such as high stringency PCR, DNA sequencing, single strand conformational polymorphism analysis, and in situ hybridization. In contrast to northern and Southern hybridizations, these techniques are usually performed with relatively short probes (e.g., usually 16 nucleotides or longer for PCR or sequencing, and 40 nucleotides or longer for in situ hybridization). The high stringency conditions used in these techniques are well known to those skilled in the art of molecular biology, and examples of them can be found, for example, in Ausubel et al., Current Protocols in Molecular Biology , John Wiley & Sons, New York, N.Y., 1998, which is hereby incorporated by reference. By “sample” is meant a tissue biopsy, amniotic fluid, cell, blood, serum, urine, stool, or other specimen obtained from a patient or a test subject. The sample can be analyzed to detect a mutation in a nil per os gene, or expression levels of a nil per os gene, by methods that are known in the art. For example, methods such as sequencing, single-strand conformational polymorphism (SSCP) analysis, or restriction fragment length polymorphism (RFLP) analysis of PCR products derived from a patient sample can be used to detect a mutation in a nil per os gene; ELISA and other immunoassays can be used to measure levels of a nil per os polypeptide; and PCR can be used to measure the level of a nil per os nucleic acid molecule. By “nil per os-related disease,” “npo-related disease,” “nil per os-related condition,” or “npo-related condition” is meant a disease or condition that results from inappropriately high or low expression of a nil per os gene, or a mutation in a nil per os gene (including control sequences, such as promoters) that alters the biological activity of a nil per os nucleic acid molecule or polypeptide. Nil per os-related diseases and conditions can arise in any tissue in which nil per os is expressed during prenatal or post-natal life. Nil per os-related diseases and conditions can include diseases or conditions of a digestive organ (e.g., intestine, liver, bile duct, pancreas, gall bladder, stomach, or esophagus) or cancer. The invention provides several advantages. For example, using the diagnostic methods of the invention it is possible to detect an increased likelihood of diseases or conditions of the digestive system or cancer in a patient, so that appropriate intervention can be instituted before any symptoms occur. This may be useful, for example, with patients in high-risk groups for such diseases or conditions. Also, the diagnostic methods of the invention facilitate determination of the etiology of an existing disease or condition of the digestive system or cancer in a patient, so that an appropriate approach to treatment can be selected. In addition, the screening methods of the invention can be used to identify compounds that can be used to treat or to prevent these diseases or conditions. The invention can also be used to treat diseases or conditions (e.g., digestive organ failure) for which, prior to the invention, the only treatment was organ transplantation, which is limited by the availability of donor organs and the possibility of organ rejection. Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims. |
Antitheft system |
The present invention provides a burglar alarm system capable of accurately discriminating external noises approximate to an alarm such as background music, ambient noises, etc. and reverberations of an alarm attributable to an architectural design of the interior of a store, etc. from a legitimate alarm. A burglar alarm system of the present invention comprises an alarm unit attached to an object to be protected from theft to emit an alarm of a certain frequency in response to illegal conduct and an alarm sensor for emitting an alarm signal upon sensing an alarm from said alarm unit, wherein said alarm sensor has first and second determination means for evaluating an input signal containing an alarm from the alarm unit and external noises, and the first determination means evaluates a random external noise contained in the input signal (S4, S5) whereas the second determination means evaluates a reflected sound caused by reverberations of the alarm (S6, S7) to emit an alarm signal (S9). |
1. In a burglar alarm system comprising an alarm unit attached to an object to be protected from theft to intermittently emit an alarm of a certain frequency in response to illegal conduct and an alarm sensor for emitting an alarm signal upon sensing an alarm from said alarm unit, said alarm sensor comprising first and second determination means for an input signal containing said alarm and external noises, wherein the first determination means determines if the input signal contains random external noises while the second determination means determines if the input signal contains reflected sound generated as a result of a reverberation of said alarm; and if the first and second determination means both determine that the input signal is a legitimate alarm, an alarm signal is generated. 2. The burglar alarm system as defined in claim 1, wherein said first determination means measures the frequency of said input signal. 3. The burglar alarm system as defined in claim 1, wherein said second determination means measures a fluctuation cycle of a sound pressure value of said input signal that is generated in each intermittent cycle of said alarm. 4. In a burglar alarm system comprising an alarm unit attached to an object to be protected from theft to emit an alarm of a certain frequency in response to illegal conduct and an alarm sensor for emitting an alarm signal upon sensing an alarm from said alarm unit, said alarm sensor comprising: a detector circuit for generating detected signals for a predetermined time period if a sound pressure value of an input signal containing said alarm and an external noise is not less than a predetermined value; a first determination means; and a second determination means, wherein when receiving an input signal, said first determination means measures a time more than once required for a predetermined number of waveforms to be counted and determines, based on whether or not each differential between the measured times is within a predetermined time, whether or not the input signal is a random external noise; and said second determination means measures a rise time of each of the detected signals generated in each cycle of intermittent waveform of said alarm, measures a rise cycle of each of the detected signals, determines whether or not the detected signals are a reflected sound generated as a result of a reverberation of said alarm and issues an alarm signal. 5. The burglar alarm system as defined in claim 4, wherein it is determined by repeating a determination operation by said first and second determination means whether or not the input signal is an alarm. 6. A burglar alarm method comprising the steps of: evaluating a random external noise contained in an input signal containing an alarm, which intermittently generates a certain frequency in response to illegal conduct, and external noises; evaluating a reflected sound caused by reverberation of said alarm; and emitting an alarm signal when the results of the both evaluations indicate that said input signal is an alarm. 7. The burglar alarm method as defined in claim 6, wherein the step of evaluating said external noise includes a step of measuring the frequency of said input signal. 8. The burglar alarm method as defined in claim 6, wherein the step of evaluating said reflected sound includes a step of measuring a fluctuation cycle of a sound pressure value of said input signal that is generated in each intermittent cycle of said alarm. 9. A burglar alarm method comprising the steps of: generating a detected signal for a predetermined time period when a sound pressure value of an input signal containing an alarm that intermittently generates a certain frequency in response to illegal conduct and an external noise is not less than a predetermined sound pressure value; determining whether or not a received input signal is a random external noise, by measuring a time more than once required for a predetermined number of waveforms of the input signal to be counted and determining whether or not each differential between the measured times is within a predetermined time; determining, by measuring rise time of one or more detected signals generated in each cycle of the intermittent waveforms of said alarm and measuring a rise cycle of each of the detected signals, whether or not the input signal is a reflect sound caused by reverberation of the alarm; and generating an alarm signal. 10. The burglar alarm method as defined in claim 9, wherein said determining steps are repeated more than once, thereby determining whether or not the input signal is a legitimate alarm. |
<SOH> BACKGROUND OF THE INVENTION <EOH>An anti-theft system such as that illustrated in FIG. 4 has been commonly employed to prevent shop lifting, etc. at retail stores where compact disk cassettes, magnetic tape cassettes, clothing garments, etc. are displayed in such a manner as to be readily accessible to customers. Such a conventional anti-theft system consists of a theft prevention gate 30 installed near an exit 40 of the store and an alarm unit 20 attached to an article 50 . The theft prevention gate 30 incorporates a circuit board 31 and a transmission antenna (not shown), and the circuit board 31 is provided with a transmitting circuit (not shown) which transmits an alarm activation signal to the alarm unit 20 . As indicated in FIG. 5 , the alarm unit 20 has a buzzer 22 , a circuit board 23 , a battery (not shown), etc., each of which is housed in a casing 21 , and in the surface of which casing a plurality of alarm emitting holes 24 are provided and an alarm activation switch piece 25 is embedded. The buzzer 22 , which is controlled by the circuit board 23 , is designed to be activated when the alarm unit 20 is removed from the article 50 or passes through the theft prevention gate 30 . As an alternative to providing the alarm unit 20 with the alarm activation switch piece 25 , the alarm unit 20 may be attached directly to the article 50 by means of a wire, in which case when the alarm unit 20 senses that the wire has been removed or cut or passed through the theft prevention gate 30 , the buzzer 22 is activated. At a retail store, the article 50 with the alarm unit 20 affixed thereto is displayed on a rack. When a sales clerk sells the article 50 to a customer (s), he first sends a specified reset signal from an alarm deactivation device (not shown) to the circuit board 23 of the alarm unit 20 to set the alarm unit such that the buzzer 22 will not be activated, then removes the alarm unit 20 from the article 50 and hands the article 50 to the customer when payment is made. On the contrary, when the alarm unit 20 is removed from the article 50 by a customer, the alarm activation switch piece 25 is also removed and the buzzer 22 is activated. Further, in a case that a customer leaves a store premises taking away the article 50 with the alarm unit 20 still attached thereto, the circuit board 23 of the alarm unit 20 receives an alarm activation signal from the transmission antenna of the theft prevention gate 30 , in response to which the buzzer 22 is activated. However, a checkout counter is usually at the back of a store, far away from the exit 40 of the store where the theft prevention gate 30 is installed and therefore, an alarm from the buzzer 22 that goes off at the exit 40 may not be readily audible from the checkout counter, especially in an environment where the back ground music is present or where many customers are present, etc. With a view to solving the problem of the anti-theft device described above, a burglar alarm system such as illustrated in FIG. 6 is used as a supplementary device in which an alarm sensor device 10 provided with a microphone 11 is disposed near the theft prevention gate 30 or in a fitting room and the like where a customer could remove and destroy or conceal the alarm unit 20 so that an alarm sensor device 10 senses an alarm 12 from the alarm unit 20 and issues an alarm signal 13 , which is sent through a wire or by wireless connection to a speaker 15 or lamp 16 disposed at a checkout counter 14 to alert store personnel at the checkout counter that the alarm has been activated. Such a system as described above, however, suffers from a problem in that it may not be able to discriminate an external noise similar to that generated by an alarm, such as background music, ambient noise, etc. from a legitimate alarm or may mistake reverberations of an alarm for an external noise. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a burglar-alarm system capable of accurately identifying an external noise approximate to an alarm such as background music, ambient noise, etc. and reverberations of an alarm attributable to an architectural design of an interior of a store, etc. A burglar-alarm system of the present invention comprises an alarm unit attached to an object to be protected from theft to intermittently emit an alarm of a certain frequency upon the occurrence of theft and an alarm sensor for sensing an alarm from the alarm unit and generating an alarm signal, wherein said alarm sensor has first and second determination means for evaluating an input signal containing said alarm and external noises and the first determination means determines if the input signal contains random external noises while the second determination means determines if the input signal contains reflected sound caused by reverberation of said alarm and if the first and second determination means both determine that the input signal is a legitimate alarm, an alarm signal is generated. |
Device for vaporising and diffusing oils |
A device which uses a heat source for heating and diffusing fragrant oils or insecticidal oils. The oil is contained in a reservoir remote from the heat source and the device comprises means for transferring the oil from a reservoir to a surface of a heat-absorbing means wherein heat from the heat source is transferred by conduction to said surface to volatilise the oil. |
1. A device for vaporising and diffusing oils comprising: a heat source and a reservoir containing fragrant oil and/or insecticide oil, a wick having a first portion in communication with oil in the reservoir, and a second portion which abuts a first surface of a heat-absorbing body thereby to transfer oil to said first surface, wherein the heat-absorbing body is disposed between the wick and the heat source such that heat from the heat source is transferred by conduction to said first surface to volatilise the oil contained within the wick. 2. A device for vaporising and diffusing oils, e.g. fragrant oils or insecticide oils comprising: a container made of heat-conducting material including a wall having inside and outside surfaces, the inside surface defining a cavity to receive a radiant heat source, a reservoir suitable for containing a fragrant oil or insecticide oil, and a porous rod that communicates with the reservoir and the outside surface of the container wall thereby to carry oil from the reservoir to the outside surface. 3. A device according to claim 2 wherein the container is surrounded by a shroud which prevents the porous rod being contacted by a user. 4. A device according to claim 1 wherein the heat source is a candle or a light bulb. 5. A device according to claim 1 wherein the reservoir and the container are detachable. 6. A device according to according to claim 1 in the form of a lantern comprising a container, a reservoir serving as a base for the container, and a shroud surrounding the container. 7. A reservoir for use in a device according to claim 1. 8. (cancelled) 9. A device according to claim 2 wherein the reservoir and the container are detachable. 10. A device according to claim 2 in the form of a lantern comprising a container, a reservoir serving as a base for the container, and a shroud surrounding the container. 11. A device according to claim 4 wherein the heat source is a light bulb. 12. A device according to claim 4 wherein the heat source is a candle. |
Magnesium-enriched compositions |
The invention concerns a special dairy food composition consisting of processed mammal milk (that is having been subjected to a substantial modification of its original state) by acidification at pH less than 5.2 either by lactic acid fermentation or by direct input of lactic acid and antacid substances containing magnesium in the form of magnesium oxide or magnesium hydroxide or magnesium salts of weak organic or mineral acids or a mixture thereof, the final pH of the composition ranging between 4.5 and 7.5 and the total amount of magnesium contained in the composition ranging between 10 and 3000 milligrams per litre. |
1. Process allowing the reduction in acidity of special dairy compositions food made up of one or more milks of mammals, pure or mixed or skimmed entirely or partly or possibly added with various substances (such of dried milk, milk proteins, flavors, gelling and stabilizing agents, thickeners, the present list not being restrictive), having been transformed beforehand (i.e. having undergone a substantial change of their state of origin) by acidification, under the action either of one or more lactic fermentations, or of a direct contribution of lactic acid, this process of elevating the pH of the special dairy food composition being characterized by the fact that the reduction in acidity is obtained by the addition to these beforehand processed milks, after their temperature was lowered to 4 centigrade degrees, of substances containing magnesium in an antiacid form such as magnesium oxide, magnesium hydroxide or one of magnesium salts of the weak mineral or organic acids or a mixture of these various magnesian substances, this addition having the effect of decreasing the acidity of these compositions until pH in the range 4.3 to 7.5, this addition of magnesian substances being able to be split and intermixed in the course of manufacture with complementary additions with lactic acid, these additions, moreover, being followed by very slow mixing intended to preserve the gelled structure of the casein coagulated by the preliminary action of the acidification and the cold, this process being also characterized by the fact that the compositions obtained are magnesium enriched, and being characterized moreover by the fact that there is not rigorous match between pH and magnesium content of the compositions prepared, these pH possibly varying 0.2 more or less for one same magnesium content, depending on the milks used. 2. Process, according to the preceding claim, of reduction in acidity of a milk processed by lactic fermentation obtained with the bacteria Streptococcus thermophilus and lactobacillus delbruecki bulgaricus, (having a pH from approximately 3.9 to 4.5), i.e. a milk in the state of yogurt, characterized by the fact that one liter of yogurt is lowered to a temperature of 4 degrees and that one gram with magnesium oxide of light quality (container 600 milligrams of magnesium) is added to this yogurt, which is closely mixed with yogurt by a slow mixing, which confers on the special dairy food composition thus obtained a pH of 5.3 which pH can be obtained with a good precision when one adds 1 gram (or a little more or a little less) of oxide of magnesium to yogurt under control of an electronic pH-meter, this process being also characterized by the fact that it makes it possible to manufacture a special dairy food composition containing approximately 600 milligrams of magnesium per liter i.e. approximately 75 milligrams per 125 milliliters serving, this process being furthermore characterized by the fact that the match of the pH and the magnesium content indicated above is not rigorous but depends on the quality of the milks used, these pH possibly varying 0.2 more or less for one same magnesium content. 3. Process, according to the claim first, of reduction in the acidity of a milk processed by lactic fermentation fermentary obtained with the bacteria Streptococcus thermophilus and lactobacillus delbrueckiibulgaricus, (having a pH approximately 3.9 to 4.5), i.e. yogurt, characterized by the fact that one liter of yogurt is lowered to the temperature of 4 degrees and that to this yogurt are added magnesium oxide (preferentially of light quality) then lactic acid then again magnesium oxide, and this according to following process: in a first stage one adds to yogurt 400 mgs magnesium oxide and one mixes by slow mixing by adjusting the pH with 4.6 by split additions of magnesium oxide under the control of an electronic pH-meter in a second stage one adds 200 milligrams magnesium oxide while proceeding as at the time of stage 1 with adjustment of the pH to 4.7 in a stage 3, one adds 200 oxide mgs of magnesium and one proceeds as in stage 1 by adjusting the pH to 4.9; in a stage 4, one adds 200 oxide mgs of magnesium and one proceeds as in 1 stage 1 by adjusting the pH to 5.3; in a stage 5, one adds 400 oxide mgs of magnesium and one proceeds as in stage 1 by adjusting the pH to 5.8; in a stage 6, one adds 400 oxide mgs of magnesium and one proceeds as in stage 1 by adjusting pH to 6.1; in a stage 7, one adds 400 oxide mgs of magnesium and one proceeds as in stage 1 by adjusting the pH to 6.5; in a step 8, one adds 0.9 gram of lactic acid by fractions and one mixes, the pH being adjusted to 6.1; in a stage 9, one proceeds as in stage 8, the pH being adjusted to 5.8; in a stage 10, one proceeds as in stage 8, the pH being adjusted to 5.3; in a stage 11, one proceeds as in stage 8, the pH being adjusted to 4.8; in a stage 12, one adds 400 magnesium oxide mgs in a split way, by adjusting pH to 5.0 under control of a pH measures; in a stage 13, one adds 400 magnesium oxide mgs in a split way, while adjusting the pH with 5.3 under control of a pH-meter, these various stages leading to the production of specialized dairy food compositions having the following properties 1 pH=4.6—magnesium per liter=240 milligrams. 2 pH=4.7—magnesium per liter=360 milligrams. 3 pH=4.9—magnesium per liter=480 milligrams. 4 pH=5.3—magnesium per liter=600 milligrams. 5 pH=5.8—magnesium per liter=480 milligrams. 6 pH=6.1—magnesium per liter=600 milligrams. 7 pH=6.5—magnesium per liter=840 milligrams. 8 pH=6.1—magnesium per liter=1080 milligrams. 9 pH=5.8—magnesium per liter=1320 milligrams. 10 pH=5.3—magnesium per liter=1320 milligrams. 11 pH=4.8—magnesium per liter=1320 milligrams. 121 pH=5.0—magnesium per liter=15é0 milligrams. 13 pH=5.3—magnesium per liter=1800 milligrams. said process being also characterized by the fact that it makes it possible to manufacture special dairy food compositions having for the same acidity of the different contents magnesium, the magnesium content possibly varying from 10 to 3000 milligrams per liter and the pH ranging from 4.1 to 7.5 according to the types of milks used and respective quantities of magnesium oxide and lactic acid implemented, this process being further characterized by the fact that the match of the pH and the magnesium content indicated above is not rigorous but depends on the quality of the milks implemented, these pH possibly varying 0.2 more or less for the same magnesium content. 4. Process, according to claim 1, of reduction in acidity, of milks processed beforehand by chemical acidification and lactic fermentation, characterized by the fact that to one liter of milk whose casein was beforehand gelled by action of rennet and calcium chloride and temperature of 30 degrees and presence of the bacteria Stretrococcus Thermophilus and lactobacillus delbruecki bulgaricus and whose pH is approximately 4.7, one adds, after cooling to 4 centigrade degrees: in a first stage 2 grams of lactic acid which after careful mixture, intended to preserve the structure of the casein gel, confers on the mixture an approximate pH of 4.2 in a second stage one adds to the composition 200 milligrams of magnesium oxide which after homogeneous mixture confers has an approximate pH of 4.4, in the following stages (stages 3 to 7) one adds each time 200 milligrams of magnesium oxide, the composition having the following successive pH: with 400 mgs MgO per liter, the pH is 4.5; with 600 mgs/l, it is 4.7; with 800 mgs/l, it is 5; with 1000 mgs/l, it is 5.3; with 1200 mgs/l, it is 5.8; these various pH being slightly variable according to the quality of milks which are used; in a stage 8, one adds 2 grs of lactic acid has the composition which takes a pH of 5; that in a stage 9, one adds 2 more grs of lactic acid, pH being 4.6; that in a stage 10, one roughly adds 1 gram of lactic acid the pH rising to 4.5; that in stages 11, 12 and 13, one add has each time 400 milligrams of magnesium oxide per liter what after mixture confers on the composition the following pH stage 11, pH 5. stage 12, pH 5.3, stage 13. pH 5.8; said process being characterized moreover by the fact that it makes it possible to prepare several special dairy food compositions containing milks processed by action of rennet and fermentation described above, having various pH and being able to contain for the same pH of the more or less large quantities of magnesium, ranging from 120 to 1.440 milligrams per liter for pH in the range of 4.2 A 5.9, said pH could possibly range from 5.8 to 7.5 if one brings complementary quantities of magnesium oxide in similar stages 14 to 19, the quantity of magnesium and lactic acid being able to be higher or lower, depending on the pH and the magnesium content of the composition that one wishes to obtain, this process being furthermore characterized by the fact that the match of the pH and the magnesium content indicated above is not rigorous but depends on the quality of the milks used, these pH possibly varying 0.2 more or less for the same magnesium content. 5. Process, according to claim 1, of reduction in acidity, in milks processed beforehand by chemical acidification by lactic acid characterized by the fact that one liter of milk whose casein was gelled in consequence of lowering its pH to 4.4 and roughly containing 7 grams of lactic acid per liter, one adds successively the following quantities of magnesium oxide which after careful mixture confers on the dairy food compositions obtained pH indicated below: magnesium oxide=200 mgs pH obtained: 4.7—Mg content=200 mgs/l magnesium oxide=200 mgs pH obtained: 4.9—Mg content=400 mgs/l magnesium oxide=200 mgs pH obtained: 5.3—Mg content=é00 mgs/l magnesium oxide=200 mgs pH obtained: 5.5—Mg content=800 mgs/l magnesium oxide=200 mgs pH obtained:.5.8—Mg content=1000 mgs/l magnesium oxide=200 mgs pH obtained: 6.1—Mg content=1200 mgs/l magnesium oxide=200 mgs pH obtained: 6.4—Mg content=1400 mgs/l said process making it possible to manufacture milks with casein gelled by acidification but whose pH is increased by the presence of magnesium, these milks having moreover high active magnesium content, compatible with the nutritional contributions recommended in the human being, this process being furthermore characterized by the fact that the match of the pH and the magnesium content indicated above is not rigorous but depends on the quality of the milks used, these pH possibly varying 0.2 more or less for the same magnesium content. 6. Process, according to claim 1, for lowering acidity, in milks processed beforehand by chemical acidification by lactic acid and the action of rennet, characterized by the fact that to one liter of milk whose casein was gelled by lowering its pH to 4.4 and roughly containing 7 grams of lactic acid per liter, one adds successively the following quantities of magnesium oxide what after careful mixture confers on the special dairy food compositions thus obtained the pH indicated below magnesium oxide=200 mgs pH obtained: 4.7—Mg content=200 mgs/l magnesium oxide=200 mgs pH obtained: 4.9—Mg content=400 mgs/l magnesium oxide=200 mgs pH obtained: 5.3—Mg content=600 mgs/l magnesium oxide=200 mgs pH obtained: 5.5—Mg content=800 mgs/l magnesium oxide=200 mgs pH obtained: 5.8—Mg content=1000 mgs/l magnesium oxide=200 mgs pH obtained: 6.1—Mg content=1200 mgs/l magnesium oxide=200 mgs pH obtained: 6.4—Mg content=1400 mgs/l said process making it possible to manufacture milks with casein gelled by acidification and the action of rennet (or chymosine) but whose pH is high by the presence of magnesium, these milks having moreover a high magnesium content, compatible with the daily nutritional contributions recommended in the human being, this process being furthermore characterized by the fact that the match of the pH and the magnesium content indicated above is not rigorous but depends on the quality of the milks used, these pH possibly varying 0.2 more or less for the same magnesium content. 7. Process, according to claim 1, of reduction in acidity, the milks processed beforehand by chemical acidification by lactic acid and the action of rennet, characterized by the fact that to one liter of milk whose casein was gelled by lowering of its pH to 4.4 and roughly containing 7 grams of lactic acid per liter, one adds 1000 milligrams of magnesium oxide which after careful mixture entrusts to this special dairy food composition thus obtained a pH of 5.8 and a magnesium content of 600 milligrams per liter compatible with the daily nutritional contributions recommended in the human being, by the fact that the match of the pH and magnesium content indicated above is not rigorous but depends on the quality of the milk used, these pH possibly varying 0.2 more or less for the same magnesium content. 8. Process, according to claim 1, of reduction in acidity, milks transformed beforehand by chemical acidification by lactic acid characterized by the fact that to one liter of milk whose casein was gelled in consequence of lowering its pH to 4.4 and roughly containing 7 grams of lactic acid per liter, one adds 800 milligrams of magnesium oxide which after careful mixture confers on the special composition food dairy obtained a pH of 5.5, this process making it possible to manufacture a curdled milk with casein gelled by the acidification but whose pH is elevated by the presence of magnesium, this milk having moreover a magnesium content compatible with the nutritional contributions recommended for human beings, this process being furthermore characterized by the fact that the match of the pH and the magnesium content indicated above is not rigorous but depends on the quality of the milk used, this pH possibly varying 0.2 more or less for the same magnesium content. 9. Process, according to claim 1, of reduction in acidity, in milks processed beforehand by chemical acidification and lactic fermentation, characterized by the fact that to one liter of milk whose casein was beforehand gelled by action of rennet and calcium chloride and temperature of 30 degrees and presence of the bacteria Stretrococcus Thermophilus and lactobacillus delbruecki bulgaricus and whose pH is approximately 4.7, one add, after cooling to 4 centigrade degrees: in a first stage 2 grams of lactic acid which after careful mixture, intended to preserve the structure of the frozen casein, confers on the mixture an approximate pH of 4.2; that in a second stage one adds to the composition 800 milligrams of magnesium oxide which after homogeneous mixture confers on that an approximate pH of 5.2, that in the third stage one adds 2 grs of lactic acid to the composition which takes a pH of 4.7, that in the fourth stage one adds 400 milligrams of magnesium oxide per liter which after mixture confers on the composition the pH of 5.8, that in the fifth stage one adds to lactic composition 2 grs of acid 15 which takes the pH of 5.5, this process being characterized by the fact that it makes it possible to manufacture according to wishes 4 compositions having the following pH and contents magnesium: magnesium content of 480 mgs per liter and a pH either of 5.2 or of 20 4.7; magnesic content of 720 milligrams per liter and a pH either of 5.8 or of 5.5; said process being still characterized by the fact that the match of the pH and the magnesium content indicated above is not rigorous but depends on the quality of the milk used, this pH possibly varying 0.2 more or less for the same magnesium content. |
<SOH> FIELD OF THE INVENTION <EOH>The present invention relates to a method of preparation of special dairy food compositions—which can be used in animal or human food—containing on the one hand one or more milks of mammals (possibly added with milk proteins as well as various other additives), having been transformed—i.e. having undergone a substantial change from their state of origin—by acidification, either direct by contribution of lactic acid, or indirect by action of lactic bacteria or yeasts or moulds or various micro-organisms, said acidification conferring on these milks a pH lower than 5 suitable to the gelification of their caseins and containing, on the other hand acidity reducing substances containing magnesium in the form of oxide or of hydroxide or magnesium salts of weak organic or mineral acids, or of composed of magnesium and organic substances, or several of these substances having deacidizing properties, and this in quantities such as the pH of these special dairy food compositions, in their final state, is increased compared to that of processed milks, in their initial state, said increase in the pH conferring on these special dairy food compositions a greater sweetness and a lower aggressiveness with respect to the digestive mucous membranes; said magnesium contribution, on its side, allowing to carry out a better magneso-calcic balance, especially favorable to the nutrition and the health of adults. The aims of this invention are thus of elaborating special dairy food compositions containing processed milks acidified then partly or entirely de-acidified, having a sweet taste and bringing to the consumers magnesium (in a organoleptically pleasurable form) of which many human beings, in the industrial societies, are deprived or sub-deficient. This manufacturing process of special dairy food moderate acidity compositions, enriched in magnesium, is based on the fact that processed milks (standard yogurt) contain approximately 9 grams of lactic acid per liter and that even a small quantity of magnesium: 1.2 grams per liter is enough to neutralize this acid. The special dairy food compositions described here can comprise various additives: various flavors, dyes, gelling, thickeners, stabilizers, amino acids, milk proteins, gelatin, taste enhancers, enzymes and possibly other complementary additions of lactic acid, in the course of manufacture process. In the present request, the term “milk” applies to any milk of any terrestrial or marine mammal. The micro-organisms implied in the preliminary transformation of milks can be of any species provided that they neither are pathogenic, nor produce toxic substances or unpleasant tastes or odors. In this request, the milks acidified by lactic fermentation or direct action of lactic acid are simply used as raw material at the time of the implementation of the manufacturing process of the special dairy food compositions by action of magnesic substances; the applicant is aware that the processes for obtaining fermented or acidified milks are not new, and therefore does not ask for their protection (the broadest references on fermented milks are in the following books: 1/Fermented milks 1989—John Libbey and company Ltd 13 Smith Yard—Summerley Street—London SW 18 4 HR England and 2/Food Microbiology Volume 2 food fermentations—1989 Technique and documentation Lavoisier, 11 Rue Lavoisier 75384 Paris cedex 08). The present request relates only to the innovation and the inventive creation of the manufacturing process of the special dairy food compositions obtained using the mixture of milks processed beforehand by acidification and magnesium in the form of oxide, hydroxide or magnesium salts of weak organic or mineral acids or substances coming from the chemical reaction of magnesium with organic substances and complementarily of acid lactic. |
Pyridin-2-yl-methylamine derivatives for treating opioid dependence |
The invention concerns compounds of general formula (I) for treating opioid drug dependence. |
1. (canceled) 2. (canceled) 3. A method of treating opioid drug dependence comprising administering to a patient in need of such treatment an effective amount of a compound of formula (I) and its therapeutically acceptable salts in which: u represents a hydrogen atom or a methyl radical with the reservation that when u is a methyl radical then v and w represent a hydrogen atom; v represents a hydrogen atom, a chlorine atom or a methyl radical with the reservation that when v is a methyl radical then u and w represent a hydrogen atom; w represents a hydrogen atom, a fluorine atom or a methyl radical with the reservation that when w is a methyl radical then U and v represent a hydrogen atom; x represents a hydrogen atom or a fluorine atom; y represents a chlorine atom or a methyl radical; z represents a hydrogen atom or a fluorine atom or a chlorine atom or a methyl radical; A represents: a hydrogen atom or a fluorine atom or a chlorine atom; an alkyl radical in C1-C5, i.e. a saturated aliphatic hydrocarbon remainder with straight or branched chain, containing from 1 to 5 atoms of carbon such as methyl, ethyl, propyl, butyl, pentyl, isopropyl, 1-methyl-ethyl, 1-methyl-propyl, 1-methyl-butyl, 2-methyl-propyl, 2-methyl-butyl or 3-methyl-butyl, 1-ethyl-propyl, 2-ethyl-propyl; a fluoroalkyl radical such as monofluoromethyl (—CH2F) or difluoromethyl (—CHF2) or trifluoromethyl (—CF3) or 1-fluoro-1-ethyl (—CHFCH3) or 1,1-difluoro-1-ethyl (—CF2CH3); a cyclopropyl or cyclobutyl or cyclopentyl radical; an aromatic heterocyclic group with 5 links, substituted or not, containing 1, 2, 3 or 4 heteroatoms chosen from amongst nitrogen, oxygen and sulphur but nonetheless without more than one oxygen and/or sulphur atom being present in the heterocycle A; an alkoxy (R1O—) or alkylthio (R1S—) group in which the R1 radical represents: an alkyl radical in C1-C5 such as defined above; a monofluoromethyl or trifluoromethyl radical; a cyclopropyl or cyclobutyl or cyclopentyl radical; an amino group of type II in which R2 and R3, identical or different, represent hydrogen or an alkyl radical in C1-C5 such as defined above or a cyclopropyl group or a trifluoromethyl group; a saturated cyclic amino group of type III in which n can take the whole numbers 1 or 2; an alkoxycarbonyl group. 4. The method according to claim 3, wherein A represents a hydrogen atom, u represents a methyl, v and n represent a hydrogen atom, x represents a fluorine, y represents a chlorine and z represents a fluorine. 5. A method according to claim 3, wherein said alkoxycarbonyl group is a methoxy-carbonyl (CH3OCO—) group or an ethoxycarbonyl (CH3CH2OCO) group. |
Cell and transgenic animal modelling human antigenic presentation and their uses |
The invention concerns an isolated animal cell comprising at least a transgene including at least a nucleotide sequence coding for at least a human polypeptide involved in the recognition and/or antigenic activation by T cells. The invention is characterised in that said cell, or a progeny of said cell, expresses at least all or part of the or said human polypeptide(s), and the homologous endogenous animal gene coding for an animal polypeptide homologous with said human peptide is invalid. The invention also concerns the corresponding transgenic animal. The cell and the transgenic animal of the invention can be used in a method for screening compounds which modulate an immune response in humans. The invention further concerns the use of the inventive cell as cell rendered autologous or tolerated by the immune system. |
1. An isolated animal cell comprising at least one transgene comprising at least one nucleotide sequence encoding all or at least part of one human polypeptide involved in antigenic recognition and/or cell activation of T cells, characterised in that said cell, or a progeny of said cell, expresses all or at least some of said human polypeptide(s), and characterised in that said nucleotide sequence is integrated into the genome of said cell in a stable manner by a targeted insertion by homologous recombination (Knock-in) at at least one allele of said endogenous animal gene, the integration of said sequence invalidating said homologous endogenous animal gene. 2. The cell according to claim 1, characterised in that the human polypeptide involved in the antigenic recognition and/or cell activation of T cells is selected in the group composed of the antigens of the major histocompatibility complex (HLA), β2-microglobulin, T cell receptor (TCR) chains, polypeptides of the CD3 complex, co-receptors CD4 and CD8, the co-stimulating molecules ICAM-1, CD80, CD86, CD40, CTLA-4, CD28, and LFA-3. 3. The cell according to claim 2, characterised in that said antigen in the major histocompatibility complex is selected in the group composed of type I, type II and type III HLA antigens in the major histocompatibility complex. 4. The cell according to claim 3, characterised in that the said nucleotide sequence is operationally linked to expression regulation sequences of said homologous endogenous animal gene. 5. The cell according to claim 3, characterised in that said nucleotide sequence is operationally linked to exogenous expression regulation sequences. 6. The cell according to claim 5, characterised in that said exogenous expression regulation sequences are the regulation sequences for expressing the human gene encoding the human polypeptide. 7. The cell according to claims 1 to 6, characterised in that it also includes at least one transgene also comprising at least all or part of a nucleotide sequence encoding at least all or part of a human polypeptide involved in antigenic recognition and/or cell activation of T cells present in said cell in episomal form, and in that said homologous endogenous animal gene is invalidated in said cell. 8. The cell according to claim 7, characterised in that said homologous endogenous animal gene is invalidated by targeted homologous recombination (Knock-Out). 9. The cell according to any one of claims 1 to 6, characterised in that said nucleotide sequence(s) encodes all or part of a human class I HLA antigen and is (are) inserted by targeted insertion by homologous recombination (Knock-In) at the homologous animal gene(s) encoding the animal antigens of the class I major histocompatibility complex (MHC I). 10. The cell according to any one of claims 1 to 6, characterised in that said nucleotide sequence(s) encode(s) all or part of class II HLA molecules and is (are) inserted by targeted insertion by homologous recombination (Knock-In) at the homologous animal gene(s) encoding the animal antigens of the class II major histocompatibility complex (MHC II). 11. The cell according to any one of claims 1 to 6, characterised in that said nucleotide sequence(s) encode(s) all or part of class I and class II HLA molecules and is (are) inserted by targeted insertion by homologous recombination (Knock-In) at the homologous animal gene(s) encoding the animal antigen(s) of the class I major histocompatibility complex (MHC I) and class II major histocompatibility complex (MHC II). 12. The cell according to one of claims 9 and 11, characterised in that said human class I HLA antigen is selected in the group composed of HLA-A2, HLA-A24, HLA-A1, HLA-A3, HLA-B7, HLA-B27, HLA-B44, HLA-B8, HLA-B35, HLA-CW7, HLA-CW3 and characterised in that said MHC I animal antigen is chosen from among H2K, H2D and H2L. 13. The cell according to one of claims 10 and 11, characterised in that the said human class II HLA antigen is chosen from among the group composed of HLA-DR4, HLA-DR1, HLA-DR11, HLA-DR7, HLA-DR2, HLA-DR3, HLA-DQ8, HLA-DQ3, HLA-DP4, and characterised in that said MHC II animal antigen is chosen from among I-A alpha, I-A beta and I-E alpha and I-E beta. 14. The cell according to any one of claims 1 to 6, characterised in that the said nucleotide sequence encodes all or part of the human β2-microglobulin, and is inserted by targeted insertion by homologous recombination (knock-in) at the homologous animal gene encoding β2-microglobulin. 15. The cell according to any one of claims 1 to 6, characterised in that said nucleotide sequence(s) encode(s) all or part of at least one of the polypeptides of the human CD3 complex and is or are inserted by targeted insertion by homologous recombination (knock-in) at the homologous animal gene(s) encoding the polypeptide(s) in the CD3 complex. 16. The cell according to any one of claims 1 to 6, characterised in that said nucleotide sequence encodes all or part of the human CD4 polypeptide and is inserted by targeted insertion by homologous recombination (knock-in) at the homologous animal gene encoding the CD4 polypeptide. 17. The cell according to any one of claims 1 to 6, characterised in that said nucleotide sequence encodes all or part of the human CD8 polypeptide and is inserted by targeted insertion by homologous recombination (knock-in) at the homologous animal gene encoding the CD8 polypeptide. 18. The cell according to any one of claims 9 to 13, characterised in that it also comprises: a) said nucleotide sequence encoding all or part of human β2-microglobulin, inserted by targeted insertion by homologous recombination (knock-in) at the homologous animal gene encoding β2-microglobulin; and/or b) said nucleotide sequence encoding for all or part of the human CD4 polypeptide inserted by targeted insertion by homologous recombination (knock-in) at the homologous animal gene encoding the CD4 polypeptide; and/or c) said nucleotide sequence encoding all or part of the human CD8 polypeptide, inserted by targeted insertion by homologous recombination (knock-in) at the homologous animal gene coding for the CD8 polypeptide. 19. The cell according to any one of claims 16 to 18, characterized in that only the extracellular part of the CD4 and CD8 polypeptides is humanised. 20. The cell according to any one of claims 1 to 19, selected from the group composed of mouse, rat, hamster, guinea pig, lagomorphs, primates (including human), porcine, ovine, caprinae, bovine, horse cells. 21. The mouse cell according to claim 20. 22. The cell according to claim 21, characterised in that they are selected from the cells of inbred murine lines (129Sv, 12901a, C57B16, BalB/C, DBA/2, but also in outbred lines or hybrid lines). 23. The cell according to claims 1 to 22, characterised in that said cell is selected from the cells of the immune system, professional and non-professional antigen presenting cells, hematopoietic stem cells, embryonic stem cells. 24. The cell according to claim 23, characterised in that said cell in the immune system is selected from all types of mature and immature T lymphocytes, thymocytes, dendritic cells, intra-epithelial lymphocytes, NK cells, B cells, monocytes, professional and non-professional antigen presenting cells. 25. The stem cell according to claim 23, characterised in that said stem cell is subsequently differentiated as a cell selected from the immune system cells according to claim 23. 26. A transgenic non-human animal, comprising at least one cell according to claims 1 to 25. 27. The animal according to claim 26, characterised in that it is selected from among mouse, rat, hamster, guinea pig, rabbit, primates, porcines, ovines, caprinae, bovines, horse. 28. The animal according to claim 27, characterised in that the animal is a mouse. 29. The animal according to claims 26 to 28, characterised in that the cells of its immune system express at least one functional human HLA antigen. 30. The animal according to claim 29, characterised in that the cells of its immune system also express humanised and functional co-receptor and co-stimulating molecules. 31. A process for screening a compound modulating an immune response in humans, characterised in that it comprises the following steps: a) contacting a cell according to claims 1 to 25, and/or an animal according to claims 26 to 30 with an immunogen responsible for initiating an immune response; b) contacting a cell according to claims 1 to 25 and/or an animal according to claims 26 to 30 with an immunogen responsible for initiating an immune response, and, either simultaneously or later, with the said compound; c) qualitatively and optionally quantitatively determining and evaluating whether or not an immune response occurs; d) then identifying the compound that selectively induces the immune response. 32. The process according to claim 31, characterised in that determining and/or evaluating said immune response is realised using a technique selected from among: a) determination of the production of soluble factors such as chemokines and cytokines, b) determination of the presence of receptors on the cell surface, c) determination of cell proliferation, d) determination of T cell effector functions (CTL, Helper, etc.), e) determination of the production of antibodies by B cells. 33. The process according to claim 31, characterized in that determining and/or evaluating said immune response is realised by measuring the expression ratio of a reporter gene. 34. Use of a cell according to claims 1 to 25, and/or an animal according to claims 26 to 30 for analysis, study and modelling of molecular, biological, biochemical, physiological and/or physiopathological mechanisms of the immune response in humans. 35. The use of a cell according to claims 1 to 25, and/or an animal according to claims 26 to 30 for screening compounds modulating the human immune response. 36. The use of a cell genetically modified ex vivo according to claims 1 to 25 for preparation of a cell and/or tissue graft for preventive or curative treatment of a human or animal necessitating such a treatment, characterised in that when an allogeneic host is transplanted with said cell, this cell is less strongly rejected or better tolerated than a cell that was not genetically modified, by the immune system of said host. 37. The use according to claim 36, characterised in that said cell is a mouse, pig, bovine or primate cell. 38. The use according to claims 36 and 37, characterised in that said cell according to claims 1 to 25 also expresses at least one protein for preventive and curative treatment of a human or animal requiring such a treatment, the said protein being preferably selected from the group composed of cytokines, interleukins, chemokines, growth factors, hormones, antibodies. |
Recombinant vectors derived from adeno-associated virus expressing tam67 for gene therapy |
The present invention is related to a recombinant adeno associated viral construct comprising at least: a first terminal repeat of an Adeno Associated Virus a strong heterologous promoter an heterologous DNA corresponding to the gene encoding for the c-jun mutant protein TAM67, said gene being under the control of said promoter a polyadenylation signal, and a second terminal repeat of an Adeno Associated Virus |
1. A recombinant adeno associated viral construct comprising at least: a first terminal repeat of an Adeno Associated Virus a strong heterologous promoter an heterologous DNA corresponding to the gene encoding for the c-jun mutant protein TAM67, said gene being under the control of said promoter a polyadenylation signal, and a second terminal repeat of an Adeno Associated Virus 2. The recombinant adeno associated viral construct of claim 1 wherein said construct is comprised in a plasmid and/or a recombinant viral particle. 3. The recombinant adeno associated viral construct of claim 1 further comprising nucleotide sequences encoding suitable regulatory elements so as to effect expression of the c-jun mutant protein TAM67 in a suitable host cell. 4. A host cell genetically transformed by the construct according to claim 1. 5. The host cell according to claim 4, characterized in that said host cell is a human tumor cell. 6. A pharmaceutical composition comprising the recombinant adeno associated viral construct of claim 1 or a host cell genetically transformed by the construct and a pharmaceutically acceptable carrier. 7. The pharmaceutical composition as in claim 6 wherein the construct is comprised in a plasmid and/or a recombinant viral particle. 8. A method for inhibiting the proliferation of cells, comprising at least the step of transferring a sufficient amount of the recombinant adeno associated viral construct according to claim 1 into said cells. 9. The method of claim 8, characterized in that said cells are epithelial cells. 10. The method according to claim 9, characterized in that said epithelial cells are mammary epithelial cells, ovarian epithelial cells or lung epithelial cells. 11. The method according to claim 9, characterized in that said cells are cancer cells. 12. The method according to claim 11, characterized in that said cancer cells are selected from the group consisting of human melanoma cells, human mammary tumor cells, human ovarian tumor cells, lung tumor cells, human sarcoma cells and carcinoma cells. 13. Use of a sufficient amount of the pharmaceutical composition according to claim 6 for the preparation of a medicament in the treatment and/or the prevention of cancers. 14. Non-human animal, genetically modified by the recombinant adeno associated viral construct according to claim 1. |
<SOH> BACKGROUND OF THE INVENTION AND STATE OF THE ART <EOH>Cancer is one of the most frequent causes of death of both males and females. Many cancers are difficult to treat with current treatment methods. One such example is ovarian cancer. In over eighty percent of cases, ovarian tumors are of the epithelial type and originate from the cellular surface epithelium overlying the ovaries. In many cases these tumors are extremely difficult to treat, especially in advanced cancer with metastases. Currently available therapies include surgery, radiation therapy, chemotherapy, radioimmunotherapy, cytokine treatment and hyperthermia. All these treatment modalities have important limitations and disadvantages. For example, surgery can only be performed on localised accessible tumors, radiation and chemotherapy are associated with both acute and latent toxicity; radioimmunotherapy and hyperthermia have limited application and effectivity. Moreover, most of these techniques are not discriminating techniques and destroy not only tumor cells but also normal cells, with various side-effects on patients. However, in general, the efficiency of said therapies and their combinations is still unsatisfactory. More recently, gene therapy has been proposed as a novel approach to treat malignancies. Gene therapy consists of correcting a deficiency or an abnormality or of ensuring the expression of a protein of therapeutic interest, by introducing genetic information into the cell or organ concerned. This genetic information can be introduced either in vitro into a cell extracted from the organ, with the modified cell then being introduced into the organism, or directly in vivo in the appropriate tissue. The introduction of a molecule carrying genetic information can be achieved by various methods known in the art. Preferred methods use gene delivery vehicles derived from viruses, including adenoviruses, retroviruses, vaccina viruses and adeno associated viruses. Among these viruses, adeno-associated viruses (AAV) offer certain attractive properties for gene therapy. The wild type AAVs are DNA viruses of relatively small size which integrate, in a stable and site-specific manner, into the genome of the cells they infect. They are able to infect a wide spectrum of cell species and tissue types, without having any effect on cell growth, on cell morphology or on cell differentiation. In particular, they can express a transgene to high levels in epithelial cells. Moreover, so far no human disease has been found to be associated with AAV infection. Examples of the use of vectors derived from AAVs for transferring genes in vitro and in vivo have been described in the literature: see in particular, documents WO 91/18088, WO 93/09239, U.S. Pat. No. 4,797,368, U.S. Pat. No. 5,139,941 and EP 488 528. However, the genetics of cancer and the molecular mechanisms operating in cells for maintaining the integrity of tissues are so complicated, that the potential ways for treating cancer are numerous and it is impossible to predict a priori the efficacy of a potential way of treatment. Different treatment strategies are currently under investigation. For example, one treatment strategy involves the enhancement of immunogenicity of tumor cells in vivo by the introduction of cytokine genes. Another treatment involves the introduction of genes that encode enzymes capable of conferring to the tumor cells sensitivity to chemotherapeutic agents. Another strategy involves the delivery of normal tumor suppressor genes and/or inhibitors of activated oncogenes into tumor cells. Considering the importance of the challenge for obtaining an efficient gene transfer system, efforts to build up new constructs to be tested in vivo are always appreciated. The AP1 complex is a transcriptional complex composed of the Jun and Fos family of DNA binding protooncoproteins. The activation of the AP1 complex has been implicated in numerous biological processes such as cell proliferation, cell differentiation and apoptosis Also, cellular transformation by many oncogenes results in an elevation of AP1 activity. Study on the expression pattern of the family of jun genes in normal ovarian epithelium and epithelial ovarian cancer identified a high level of c-jun expression in epithelial tumors and cultured ovarian cancer cells that is induced by serum and TPA (Neyns et al., Oncogene 12, 1247-1257 (1996)). In cultured breast cancer cells comparable observations were made (Chen et al., Mol. Carcinog. 15, 215-226 (1996)). A truncated form of the c-Jun molecule, the TAM 67 molecule, has been shown to inhibit wild-type C-Jun mediated transcription and transformation by quenching endogenous Jun and Fos proteins (Brown et al., Oncogene 8, 877-886 (1993)). This trans-dominant negative c-Jun mutant lacks the c-Jun 5′ transactivating domain of c-Jun but possesses a functional c-jun leucine zipper and a DNA binding domain. Malignant transformation of rat embryo cells and NIH3T3 fibroblasts by high level of c-jun expression and tumorigenicity of malignant mouse epidermal cells could also be inhibited by TAM 67 (Rapp et al., Oncogene 9, 3493-3498 (1994); Domann et al., Cell Growth Differ. 5, 9-16 (1994)). It has also been shown that TAM 67 expression inhibits AP1-transactivating activity and colony formation of MCF human breast cancer cells in vitro (Kameda et al., 1993). However, no construct designed for gene therapy purposes and containing the gene encoding for TAM 67 and being able after transfection to inhibit growth of epithelial tumor cells, in particular of epithelial ovarian tumor cells has been proposed until now. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is related to a recombinant adeno associated viral construct comprising at least: a first terminal repeat of an Adeno Associated Virus a strong heterologous promoter an heterologous DNA corresponding to the nucleotide sequence encoding for the c-jun mutant protein TAM67, said gene being under the control of said promoter a polyadenylation signal, and a second terminal repeat of an Adeno Associated Virus With construct is meant a genetic construct, which can be comprised in a plasmid and/or in a recombinant viral particle. The recombinant adeno associated viral construct may further comprise nucleotide sequences encoding suitable regulatory elements so as to effect expression of the c-jun mutant protein TAM67 in a suitable host cell. The present invention is also related to a host cell genetically transformed by said construct. Preferably, said host cell is a human tumor cell. The present invention is also related to a pharmaceutical composition comprising said recombinant adeno associated viral construct or said cell alone or with a pharmaceutically acceptable carrier. Preferably, the recombinant adeno associated viral construct is comprised in a recombinant viral particle. The present invention is also related to a method for inhibiting the proliferation of cells, comprising at least the step of transferring a sufficient amount of the recombinant adeno associated viral construct according to the invention into said cells. Preferably, said cells are epithelial cells. Preferably, said epithelial cells are mammary epithelial cells, ovarian epithelial cells or lung epithelial cells. Preferably, said cells are cancer cells. Preferably, said cancer cells are selected from the group consisting of human melanoma cells, human mammary tumor cells, human ovarian tumor cells, lung tumor cells, human sarcoma cells and carcinoma cells. The present invention is also related to the use of a sufficient amount of the pharmaceutical composition according to the invention for the preparation of a medicament in the treatment and/or the prevention of cancers. The present invention is also related to a non-human animal, genetically modified by the recombinant adeno associated viral construct according to the invention. |
Method for modification of data on a memory card on a transaction |
A method for modification of data in a card transaction system having a memory card and a reader for reading the card. The card has a first memory (RAM) and a second memory (EEPROM) with data locations occupied by data recordings. Each transaction involves the modification of at least one of the data locations or the addition of a new recording. The method reads the address of a free location from a previous control register located in a first fixed location in the EEPROM memory, writes the new modified recording or addition in the free location, repeat the steps for each new recording to be modified or added, and writes in a second fixed location in the EEPROM memory a new control register containing the addresses of free locations within the EEPROM memory to use in the next transaction. |
1. A process for modifying data in a card-based transaction system featuring a memory card and a reader capable of reading said card when the latter is located in a position determined in relation to the reader, said card featuring a first RAM memory and a second nonvolatile erasable and re-writable memory, the latter including locations containing data records related to the transactions made by said card, each transaction leading to the modification of at least one of said data locations or the addition of a new record, said process being characterized by the following steps: a. for a data record to be modified or a record to be added, reading the address of a free location of said second memory in a previous check register located in a first determined location of said second memory, b. writing the new modified or added record in said free location, c. repeating steps a) and b) for each of the new records to be modified or added, and d. writing, in a second determined location of said second memory, a new check register containing the addresses of the free locations of said second memory to be used in the next transaction. 2. The process according to claim 1, also including the following step carried out prior to step a), writing the number of the location where the record to be modified or added is placed in said first RAM. 3. The process according to claim 2, in which said location number written in said first RAM is a virtual number when the operation to be performed concerns a record to be added. 4. The process according to claim 1, in which two variables are recorded in memory when the transaction is initialized, a first variable (NbUpdates) being set to zero so that it can be incremented at each record modification or additional operation and a second variable (TransactionLevel) being set to zero so that it can be changed from 0 to 1 wherein the transaction is in progress. 5. The process according to claim 1, in which the locations containing the records that were modified or added are erased when the transaction is closed. 6. The process according to claim 5, in which said first determined location containing the previous check register is erased when the transaction is closed. 7. The process according to claim 6, in which the numbers of said locations containing the records which were modified or added are written in said new check register from said first RAM when the transaction is closed. 8. The process according to claim 7, in which said variables of said first RAM are rest to zero when the transaction is closed. 9. The process according to claim 8 in which a field (FlagClose) of said new check register is rest to zero before said previous check register and said records which were modified or added, are erased and set to another value after these operations have been performed. 10. A system including means adapted to implement the steps of the process according to claim 1. |
<SOH> BACKGROUND <EOH>Memory cards, still referred to as smart cards, are being used increasingly as a support for data associated with cardholders. Among these cards, contactless cards, which exchange information by remote electromagnetic coupling between an antenna lodged in the card and an associated reader, were developed as a means of access into controlled access zones, a means of personal identification or even electronic wallets. The memory containing the data in a smart card is generally a nonvolatile, erasable and re-writable memory, preferably of EEPROM type. This type of memory is divided into a plurality of locations containing data records of a determined length, 32 bytes for example. During a transaction, several records contained in the card's memory must generally be modified in order to be adapted to the new conditions resulting from the transaction. Unfortunately, the memory, particularly if it is of EEPROM type, requires a relatively long erase/write time (5 ms, for example), during which a power failure may result in the loss of the data contained in the record. In addition, the various transaction operations, during which several data records are modified, operate in on/off mode. For transaction security reasons, all transactions must be repeated if a power outage occurs during the operation, at the risk of permanently losing sensitive data (for example, the credit balance of an electronic wallet) if it is not possible to repeat the transaction from the beginning. In order to respond to the problem mentioned above, an attempt was made to memorize the data record in a buffer memory before modifying it. When all changes are completed, the old records are erased from the buffer memory. Unfortunately, this method requires that 4 operations be performed for each record, namely saving into in the buffer zone, erasing the record to be modified, re-writing, and then erasing the old record in the buffer zone. When the transaction includes many operations, 8 operations for example, as may be the case, the transaction can take a long time and be incompatible with the time taken by the cardholder to present the card to the reader. In addition, the major drawback of this method is that the records in the data zone are always located in the same locations and that the buffer zone is located at a fixed place in the memory; this increases the “stress” on the memory and limits the number of transactions which may be carried out insofar as the number of operations is limited to a given value guaranteed by the silicon manufacturer. |
<SOH> SUMMARY OF THE INVENTION <EOH>Another problem that makes the above-mentioned problems more difficult relates to the designation of free sectors for positioning the modified records. The traditional method comprises scanning the memory in order to find a free sector. This operation takes time which adds to the time necessary for the already-mentioned operations for recording and erasing. However this time becomes very important and incompatible with the time of the transaction when there are several records to be modified and therefore several searches for free sectors. This is why the object of the invention is to create a process for modifying memory card data during a transaction which allows the on/off mode synchronization of the modifications to be made during the transaction, over a short period of time compatible with the access time limited to the card during the transaction, while avoiding the use of a portion of the memory as a buffer memory for saving data and thus avoiding memory “stress” that results from always writing data in the same memory zones. The invention thus concerns a process for modifying data in a card-based transaction system featuring a memory card and a reader capable of reading the card when it is placed in a determined position in relation to the reader. The card includes a first memory (RAM) and a second nonvolatile erasable and re-writable memory (EEPROM), the latter having locations containing data records relative to the transactions performed by the card, each transaction resulting in the modification of at least one of the data locations and the addition of a new record. This method according to the invention includes the following steps: a) for a data record to be modified or a record to be added, reading the address of a free location of the second memory in a previous check register located in a first determined location of the second memory, b) writing the new modified or added record in the free location, c) repeating steps a) and b) for each of the new records to be modified or added, d) writing, in a second determined location of the second memory, a new check register containing the addresses of the free locations of the second memory to be used in the next transaction. |
Isolated human secreted proteins, nucleic acid molecules encoding human secreted proteins, and uses thereof |
The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the secreted peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the secreted peptides, and methods of identifying modulators of the secreted peptides. |
1. An isolated peptide consisting of an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown, in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids. 2. An isolated peptide comprising an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids. 3. An isolated antibody that selectively binds to a peptide of claim 2. 4. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d). 5. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d). 6. A gene chip comprising a nucleic acid molecule of claim 5. 7. A transgenic non-human animal comprising a nucleic acid molecule of claim 5. 8. A nucleic acid vector comprising a nucleic acid molecule of claim 5. 9. A host cell containing the vector of claim 8. 10. A method for producing any of the peptides of claim 1 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence. 11. A method for producing any of the peptides of claim 2 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence. 12. A method for detecting the presence of any of the peptides of claim 2 in a sample, said method comprising contacting said sample with a detection agent that specifically allows detection of the presence of the peptide in the sample and then detecting the presence of the peptide. 13. A method for detecting the presence of a nucleic acid molecule of claim 5 in a sample, said method comprising contacting the sample with an oligonucleotide that hybridizes to said nucleic acid molecule under stringent conditions and determining whether the oligonucleotide binds to said nucleic acid molecule in the sample. 14. A method for identifying a modulator of a peptide of claim 2, said method comprising contacting said peptide with an agent and determining if said agent has modulated the function or activity of said peptide. 15. The method of claim 14, wherein said agent is administered to a host cell comprising an expression vector that expresses said peptide. 16. A method for identifying an agent that binds to any of the peptides of claim 2, said method comprising contacting the peptide with an agent and assaying the contacted mixture to determine whether a complex is formed with the agent bound to the peptide. 17. A pharmaceutical composition comprising an agent identified by the method of claim 16 and a pharmaceutically acceptable carrier therefor. 18. A method for treating a disease or condition mediated by a human secreted protein, said method comprising administering to a patient a pharmaceutically effective amount of an agent identified by the method of claim 16. 19. A method for identifying a modulator of the expression of a peptide of claim 2, said method comprising contacting a cell expressing said peptide with an agent, and determining if said agent has modulated the expression of said peptide. 20. An isolated human secreted peptide having an amino acid sequence that shares at least 70% homology with an amino acid sequence shown in SEQ ID NO:2. 21. A peptide according to claim 20 that shares at least 90 percent homology with an amino acid sequence shown in SEQ ID NO:2. 22. An isolated nucleic acid molecule encoding a human secreted peptide, said nucleic acid molecule sharing at least 80 percent homology with a nucleic acid molecule shown in SEQ ID NOS:1 or 3. 23. A nucleic acid molecule according to claim 22 that shares at least 90 percent homology with a nucleic acid molecule shown in SEQ ID NOS:1 or 3. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Secreted Proteins Many human proteins serve as pharmaceutically active compounds. Several classes of human proteins that serve as such active compounds include hormones, cytokines, cell growth factors, and cell differentiation factors. Most proteins that can be used as a pharmaceutically active compound fall within the family of secreted proteins. It is, therefore, important in developing new pharmaceutical compounds to identify secreted proteins that can be tested for activity in a variety of animal models. The present invention advances the state of the art by providing many novel human secreted proteins. Secreted proteins are generally produced within cells at rough endoplasmic reticulum, are then exported to the golgi complex, and then move to secretory vesicles or granules, where they are secreted to the exterior of the cell via exocytosis. Secreted proteins are particularly useful as diagnostic markers. Many secreted proteins are found, and can easily be measured, in serum. For example, a ‘signal sequence trap’ technique can often be utilized because many secreted proteins, such as certain secretory breast cancer proteins, contain a molecular signal sequence for cellular export. Additionally, antibodies against particular secreted serum proteins can serve as potential diagnostic agents, such as for diagnosing cancer. Secreted proteins play a critical role in a wide array of important biological processes in humans and have numerous utilities; several illustrative examples are discussed herein. For example, fibroblast secreted proteins participate in extracellular matrix formation. Extracellular matrix affects growth factor action, cell adhesion, and cell growth. Structural and quantitative characteristics of fibroblast secreted proteins are modified during the course of cellular aging and such aging related modifications may lead to increased inhibition of cell adhesion, inhibited cell stimulation by growth factors, and inhibited cell proliferative ability (Eleftheriou et al., Mutat Res 1991 March-November;256(2-6):127-38). The secreted form of amyloid beta/A4 protein precursor (APP) functions as a growth and/or differentiation factor. The secreted form of APP can stimulate neurite extension of cultured neuroblastoma cells, presumably through binding to a cell surface receptor and thereby triggering intracellular transduction mechanisms. (Roch et al., Ann N Y Acad Sci 1993 Sep. 24;695:149-57). Secreted APPs modulate neuronal excitability, counteract effects of glutamate on growth cone behaviors, and increase synaptic complexity. The prominent effects of secreted APPs on synaptogenesis and neuronal survival suggest that secreted APPs play a major role in the process of natural cell death and, furthermore, may play a role in the development of a wide variety of neurological disorders, such as stroke, epilepsy, and Alzheimer's disease (Mattson et al., Perspect Dev Neurobiol 1998; 5(4):337-52). Breast cancer cells secrete a 52K estrogen-regulated protein (see Rochefort et al., Ann N Y Acad Sci 1986;464:190-201). This secreted protein is therefore useful in breast cancer diagnosis. Two secreted proteins released by platelets, platelet factor 4 (PF4) and beta-thromboglobulin (betaTG), are accurate indicators of platelet involvement in hemostasis and thrombosis and assays that measure these secreted proteins are useful for studying the pathogenesis and course of thromboembolic disorders (Kaplan, Adv Exp Med Biol 1978;102:105-19). Vascular endothelial growth factor (VEGF) is another example of a naturally secreted protein. VEGF binds to cell-surface heparan sulfates, is generated by hypoxic endothelial cells, reduces apoptosis, and binds to high-affinity receptors that are up-regulated by hypoxia (Asahara et al., Semin Interv Cardiol 1996 Sep. 1(3):225-32). Many critical components of the immune system are secreted proteins, such as antibodies, and many important functions of the immune system are dependent upon the action of secreted proteins. For example, Saxon et al., Biochem Soc Trans 1997 May;25(2):383-7, discusses secreted IgE proteins. For a further review of secreted proteins, see Nilsen-Hamilton et al., Cell Biol Int Rep 1982 Sep. 6(9):815-36. Epidermal growth factors The novel human protein, and encoding gene, provided by the present invention is related to the epidermal growth factor (EGF) superfamily, including proteins containing EGF or EGF-like domains and other EGF-related proteins such as those containing a CUB (Cls-like) domain such as Scubel (see Grimmond et al., Genomics 70 (1), 74-81 (2000)). EGF proteins play important roles as signaling molecules, growth factors, and as part of the extracellular matrix. EGF proteins are also known to be important in vertebrate development (Grimmond et al., Genomics 70 (1), 74-81 (2000)). Scubel has been found to be highly expressed in developing gonads, nervous system, somites, surface ectoderm, and limb buds of the mouse (Grimmond et al., Genomics 70 (1), 74-81 (2000)). The protein of the present invention is expressed in pancreas adenocarcinoma (as well as in the brain), and therefore is a potential target for treating pancreatic cancer. Secreted proteins, particularly members of the epidermal growth factor protein subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of secreted proteins. The present invention advances the state of the art by providing previously unidentified human secreted proteins that have homology to members of the epidermal growth factor protein subfamily. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is based in part on the identification of amino acid sequences of human secreted peptides and proteins that are related to the epidermal growth factor protein subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate secreted protein activity in cells and tissues that express the secreted protein. Experimental data as provided in FIG. 1 indicates expression in the brain and pancreas adenocarcinoma. |
Catalytic Reactor |
A catalytic reactor (40) comprises a plurality of sheets (42) defining flow channels (44) between them. Within each flow channel (44) is a foil (46) of corrugated material whose surfaces are coated with catalytic material apart from where they contact the sheets (44). At each end of the reactor (40) are headers to supply gas mixtures to the flow channels (44), the headers communicating with adjacent channels being separate. The reactor (40) enables different gas mixtures to be supplied to adjacent channels (44), which may be at different pressures, and the corresponding chemical reactions are also different. Where one of the reactions is endothermic while the other reaction is exothermic, heat is transferred through the sheets (42) separating the adjacent channels (44), from the exothermic reaction to the endothermic reaction. The reactor (40) may be used in a compact plant to perform steam/methane reforming, obtaining the necessary heat by catalytic methane combustion, and also for Fischer-Tropsch synthesis, so that the overall process involves conversion of methane to long-chain hydrocarbons. |
1. A stacked assembly of plates, the stack having an inlet (12) and an outlet (14) for a first fluid and an inlet (24) and an outlet (34) for a second fluid, characterised in that a first portion of the length of the assembly is formed of one or more first perforated plates (26), each first perforated plate being perforated to define a first series of slots (36) spaced across the plate and a second series of slots (46) is spaced across the plate, each slot (36) of the first series being positioned between a pair of slots (46) of the second series, whereby the slots of the first series define first passageways through the first portion of the length for a first fluid and the slots of the second series define second passageways through the first portion of the length for a second fluid, the first series of passageways being connected to said inlet (12) and outlet (14) for the first fluid, a second portion of the length of the assembly being formed of one or more second perforated plates (20, 22, 20A), each second perforated plate being perforated to define a first (36A, 36B) and a second (46A, 46B) series of slots corresponding to the slots of the first plate(s) so as to provide continuing passageways in line with the first and second passageways of the first portion, each slot (46A, 46B) of the second series opening at one of its two ends into a feeder slot (50, 50B) extending across the second plate, the feeder slot(s) being connected to an inlet (24) or an outlet (34) for the second fluid. 2. A stacked assembly according to claim 1, characterised in that a third portion of the length of the assembly at the opposite end of the first portion to the second portion is formed of one or more plates (30, 32, 30A) of similar construction to the plates of the second portion with the feeder slot(s) being connected to an outlet (34) or an inlet (24) accordingly for the second fluid. 3. A stacked assembly according to claim 2, characterised in that the or each plate (30, 32, 30A) of the third portion is formed to have its feeder slot (50, 50B) extending in the opposite side of the assembly to the feeder slots (50, 50B) of the first portion, whereby the second fluid must cross the assembly from one side to the other between the second fluid inlet (24) and outlet (34). 4. A stacked assembly according to claim 1, 2 or 3, characterised in that the first series of passageways is packed with a catalyst (92). 5. A stacked assembly according to any preceding claim, characterised in that the plates (20, 26, 22) are square or rectangular in plan and the slots (36, 46, 36A, 46A, 36B, 46B) are linear. 6. A stacked assembly according to any preceding claim, characterised in that it comprises along the stack a second length portion (20, 22, 20A), a first length portion (26), a baffle plate (28), a further first length portion (26A) and then a further second length portion (30, 32, 30A). 7. A stacked assembly according to claim 6, characterised in that the baffle plate (28) comprises a series of slots (66) corresponding to and in line with the first series of passageways for the first fluid so that the first fluid has an uninterrupted flow through the assembly and a second series of staggered slots (68, 70) which partially interrupt flow through the second series of passageways whereby some at least of the second fluid must travel non-linearly between the two halves of the assembly divided by the baffle plate. 8. A stacked assembly according to any preceding claim, characterised in that it includes at one or each end thereof a perforated closure plate (18). 9. A stacked assembly according to claim 8, characterised in that the perforated closure plate (18) has a first series of slots (86) corresponding to the first passageways, whereby the first fluid can flow uninterrupted through the closure plate, but no slot corresponding to the second passageways, whereby the second fluid is diverted into the inlet (24) and/or outlet (34) provided in the second plate(s). 10. A stacked assembly according to any preceding claim, characterised in that it includes a plate (100) having a series of slots (102) corresponding to the first series of slots (36), wherein each slot (102) lies between a pair of injection grooves (104), the injection grooves (104) opening into a feeder groove (106) connected to an injection inlet (108), and the grooves (104) giving access into the slots at a plurality of positions (112) along each slot (102), whereby fluid may be injected into each slot (102), the plate (100) being positioned next to a plate having solid closure regions corresponding to and covering the grooves (104, 106, 108, 112). 11. A stacked assembly according to claim 10, characterised in that restrictions (104A) are provided in some of the grooves (104), the restriction (104A) being greater the nearer the groove is to the injection inlet (108). 12. A stacked assembly according to any one of claims 1 to 9, characterised in that it includes a modified first plate (200) in which one end of each first slot (202) is connected (212) to a feeder groove (210) extending across the plate, the feeder groove (210) being connected to an injection inlet (208). 13. A stacked assembly according to claim 12, characterised in that it includes a pair of identical modified first plates (200) stacked together but rotated through 180° with respect to each other. 14. A stacked assembly according to any one of claims 10 to 13, characterised in that the grooves (104, 106, 108, 112, 208, 210, 212) are of depth equal to about half the thickness of their respective plates (100, 200). 15. A stacked assembly according to any preceding claim, characterised in that all the plates are of the same external dimensions. 16. A stacked assembly according to any preceding claim, characterised in that the plates are from 1 mm to 12 mm in thickness. 17. A stacked assembly according to any preceding claim, characterised in that adjacent solid regions of a plate extending between adjacent pairs of first slots are connected together by one or more strengthening tie bars (54, 54B, 206, 258, 278, 298, 308) extending across an intervening second slot (46A, 46B, 204, 254, 274, 294,304). 18. A stacked assembly according to any one of claims 5 to 17, characterised in that the feeder slots (50, 50B) extend almost the entire length of one side (38A, 38B) of their respective plates (20, 22) and are defined inside a solid edge portion (38A′, 38B′) at that side of the plate. 19. A stacked assembly according to claim 18, characterised in that the second fluid is fed into the feeder slot (50, 50B) of a second plate (20, 22) through a gap (60, 60B) in a solid edge portion of the plate (42A′, 42B′) and a tie bar (56, 56B) is provided adjacent the gap to connect the solid edge portion to a solid region extending across the plate between an adjacent pair of slots (36A, 46A; 36B, 46B). 20. A stacked assembly according to claim 19, characterised in that it comprises one or more adjacent pairs of second plates (20, 22), the plates of the pair having essentially the same slotted construction whereby their feeder slots (50, 50B) and first and second passageways are in alignment but their tie bars (56, 56B) are offset. 21. A stacked assembly according to any preceding claim, characterised in that in at least some of the plates (250, 270, 290, 290A) pressure equalisation means are provided between the first passageways defined by the first series of slots (252, 272, 292, 292A) in each of those plates. 22. A stacked assembly according to claim 21, characterised in that the rows of slots (252) of the first series are joined together at their ends (253). 23. A stacked assembly according to claim 21, characterised in that venting is provided through venting channels (280, 296, 296A) in tie bars (278, 298, 298A) spaced along the length of each second slot (274, 294, 294A), each tie bar running across its second slot from one first slot (274, 292, 299A) to an adjacent first slot (272, 292, 292A) and having its venting channel formed partially through its thickness. 24. A stacked assembly according to any preceding claim, characterised in that the plates have one or more holes (328, 330) in a margin (332, 334) outside the slots (326), the holes aligning in the stack (320) to provide one or more passages (336, 338) through which a reactant may be injected into the stack, the passages connecting to first passageways through the stack at different levels in the stack. 25. A heat exchanger, characterised in that it comprises a stacked assembly (103, 320) of plates according to any one of the preceding claims. 26. A perforated plate for a heat exchanger, characterised in that the plate (20, 22) is perforated to define a first series of slots (36A, 36B) spaced across the plate and a second series of slots (46A, 46B) spaced across the plate, each slot. (36A, 36B) of the first series being positioned between a pair of slots (46A, 46B) of the second series and each slot (46A, 46B) of the second series opening at one of its two ends into a feeder slot (50, 50B) extending across the second plate, the feeder slot thereby connecting those first ends together. |
Unique properties of a stem cells |
The present invention is directed to unique properties of stem cells, including methods to identify stem cell markers by identifying molecules associated specifically with chromosomes in stem cells. More particularly, we have discovered that somatic stem cells repeatedly inherit an entire complement of chromosomes that contain the same parental template DNA strands from one generation to the next. The present invention also provides methods related to diagnosis, prognosis, and treatments for cancer and aging in mammalian tissus, including human. |
1. A method for identifying a stem cell chromosome which comprises a) labeling cells undergoing cell cycle division by exposing said cells to a detectable nucleotide analogue for the duration of one cell cycle; b) removing the nucleotide analogue and growing the cells in the absence of said analogue for the duration of another cell cycle; c) treating the cells to allow the identification and comparison of two sister nuclei which share a common parental nucleus; and d) identifying those unlabeled nuclei which contain only unlabeled chromosomes which have not incorporated said detectable nucleotide analogue, but which have a labeled sister nucleus which does contain labeled chromosomes; wherein the unlabeled chromosomes in said unlabeled nuclei are the stem cell chromosomes. 2. The method of claim 1, further comprising isolating the identified stem cell chromosomes. 3. The method of claim 1, wherein the detectable nucleotide analogue is selected from the group consisting of 5-bromo-deoxyuridine (BrdU), 3H-thymidine, halogenated nucleotide analogues, and iodo-uridine. 4. The method of claim 3, wherein the detectable nucleotide analogue is BrdU. 5. The method of claim 1, wherein the treatment of cells which allows the identification and comparison of two sister nuclei which share a common parental nucleus is selected from the group consisting of cytochalasin D, colcemid, chochicine, mitotic shake-off, nocodazole, cell synchronization, expression of specific mitotic arrest genes, and repression of specific mitotic promoting genes. 6. The method of claim 5, wherein the treatment is cytochalasin D. 7. The method of claim 1, further comprising the step of isolating mitotic cells prior to exposing cells to a detectable nucleotide analogue. 8. The method of claim 7, wherein said mitotic cells are isolated by mitotic shake-off, wherein mitotic cells are isolated every hour by shaking cell cultures to loosen cells undergoing mitosis and collecting the culture medium. 9. The method of claim 8, wherein the mitotic cells from the first two collections are discarded. 10. The method of claim 7, where the mitotic cells are maintained at 4° C. prior to exposing cells to the nucleotide analogue. 11. The method of claim 1, wherein said stem cell chromosomes contain alterations selected from the group consisting of modifications and structural conformations. 12. The method of claim 10, wherein the modification is selected from the group consisting of methylation, deamination, glycosylation, acetylation, phosphorylation, lipidation, and previously unknown modifications. 13. The method of claim 10, wherein the structural conformation alteration is selected from the group consisting of a region of single-stranded DNA and a region of double-stranded DNA. 14. The method of claim 13, wherein the structural conformation alteration is a region of single-stranded DNA. 15. The method of claim 13, wherein the structural conformation alteration is a region of double-stranded DNA. 16. A method of identifying stem cell markers associated specifically with chromosomes in stem cells, comprising: a) exposing cells undergoing cell cycle division to a detectable nucleotide analogue for at least one cell cycle; b) detecting incorporation of said nucleotide analogue into said chromosomes; c) identifying unlabeled chromosomes which do not incorporate said nucleotide analogue; and d) comparing said unlabeled chromosomes with corresponding labeled chromosomes from said cells to identify differences between the chromosomes, wherein said differences are the stem cell markers. 17. The method of claim 16, wherein the stem cell marker is selected from the group consisting of proteins, DNAs, RNAs, antibodies, ligands, methylation, and chemical reagents. 18. The method of claim 16, further comprising identifying molecules that specifically associate with the stem cell marker. 19. The method of claim 18, wherein the molecule that specifically associates with the stem cell marker is selected from the group consisting of proteins, DNAs, RNAs, antibodies, and chemical reagents. 20. The method of claim 19, wherein the molecule that specifically associates with the stem cell marker is an antibody. 21. The method of claim 20, wherein said antibody is generated by using said unlabeled chromosomes as antigenic material using standard antibody technology. 22. The method of claim 21, wherein the antibody technology is selected from the group consisting of hybridoma technology, 2-dimensional gel isolation of proteins for raising anti-peptide antibodies, and microarray technology to identify candidate genes to raise anti-peptide antibodies. 23. An antibody which specifically binds to immortal strands of DNA. 24. A method for isolating stem cells using the antibody of claim 20, wherein flow cytometry of a population of cells exposed to said antibody is used to separate stem cells from non-stem cells. 25. A method for detecting stem cells in a biological sample, wherein said stem cells are detected using the method of claim 1. 26. The method of claim 25, wherein said nucleotide analogue is BrdU which is detected using immunofluorescence with anti-BrdU antibodies. 27. The method of claim 24, wherein said stem cells are detected using immunofluorescence with an antibody which specifically binds to immortal strands of DNA. 28. The method of claim 24, wherein said stem cells are detected using a fluorescent label, and quantified using image-programmed laser scanning cytometry. 29. A method of assessing the age of a tissue, comprising isolating somatic stem cells from said tissue and measuring damage to the chromosomes of said cells by comparing said chromosomal damage with chromosomal damage in a control population of cells. 30. A method of prognosis for aging, comprising assessing the underlying state of disease using the method of claim 29. 31. A method to rejuvenate a tissue in an animal by inducing synthesis and selection of new immortal DNA strands in somatic stem cells. 32. A method of enumerating somatic stem cells in a biological sample, comprising counting the number of stem cells in said animal using the molecule that specifically associates with the stem cell marker of claim 17. 33. The method of claim 32, wherein the biological sample is selected from the group consisting of an animal, an organ, a tissue sample, a blood sample, a bone marrow sample, an in vitro cell culture, biopsy material, surgical resections, needle aspirations, and lavages. 34. The method of claim 33, wherein the biological sample is a bone marrow sample. 35. A method of predicting bone marrow transplant engraftment success, wherein the number of hematopoetic stem cells present in the transplanted sample is predictive of transplantation success comprising enumerating the hematopoetic stem cells present in the transplanted sample by: a) preparing a bone marrow sample for transplantation in a patient in need thereof; b) removing a prognostic fraction of the bone marrow sample prior to transplantation for enumeration; and c) estimating the number of somatic stem cells present in the bone marrow sample by counting the number of somatic stem cells in said prognostic fraction using the stern cell marker of claim 15. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Considerable attention has focused on stem cells and their uses in a range of therapies. The availability of somatic stem cells from adult tissues would greatly contribute to cell replacement therapies such as bone marrow transplants, gene therapies, tissue engineering, and in vitro organogenesis. Production of autologous stem cells to replace injured tissue would also reduce the need for immune suppression interventions. Somatic stem cells, also known as adult stem cells, are stem cells derived from adult tissues, in contrast to other sources of stem cells such as cord stem cells and embryonic stem cells, which may originate from a variety of sources of embryonic tissue. Somatic stem cells are particularly attractive for a range of therapies in light of the ongoing controversies surrounding the use of embryonic stem cells. Somatic stem cells possess the ability to renew adult tissues (Fuchs and Segre, 2000). Cell growth is a carefully regulated process that responds to the specific needs of the body in different tissues and at different stages of development. In a young animal, cell multiplication exceeds cell loss and the animal increases in size; in an adult, the processes of cell division and cell loss are balanced to maintain a steady state. For some adult cell types, renewal is rapid: intestinal cells and certain white blood cells have a half-life of a few days before they die and are replaced. In contrast, the half-life of human red blood cells is approximately 100 days; healthy liver cells rarely die, and in adults, there is a slow loss of brain cells with little or no replacement. Somatic stem cells may also play an important role during aging. As an animal ages, cellular changes that occur in tissues are also likely to reflect alterations in the number and function of somatic stem cells. One model which has been proposed is that alterations in a cell's DNA reflects the relative age of the cell. These alterations could include stable covalent base modification (e.g., methylation) or poorly repaired forms of oxidative and other chemical damage (production of hypoxanthine and xanthine bases via deamination), as well as base pair errors introduced during replication. Accumulation of these defects over time would eventually lead to declines in stem cell number and function to the ultimate demise of tissue function and life. Thus, it would be desirable if there was a way to counter some of the aforementioned effects of aging to rejuvenate aged tissues. Beyond their potential therapeutic applications, homogenous preparations of somatic stem cells would have another important benefit, the ability to study their molecular and biochemical properties. The existence of stem cells in somatic tissues is well established by functional tissue cell transplantation assays (Reisner et al, 1978). However, their individual identification has been difficult to accomplish. Even though their numbers have been enriched by methods such as immuno-selection with specific antibodies, there are no known markers that uniquely identify stem cells in somatic tissues (Merok and Sherley, 2001). Secondly, somatic stem cells are often present in only minute quantities, are difficult to isolate and purify, and their numbers may decrease with age. For example, brain cells from adults that may be neuronal stem cells have only been obtained by removing a portion of the brain of epileptics, not a trivial procedure. Thus, there is a need to develop simple and reliable methods for the identification of stem cell-specific markers for the development of stem cell-specific molecular probes. Such stem cell markers could then be used to develop methods to identify stem cells in tissues and to isolate them directly from tissues. The ability to readily obtain stem cells in human tissues would permit easy harvesting of those cells as well as add greatly to our understanding of tissue cell physiology. An understanding of the mechanisms which control stem cell number can suggest new therapeutic strategies for cancer prevention and treatment, and for reducing morbidity associated with aging. Accordingly, methods to isolate and expand stem cells from somatic tissue, particularly without significant differentiation, are highly desirable. Attempts at isolating somatic stem cells have encountered a number of significant difficulties. Attempts at somatic stem cell isolation have been described, for example, in studies to enrich for hematopoietic stem cells (HSCs; Phillips et al., 2000). However, although high degrees of enrichment have been reported, so far HSCs (and other somatic stem cells) have neither been identified nor purified to homogeneity. A major obstacle to these two challenges is the lack of stem cell-specific molecular probes. Thus, despite the need for methods to isolate somatic cells from an individual, it has not been possible to readily do so. Accordingly, it would be desirable to have a method to identify markers associated with somatic stem cells in mammalian tissues. It would also be desirable to have methods to detect aging in mammalian tissues, including humans. Finally, it would be desirable to have methods to reduce or reverse at least some of the consequences of such aging in adult mammalian tissues, including humans. |
<SOH> SUMMARY OF THE INVENTION <EOH>We have now discovered methods for identifying stem cell chromosomes, by exposing cells undergoing cell cycle division to a detectable nucleotide analogue, and identifying those chromosomes which do not incorporate the nucleotide analogue. Preferably, the nucleotide analogue is 5-bromo-deoxyuridine. In a preferred embodiment, mitotic cells are isolated using mitotic shake-off after exposing the cells to the nucleotide analogue labeling regimens to enrich for cells undergoing cell cycle division. In a further preferred embodiment, cytochalasin D is used to generate bi-nucleated single cells to improve detection of stem cell chromosomes. The invention also provides methods to identify stem cells markers by identifying molecules associated specifically with chromosomes in stem cells; identification of markers that are specific to such stem cells permit the development of stem cell-specific molecular probes. The stem cell markers of the present invention are associated with alterations of the stem cell chromosomes compared to non-stem cell chromosomes. For example, stem cell chromosomes may have specific molecules, such as proteins, RNA, or DNA, associated with them. Alternatively, the chromosomes of stem cells may have characteristic alterations, such as modifications (including methylation, deamination, glycosylation, acetylation, phosphorylation, lipidation, and previously unknown modifications) or specific structural conformities (including regions of single-stranded DNA). In a preferred embodiment, the stem cell marker is an antibody which specifically recognizes stem cell chromosomes. The present invention also provides methods to generate such antibodies, by isolating stem cell chromosomes and using hybridoma technology to raise monoclonal antibodies against them. An alternative approach is to identify protein(s) uniquely associated with stem cell chromosomes using 2-D gel analysis, and raise anti-peptide antibodies to those proteins. A third approach is to identify stem cell chromosome-specific genes using microarray analysis, and to raise anti-peptide antibodies to the proteins they encode. The present invention also provides a method for isolating stem cells, using for example, a means to identify the unique stem cell chromosome such as a stem cell chromosome specific antibody, with flow cytometry to separate stem cells from non-stem cells. The present invention also provides methods for detecting stem cells in a biological sample. In one embodiment, cells undergoing cell cycle division are exposed to a detectable nucleotide analogue, and stem cells are identified as those cells containing chromosomes which do not incorporate the nucleotide analogue. Preferably, the nucleotide analogue is 5-bromo-deoxyuridine (BrdU), which is detected using a fluorescent anti-BrdU antibody or quenching of bound fluorescent Hoechst DNA dyes. Methods for detecting stem cells also include the use of the stem cell markers described above, and any method which allows their detection. For example, immunohistochemistry with the stem cell antibody. In a further preferred embodiment, image-programmed laser scanning cytometry is used to detect fluorescently labeled cells. In yet another preferred embodiment, flow cytometry may be used to detect binucleated cells that contain one labeled and one unlabeled nucleus. The use of the stem cell markers permits one to enumerate the number of stem cells in an animal. A method to rejuvenate aged tissue can be accomplished by replacing the immortal DNA strand in somatic stem cells, for example by inducing their replication to eliminate stem cells of age-dependent genomic defects. In one preferred embodiment, stem cells can be harvested from an animal, expanded by symmetric replication, cryo-preserved and thawed, expanded and administered to the animal to replace the age-damaged stem cells at later stages in the life cycle. Another preferred embodiment provides a method of prognosis or diagnosis, for example of aging, by comparing the number and quality (e.g., functional immortal strand co-segregation efficiency) of somatic stem cells in a patient or tissue to a control. Another preferred embodiment provides a method of predicting bone marrow transplant engraftment success by enumerating the number and quality and quality of hematopoietic stem cells present in the transplanted sample, by removing a prognostic fraction of the bone marrow sample prior to transplantation, and estimating the number of stem cells in the transplanted sample by enumerating the number of stem cells present in the prognostic fraction. |
Data storing method, data storing system, data recording control apparatus, data recording instructing apparatus, data receiving apparatus, and information processing terminal |
A distributed storage system having n nodes (201), (202), . . . , (20n), a recording commanding device (10) for commanding recording of data in each node, and a transmission commanding device (30) for reading out the data recorded in the respective nodes. These nodes and the devices are interconnected over a network. The recording commanding device (10) applies FEC coding, with the coding rate of q/p, to data composed of p blocks, to generate q blocks, and appends a recording probability a, recorded by the node, to the data, to send out the resulting data to the network. In readout, the transmission commanding device (30) responds data to which has been appended the response probability β in responding the data. |
1. A data storage method for distributing and recording data in a plurality of information terminals interconnected over a network, comprising an encoding step for encoding blocked first data to generate blocked second data; a packetizing step of packetizing said second data; a recording probability generating step of generating a recording probability indicating whether or not the packet generated in said packetizing step is to be recorded in said plural information processing terminals; a transmission step of transmitting said packet and the recording probability to said plural information processing terminals; and a recording step of recording said packets in said information processing terminal based on said recording probability; wherein the method includes, as processing for reading out said packet recorded in one or more of said plural information processing terminals with said recording probability, a response request data generating step of generating response request data with a response probability attached thereto for indicating whether or not said packet is to be responded; a transmission step of transmitting said response request data to said plural information processing terminals; and a response step of responding said packet in said plural information processing terminals based on said response probability. 2. The data storage method according to claim 1 wherein said recording probability is included in said packet and is transmitted along with said packet to said plural information processing terminals. 3. The data storage method according to claim 1 wherein said recording probability is transmitted with a packet different from said packet. 4. The data storage method according to claim 1 wherein the number of the packets responded in said responding step is the same as or larger than the number of the blocks of said first data. 5. The data storage method according to claim 4 further comprising a packet distribution controlling step of changing the ratio of said first and second data, recording probability and the response probability to control the distribution of said packets. 6. The data storage method according to claim 4 wherein, in said packet distribution controlling step, said recording probability, response probability, the number of said information processing terminals and the number of blocks of said second data are changed so that the product of said recording probability, response probability, the number of said information processing terminals and the number of blocks of said second data is the same as or larger than the number of blocks of said first data. 7. The data storage method according to claim 6 wherein, in said packet distribution controlling step, a data loss probability in said network, said recording probability, response probability, the number of said information processing terminals and the number of blocks of said second data are changed so that the product of said data loss probability in said network, said recording probability, response probability, the number of said information processing terminals and the number of blocks of said second data is the same as or larger than the number of blocks of said first data. 8. The data storage method according to claim 4 wherein, in said packet distribution controlling step, the probability of said plural information terminals not responding, said recording probability, response probability, the number of said information processing terminals and the number of blocks of said second data are changed so that the product of the probability of said plural information terminals not responding, said recording probability, response probability, the number of said information processing terminals and the number of blocks of said second data is the same as or larger than the number of blocks of said first data. 9. The data storage method according to claim 1 wherein, in said recording probability generating step and in said response probability generating step, said recording probability, response probability or the coding rate is increased to lower the probability of overlapping of the packets responded in said responding step. 10. A data storage system for distributing and recording data in a plurality of information terminals interconnected over a network, comprising a data recording controlling device; said data recording controlling device including encoding means for encoding blocked first data to generate blocked second data; packetizing means for packetizing said second data; recording probability generating means for generating a recording probability indicating whether or not the packet generated in said packetizing means is to be recorded in said plural information processing terminals; and transmission means for transmitting said recording probability and said response request data to said plural information processing terminals; said data storage system also comprising a plurality of information processing terminals, said information processing terminals each including transmission/receiving means for transmitting/receiving said packet and the recording probability to/from said data recording controlling device; recording means for recording said packet; and controlling means for managing control whether or not the received packet is to be recorded based on said recording probability and whether or not the recorded packet is to be responded with said response probability; said means being interconnected in their entirety over a network. 11. The data storage system according to claim 10 wherein said recording probability is included in said packet and is transmitted along with said packet to the information processing terminals. 12. The data storage system according to claim 10 wherein said recording probability is transmitted with a packet different from said packet. 13. The data storage system according to claim 10 wherein the number of the packets received in said receiving means in said data receiving controlling device is the same as or larger than the number of the blocks of said first data. 14. The data storage system according to claim 13 further comprising a packet distribution controlling means of changing the ratio of said first and second data, recording probability and the response probability to control the distribution of said packets. 15. The data storage system according to claim 13 wherein, in said packet distribution controlling means, said recording probability, response probability, the number of said information processing terminals and the number of blocks of said second data are changed so that the product of said recording probability, response probability, the number of said information processing terminals and the number of blocks of said second data is the same as or larger than the number of blocks of said first data. 16. The data storage system according to claim 15 wherein, in said packet distribution controlling means, a data loss probability in said network, said recording probability, response probability, the number of said information processing terminals and the number of blocks of said second data are changed so that the product of said data loss probability in said network, said recording probability, response probability, the number of said information processing terminals and the number of blocks of said second data is the same as or larger than the number of blocks of said first data. 17. The data storage system according to claim 13 wherein, in said packet distribution controlling means, the probability of said plural information terminals not responding, said recording probability, response probability, the number of said information processing terminals and the number of blocks of said second data are changed so that the product of the probability of said plural information terminals not responding, said recording probability, response probability, the number of said information processing terminals and the number of blocks of said second data is the same as or larger than the number of blocks of said first data. 18. The data storage system according to claim 10 wherein, in said recording probability generating means and in said response probability generating means, said recording probability, response probability or the coding rate is increased to lower the probability of overlapping of the packets responded by said information processing terminals. 19. A data recording controlling apparatus for distributing and recording data in a plurality of information terminals interconnected over a network, said apparatus comprising encoding means for encoding blocked first data to generate blocked second data; packetizing means for packetizing said second data; recording probability generating means for generating a recording probability specifying whether or not the packet generated by said packetizing means is to be recorded in said plural information processing terminals; response request data generating means for generating response request data with a response probability attached thereto, said response probability indicating whether or not said packet is to be responded; transmission means for transmitting the packet including said recording probability and said response request data to said plural information processing terminals; and receiving means for receiving the packet sent from said terminals. 20. A data recording commanding apparatus for distributing and recording data in a plurality of information terminals interconnected over a network, said apparatus comprising encoding means for encoding blocked first data to generate blocked second data; packetizing means for packetizing said second data; recording probability generating means for generating a recording probability indicating whether or not the packet generated in said packetizing means is to be recorded in said plural information processing terminals; and transmission means for transmitting said packet inclusive of the recording probability to said plural information processing terminals. 21. A data receiving apparatus for receiving data from a plurality of information processing terminals interconnected over a network, said apparatus comprising response request data generating means for generating response request data with a response probability attached thereto, said response probability indicating whether or not a packet recorded in said plural information processing terminals is to be responded; transmitting means for transmitting said response request data to said information processing terminals; and receiving means for receiving the packet sent from said information processing terminals. 22. An information processing terminal comprising transmitting/receiving means for transmitting/receiving data with an external device connected thereto over a network; recording means for recording the packet; and controlling means for controlling whether or not said packet is to be recorded based on the recording probability attached to said packet and whether or not the recorded packet is to be responded with the response probability sent from said external device. |
<SOH> BACKGROUND ART <EOH>Recently, a large scale storage system, in which data is distributed and recorded in a large number of information processing terminals interconnected over a network. In this distributed storage system, a server for recording and managing data transmits data to information processing terminals or to other servers, by e.g. multi-cast, for recording on local recording mediums provided in the information processing terminals or other servers. If, in this case, data is to be taken out on demand, a large amount of data must be recorded in the recording medium. For example, if data is a motion picture, with the data capacity of approximately 2 Gbytes, and 500 of such motion pictures are to be recorded, the capacity not less than 1 terabyte is needed. On the other hand, if, as an example of providing data by streaming, the server provides data to a client which requests the data by unicast, a protocol is used, in which re-transmission of data, such as an arrival complete signal (ACK signal) of TCP/IP, is requested for achieving error-free transmission. However, this technique imposes significant load on the server, so that, in the current status of the art, services can be rendered to only hundreds of clients even if a high performance server is used. Even if a protocol, not employing the ACK, such as UDP/IP, is used, the number of clients, to whom services can be rendered, is not more than the order of thousands. Thus, if it is tried to provide data by streaming, server side costs are raised, such that the number of the clients is limited. Recently, such a system has been proposed in which FEC (Forward Error Correction) is used as a multi-cast technique and in which data is transmitted to plural clients without requesting re-transmission of data. This system is such a one in which a server repeatedly transmits a stream by multi-cast and in which a client picks up necessary signals form the stream and decodes and reproduces the so picked up data. In transmitting picture data of 500 motion pictures, each amounting to 2 Gbytes, within ten minutes, a transmission bandwidth of approximately 14.7 Gbits/sec is needed. If the same amount of the picture data is to be transmitted within one minute, the transmission bandwidth of approximately 147 Gbits/sec is needed. Although the above numerical values are theoretical values, the server that is able to withstand the capacity or the transmission system is extremely costly and, if such server is built, it is not practical. Although there is such a system in which data is distributed and recorded in a plural number of hosts, such a system is in need of increased processing for data management or data communication, because a huge amount of data has to be managed by plural servers. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 shows a structure of a distributed storage system according to the present invention. FIG. 2 shows an inner structure of a recording commanding apparatus. FIG. 3 shows a structure of a packet for storage of data for recording. FIG. 4 shows an inner structure of a node. FIG. 5 shows an inner structure of a transmission commanding apparatus. FIG. 6 shows a structure of a packet for requesting data transmission. FIG. 7 shows a structure of a packet responded from the node. FIGS. 8A and 8B show the process for generating a packet. FIG. 9 shows a transmission channel for a packet including data for recording. FIG. 10 shows a transmission channel for a packet requesting response of data. FIG. 11 shows a transmission channel for a packet responded from the node to the transmission commanding apparatus. FIGS. 12A and 12B show the process of restoring the packet transmitted from the node to original data. detailed-description description="Detailed Description" end="lead"? |
Production of soluble keratin derivaties |
A process for the preparation of soluble proteins of high molecular weight with little or no damage to the structural integrity of the proteins. The process is economically and environmentally acceptable by virtue of the cost of reagents that are used, and the recycling of some of those reagents, and is suitable for the production of soluble proteins on a large scale. The process includes a first stage using oxidative sulfitolysis followed by a second stage using mild conditions to extract the soluble protein. In the case of wool as the protein source the process leads to the production of soluble keratin proteins fractionated into the classes S-sulfonated keratin intermediate filament proteins and S-sulfonated keratin high sulfur proteins. |
1. A process for the preparation of keratin derivatives of high molecular weight, the process including a first stage digestion step of sulfonating a keratin source by oxidative sulfitolysis followed by a second stage repetitive aqueous extraction involving separation of soluble and insoluble keratin and subsequent re-extraction of the insoluble keratin to thereby produce a highly S-sulfonated keratin derivative. 2. A process according to claim 1 wherein the first stage is carried out without the use of a chaotropic agent. 3. A process according to claim 1 wherein the first stage is carried out under conditions of pH that maintains structural integrity of the protein. 4. A process according to claim 1 in which the oxidative sulfitolysis uses cuprammonium hydroxide, or a thionate, as the oxidant, and sulfite. 5. A process as according to claim 1 wherein the two stages use surfactants, heat, agitation and homogenization to control the rate of digestion in the first stage and extraction in the second stage. 6. A process as recited in claim 4 using surfactants, heat agitation and homogenization to control the rate of release of residual reagents and soluble protein, thereby allowing separation of the highly S-sulfonated keratin derivative. 7. A process for the separation of a gelatinous keratin substrate from the solution of S-sulfonated keratin produced by the process as recited in claim 1 wherein the S-sulfonated keratin derivative solution is treated by the use of a gentle, gravity fed filtration system followed by centrifugal separation. 8. A process as recited claim 1 that uses a combination of engineering solutions to allow continuous preparation of S-sulfonated keratin derivatives. 9. A process as recited in claim 1 wherein the solution of S-sulfonated keratin is purified using chelating agents, such as EDTA, to sequester metal ions, such as copper. 10. A process as recited in claim 1 wherein the solution containing S-sulfonated keratin derivatives is purified using ion exchange media to remove residual reagents, including metals such as copper. 11. A process as recited in claim 10 wherein the solution is concentrated prior to an ion exchange treatment through the use of ultrafiltration membranes, or similar. 12. A process as recited in claim 11 in which reagents such as copper are isolated and reused in subsequent processes. 13. A highly S-sulfonated solution of keratin derivatives produced by the process of claim 1. 14. A process or isolation of highly S-sulfonated keratin intermediate filament proteins from the solution of keratin claimed in claim 13 wherein the highly S-sulfonated keratin intermediate filament protein is isolated by isoelectric precipitation at acidic pH. 15. A highly purified proteinaceous product produced by the process of claim 14. 16. A proteinacious product as recited in claim 15 which is purified by the dissolution of the S-sulfonated keratin intermediate filament proteins in base in the presence of EDTA, and subsequently precipitated at acidic pH. 17. A water soluble form of S-sulfonated keratin intermediate filament protein produced by spray drying an aqueous solution of the polymeric product as recited in claim 15. 18. A process for the production of highly S-sulfonated keratin high sulphur proteins, produced by spray drying the solution of S-sulfonated keratin derivatives purified as claimed in claim 9, and after having had the intermediate filament proteins removed. 19. A proteinaceous product produced by the process of claim 18. 20. A process for the production of soluble keratin peptides through the action of hydrogen peroxide solutions on the gelatinous residue produced by the process as recited in claim 1. 21. A proteinaceous product produced by the process recited in claim 20. 22. A process for the production of soluble keratin peptides through the action of sodium sulfide solution on the gelatinous residue produced by the process as recited in claim 1. 23. A proteinaceous product produced by the process recited in claim 22. 24. A process for the production of soluble keratin peptides through the action of proteolytic enzymes, such as those from the subtilisin, papain or trypsin families, on the gelatinous residue produced by the process as recited in claim 1. 25. The proteinaceous product produced by the process recited in claim 24. 26. A process for the treatment of copper-rich solutions produced by the process as recited in claim 4, in which wool is used as a filter media to bind copper and remove it from the solution. 27. A subsequent protein extraction process for the treatment of the copper-laden wool produced by the process recited in claim 26. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Keratins are a class of structural proteins widely represented in biological structures, especially in epithelial tissues of higher vertebrates. Keratins may be divided into two major classes, the soft keratins (occurring in skin and a few other tissues) and the hard keratins, forming the material of nails, claws, hair, horn, feathers and scales. The toughness and insolubility of hard keratins, which allow them to perform a fundamental structural role in many biological systems, are also desirable characteristics in many of the industrial and consumer materials currently derived from synthetic polymers. In addition to possessing excellent physical properties, keratin, as a protein, is a material with a high degree of chemical functionality and, consequently, exhibits many properties that synthetic materials cannot achieve. Keratin is, therefore, well suited to the development of products with high-value, niche-market applications. Keratin is also an environmentally acceptable polymer produced from a sustainable resource and therefore has environmental benefits over synthetic materials. Following the global trend of developing materials from renewable sources produced in a sustainable process, a range of materials has been produced from keratin, most commonly in the form of keratin films. At the core of a new industry producing biopolymer materials from keratin it is essential to have a process for extracting keratin from its source that is economically viable, sustainable from an environmental perspective, and produces a stable and versatile product. Methods used to date for the extraction of keratin that maintain the integrity of the individual proteins have been designed for the purpose of protein analysis and characterisation and consequently are not viable on an industrial scale, from an economic and environmental viewpoint. Methods used to date for the economic dissolution of keratin have significantly degrading effects on the protein, and consequently the dissolved protein retains few of the physicochemical properties that lead to the desirability of keratin as a biopolymer, such as the ability to reconstitute into tough materials. It is an object of the invention to go some way in overcoming the disadvantages with known processes or at least provide the public with a useful choice. In at least one embodiment the invention strives to provide an economic and environmentally acceptable process for the dissolution of keratin proteins that maintains the structural integrity and chemical functionality of the proteins during the dissolution process and leads to a stable and versatile keratin derivative product for the development of biopolymer materials. |
<SOH> SUMMARY OF THE INVENTION <EOH>According to a first aspect the invention provides a dissolution process for producing a range of stable, soluble keratin derivatives of high molecular weight, the molecular weight being similar to or greater than that of proteins originally expressed in the keratin source, with little or no damage to the structural integrity of the constituent proteins. The dissolution occurs in a two-stage process. According to a preferred aspect the invention provides a process for the preparation of keratin derivatives of high molecular weight, the process including a first stage digestion step of S-sulfonating a keratin source by oxidative sulfitolysis followed by a second stage extraction step using controlled washing with water to thereby obtain a highly S-sulfonated keratin derivative. The conversion of highly S-sulfonated keratin from a solid state into solution is without the use of chaotropic agents, by controlled, gradual washing of the sulfonated keratin with water in order to wash out the residual chemical reagents from the extraction procedure and alter the ionic strength of the extraction solution. The first stage involves oxidative sulfitolysis to convert cystine groups present in the protein to S-sulfocysteine, using industrially acceptable concentrations of inexpensive reagents for the purpose of sulfonation (eg. sodium sulfite) and oxidation (eg. cupraammonium hydroxide). According to another aspect, the invention provides a process for the separation of a gelatinous keratin product from a solution of S-sulfonated keratin produced by the above process, wherein the S-sulfonated keratin derivative solution is treated by the use of a gentle, gravity fed filtration system followed by separation. Preferably the separation is centrifugal. According to another aspect of the invention, a liquid stream remaining after the gelatinous keratin is removed is processed by passing over scoured wool, thereby removing residual chemicals from the solution and preparing the wool for subsequent protein extraction processes. Following conversion of the cystine groups, the second stage of the process is one in which the highly S-sulfonted keratin derivative is brought from a solid or gelatinous state into solution by extensive dilution with water. The rate and extent of dissolution can be controlled by the use of heat, surfactants, gentle agitation and vigorous chopping or homogenisation. By controlling the rate of dissolution, reaction solutions can be isolated, for example if a copper oxidant is used a reaction solution rich in copper is produced but it contains little or no dissolved protein, or are rich in protein but contain little or no copper. According to another aspect of the invention, a liquid stream resulting from the second stage of the process, which contains residual chemicals such as copper sulfate and sulfite, as well as S-sulfonated keratin derivatives, is processed using any one or more of a variety of methods that allow the recycling of reagents from the solution and the separate isolation of purified S-sulfonated Keratin Intermediate Filament Protein(s) (SIFP) and S-sulfonated Keratin High Sulfur Protein(s) (SHSP). This is achieved through the use of chelating agents, such as ethylenediamine tetraacetic acid, or chelating ion exchange resins, such as those containing the iminodiacetic functional group, and the use of isoelectric precipitation to separate protein types. Ultrafiltration can be used at several stages in the process to improve the efficiency of reagent removal or protein separation. Metallic impurities in the protein products can be further reduced by the washing of the protein derivative(s) following precipitation with dilute acids, water or chelating agents. Once separated, the protein products can be dried by a range of methods such as fluid bed, spray or freeze drying. Another aspect of the invention is the further processing of residual keratin not dissolved by the two stage sulfitolysis process, through the use of other reagents, such as hydrogen peroxide, sodium sulfide or proteolytic enzymes, to produce keratin peptides. Another aspect of the invention is the provision of a method for large scale recovery of proteins from a natural source, including subjecting said natural protein source to a treatment sufficient to render at least some of the protein(s) water soluble, and subsequently separating the water soluble protein(s). Another aspect of the invention is the provision of an installation for large scale recovery of proteins from a natural source, a treatment vessel to contain and subject a large quantity of natural protein source to a treatment sufficient to render at least some of the protein(s) contained in said feed, water soluble, and a separation apparatus to subsequently separate the water soluble protein(s). Another aspect of the invention is a method of selectively solubilising a protein having plurality of disulfide bonds from a mixture of proteins including subjecting said mixture of proteins to oxidative sulfitolysis to produce a soluble S-sulfonated protein fraction. The oxidative sulfitolysis is preferably effected in the absence of chaotropic agents with little or no damage to the structural integrity of the protein. Another aspect of the invention is method for obtaining a purified protein from an impure protein source with little or no damage to the structural integrity of the protein including subjecting said protein source to a treatment sufficient to render at least some of the protein(s) water soluble, and subsequently separating the water soluble protein(s) in the absence of chaotropic agents. detailed-description description="Detailed Description" end="lead"? |
Dry products comprising a sheet and two phases |
This invention concerns products for cleansing and other applications, which products comprise a sheet of absorbent material, in particular of a non-woven material, such as a wipe, to which a lipid and aqueous phase have been applied and which products have been dried afterwards. The invention further concerns the manufacture and use of such products. |
1. A product comprising a porous or absorbent sheet whereto an aqueous and a lipid phase have been applied and which has been dried. 2. A product according to claim 1 wherein the melting point or melting range of the lipid phase is above or equal to 25° C. 3. A product according to claim 2 wherein the melting point or melting range of the lipid phase is in the range of 32 to 40° C. 4. A product according to claim 1 wherein the lipid phase comprises mono-, di- or triglcerides. 5. A product according to claim 4 wherein the lipid phase comprises mono-, d-, or triglycerides derived from or present in natural oils. 6. A product according to claim 4 wherein the lipid phase comprises fatty acid mono-, di- or triglycerides wherein the fatty acids contain from 12 to 24, preferably from 16 to 20 carbon atoms. 7. A product according to claim 4 wherein the lipid phase comprises triglycerides selected from glyceryl stearate, glyceryl oleate, glyceryl laurate, glyceryl myristate, cocoglycerides, or hydrogenated palm oil glycerides, hydrogenated castor oil, or hydrogenated rapeseed oil. 8. A product according to claim 4 wherein the lipid phase comprises mono, di- or triglycerides in an amount of at least 50%, preferably at least 70%, more preferably at least 90%, w/w of the total amount of components making up the lipid phase. 9. A product according to claim 1 wherein the lipid phase contains fatty alcohols. 10. A product according to claim 9 wherein the lipid phase contains C12-C50-fatty alcohols, in particular the C12-C24-fatty alcohols. 11. A product according to claim 10 wherein the fatty alcohols are selected from myristyl alcohol, 1-pentadecanol, cetyl alcohol, lauryl alcohol, oleyl alcohol, palmityl alcohol, 1-heptadecanol, stearyl alcohol, cetearyl alcohol, 1-nonadecanol, arachidyl alcohol, 1-heneicosanol, behenyl alcohol, brassidyl alcohol, lignoceryl alcohol, ceryl alcohol or myricyl alcohol and C16/C18-Guerbet alcohols. 12. A product according to claim 9 wherein the fatty alcohols are present in the lipid phase, in an amount relative to the total weight amount of the lipid phase, which is in the range of 1-40%, preferably 1-30% (w/w), more preferably of 1-20% (w/w), still more preferably from 1-10% (w/w). 13. A product according to claim 1 wherein the lipid phase contains fatty acids. 14. A product according to claim 13 wherein the fatty acids are C14-C40-fatty acids or in particular are C16-C30-fatty acids. 15. A product according to claim 13 wherein the fatty acids are selected from myristic-, pentadecanoic-, palmitic-, margaric-, stearic-, nonadecanoic-, arachic-, behenic-, lignoceric-, cerotic-, melissic-, erucaic-, elaeostearic, oleic, linoleic, lauric acid and hydroxy-substituted fatty acids. 16. A product according to claim 13 to wherein the total amount of the fatty acids present in the lipid phase, relative to the total weight amount of the lipid phase, is in the range of 1-30% (w/w), preferably of 1-20% (w/w), more preferably from 1-10% (w/w). 17. A product according to claim 1 wherein the lipid phase contains one or more of components (a), (b), (c), (d), (e) or (f) as defined hereafter: (a) at least 1-50% (w/w), in particular at least 1-10% of an oily or waxy component (b) 0,1-5% (w/w) of at least one active ingredient (c) 1-10% (w/w) of at least one oil (d) 0.1-10% (w/w) of at least one emulsifier (e) 5-90% (w/w) of further waxy components (f) 0-5% (w/w/) water. 18. A product according to claim 17 wherein the lipid phase contains all components (a)-(f). 19. A product according to claim 17 wherein component (a) is an oily or waxy component selected from C14-C30-dialkyl ethers, C14-C30-dialkyl carbonates, C4-C34-dicarbonic acids or C12-C30-hydroxyfatty alcohols or mixtures thereof. 20. A product according to claim 1 wherein the lipid phase comprises dialkyl(ene) ethers or -carbonates, dicarboxylic acids or hydroxy fatty alcohols, or a combination thereof. 21. A product according to claim 1 wherein the lipid or the aqueous phase contains one or more active substances. 22. A product according to claim 21 wherein the active substance(s) is or are anti-microbials, e.g. anti-bacterials and antifungals, anti-inflammatory agents, anti-irritating, anti-itching, anti-perspirant agents. 23. A product according to claims 1 wherein the lipid or the aqueous phase contains at least one moisturizer, deodorant, skin caring ingredient, plant extract, vitamin, perfume oil, dye, sunscreen filter, hydrotrope or self-tanning agent. 24. A product according to claim 1 wherein the lipid or the aqueous phase contains at least one emulsifier. 25. A product according to claim 1 wherein the lipid phase contains at least one superfatting agent, thickener, cationic polymer, anionic polymer, zwitterionic polymer, amphoteric polymer, consistency agent, anti-oxidant. 26. A product according to claims 1 wherein the lipid or the aqueous phase contains an insect repellent, a sunscreen filter, a powder or a peeling agent. 27. A product according to claim 1 which is a wipe. 28. A product according to claim 27 wherein the wipe is a non-woven wipe. 29. A method of manufacturing a product as claimed in claim 1 said method comprising contacting the sheet with a lipid phase and with an aqueous phase, either subsequently or simultaneously, and further comprising an additional drying step. 30. A method according to claim 29 wherein a lipid phase having a melting point or a melting range of above room temperature is first applied to the surface of the sheet and subsequently the aqueous phase is applied. 31. A method according to claim 29 wherein the aqueous phase is applied by spraying, drippling, immersing or running through a bath, and the lipid phase is applied by spraying, contacting, printing or a direct contact process where there is a direct contact between the sheet and an application head having slit nozzles. 32. A method according to claim 31 wherein the drying step takes place after the aqueous phase has been applied. 33. A method according to claim 29 wherein the drying step comprises the application of hot air, or by leading the wet sheet through an oven or over an heated or warmed transport roll. 34. Use of a product as claimed in claim 1 as a combined cleanser and applicator of active substances. 35. A product comprising a porous or absorbent sheet whereto a waxy lipid phase has been applied wherein (a) the lipid phase comprises one or more C12-24 fatty acid triglycerides, C12-C50-fatty alcohols, or at least one oil or wax component selected from dialkyl(ene) ethers, dialkyl(ene) carbonates, dicarboxylic acids or hydroxy fatty alcohols, or mixtures thereof; (b) the lipid phase has a water content of less than 10%; and 36. A product according to claim 35 wherein the lipid phase comprises one or more C16-20 fatty acid triglycerides and the total amount of triglyceride(s) in the lipid phase is at least 50%, preferably at least 70%, more preferably at least 90%, w/w of the total amount of components making up the lipid phase. 37. A product according to claim 36 wherein the triglycerides are selected from glyceryl stearate, glyceryl oleate, glyceryl laurate, glyceryl myristate, cocoglycerides, or glycerides present in hydrogenated palm oil, hydrogenated rapeseed oil or hydrogenated castor oil. 38. A product according to claim 35 wherein the lipid phase contains C12-C24-fatty alcohols, that are derived from natural fats, oils or waxes and the total amount of the-fatty alcohols present in the lipid phase, relative to the total weight amount of the lipid phase, is in the range of 1-30% (w/w), preferably of 1-20% (w/w) more preferably from 1-10% (w/w). 39. A product according to claim 38 wherein the fatty alcohols are selected from myristyl alcohol, 1-pentadecanol, cetyl alcohol, lauryl alcohol, oleyl alcohol, palmityl alcohol, 1-heptadecanol, stearyl alcohol, cetearyl alcohol, 1-nonadecanol, arachidyl alcohol, 1-heneicosanol, behenyl alcohol, brassidyl alcohol, lignoceryl alcohol, ceryl alcohol or myricyl alcohol and Guerbet alcohols. 40. A product according to claim 35 wherein the lipid phase is a waxy composition containing or at least one oil or wax component selected from dialkyl(ene) ethers, dialkyl(ene) carbonates, dicarboxylic acids or hydroxy fatty alcohols, or mixtures thereof. 41. A product according to claim 35 wherein the lipid phase additionally contains an active ingredient. 42. A product according to claim 35 wherein the lipid phase contains (a) at least 1-50% (w/w), in particular at least 1-10% of an oily or waxy component selected from C14-C30-dialkyl ethers, C14-C30-dialkyl carbonates, C4-C34-dicarbonic acids or C12-C30-hydroxyfatty alcohols or mixtures thereof (b) 0,1-5% (w/w) of at least one active ingredient (c) 1-10% (w/w) of at least one oil (d) 0.1-10% (w/w) of at least one emulsifier (e) 5-90% (w/w) of further waxy components (f) 0-5% (w/w) water. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Wipe products have become an important product category that has found a wide variety of applications for adults and babies. Examples include face or body cleansing wipes, wipes for skin treatment, and skin conditioning wipes. Over the last couple of decades so-called wet wipes have become successful as products particularly suited for these applications. These products typically are manufactured by impregnating sheets made of non-woven fabric with a suitable lotion. Recent innovations in the wipes area included improvements in the fabric, in the impregnating liquid as well as in product presentation. Initially, wet wipe products were made of traditional non-woven materials based on paper making technology (pulp based products). These products were well accepted but deficient in softness of the fabric material. The introduction of the ‘spunlace’ non-woven technology offered products that, compared to traditional paper based products, were superior in terms of softness. This is mainly due to (i) the use of long soft fibres (most frequently rayon and PET/PP or a mixture of these fibres) in the spunlace process and (ii) the fact that during the spunlace process no binder is added to the fabric. Another innovation was the introduction of the so-called ‘Pop-up’ technology that offered advantages as regards the dispensing of individual wipes. In addition to the above, lotions have been developed which offered skincare benefits in addition to the basic cleansing properties of the wipe. One approach was the introduction of lotions that were based on oil-in-water emulsions which delivered useful properties such as superior mildness, moisturisation, protection and skin smoothness when compared to simple aqueous cleansing formulations. Another approach encompassed the incorporation of active skincare ingredients, e.g. chamomile, into simple aqueous formulations, thereby delivering useful properties such as soothing. Current wet wipe products are still based on these approaches in that they are impregnated with either aqueous lotions or with oil in water emulsions. However, these approaches have several limitations. Firstly, only a small portion of the lotion (usually about 15%) is released from the wipes during use. Thus a large quantity of the relatively expensive lotion is not delivered to the skin providing no benefit to the consumer and is wasted when the product is discharged after use. This also prevents the use of expensive but more effective ingredients. Secondly, from a formulation point there is an apparent contradiction in the optimization of cleansing performance and skincare benefits in one single lotion, since ingredients which are effective in cleansing usually are not compatible with efficient skin care agents. Another important factor in cleansing is the fact that a number of soils are water-compatible and therefore more easily removed by water-based formulations, whereas others are lipid-compatible and therefore adequately removed by lipid or oil based formulations. A complete and effective removal of soils therefore requires the presence in or on a wipe of as well water and oil-based components. This is in particularly required in wipes for personal cleansing and in particular in wipes used for babies and infants. In the latter instance wet wipes are used for cleansing the perianal region when changing diapers. Inadequate cleaning not only results in personal discomfort but also gives rise to diaper rash and other infection related phenomena. It has been shown that the most effective way of preventing diaper rash is to cleanse the skin thoroughly and to remove the microorganisms that have been identified as causative. The source of these microorganisms is often the fecal deposits that can remain on a baby's skin while wearing the diaper. Because fecal deposits consist of both water-soluble and oil-soluble matter, however, complete removal of fecal deposits from the diaper area requires both water-based and oil-based cleansing agents. Thus, it is an object of this invention to provide a mechanism for cleansing babies' skin in order both to remove waste deposits and to reduce the number of microorganisms available to cause infection. WO 96/14835 discloses dry tissues to which a water-in-lipid emulsion has been applied and WO 99/25923 concerns a process and an apparatus for selectively coating a wipe with a water-in-lipid emulsion. WO 99/01536 discloses wipes wherein the carrier comprises two regions of different basis weight being applied with an emulsion comprised of a solidified lipid phase, a polar phase dispersed therein and an emulsifier. Other prior art in this field is WO 95/35411, WO 95/35412, WO 95/16824, WO 97/30216, DE 33 09 530 and the publication of R. E. Mathis in Nonwovens World 1999, pp. 59-65. It is an object of this invention to offer a cleansing article and in particular a wettable wipe product that allows to independently optimize the cleansing and skincare attributes of the product and at the same time improves the delivery of skincare actives onto the skin during use. It is a further object of this invention to provide products that have an improved release of the active ingredient(s) onto the skin during use. It is still a further object of the present invention to provide a product for use as a cleansing tool that effectively and completely removes oil and water compatible soils. Another object of this invention is to provide products for cleansing and other applications that allow convenient and quick application, are easy to carry, as well as an easier and more evenly distribution of the ingredients in or on the product. They moreover should be convenient for application on babies and children. These objects are attained by the products according to the present invention, which comprise a sheet that contains an aqueous and lipid phase, which product is dried. Whereas traditional wet wipes have been based on impregnation of a fabric with one phase, the products of this invention concern the application of two distinctly different phases onto a sheet. Both phases differ in terms of physical properties and may be applied on various parts or portions of the sheet. This approach allows combined optimal cleansing performance and superior skincare properties. |
<SOH> SUMMARY OF THE INVENTION <EOH>This invention relates to products that comprise a porous or absorbent sheet whereto a lipid and an aqueous phase have been applied and which products have been dried subsequently. In a different aspect, this invention relates to products that comprise a porous or absorbent sheet whereto a lipid and an aqueous phase have been applied and which products are dry or essentially dry. Dry refers to the situation where the water content is very low, i.e. lower than 1% and essentially dry means that the product contains limited amounts of water, e.g. less than 10% of the total weight of the product, preferably less than 8%, more preferably less than 5%, still more preferably less than 2%. In one embodiment the invention concerns a product comprising a porous or absorbent sheet to which an aqueous phase has been applied, after which the product is dried, and to which subsequently a lipid phase is applied. In another embodiment the invention concerns a product comprising a porous or absorbent sheet to which a lipid phase and an aqueous phase have been applied, after which the product is dried. A particular subtype of this embodiment concerns a product comprising a porous or absorbent sheet to which an aqueous phase has been applied and to which subsequently a lipid phase is applied, whereafter the product is dried. Another subtype of this embodiment concerns a product as defined herein comprising a porous or absorbent sheet to which a lipid phase has been applied and to which subsequently an aqueous phase is applied, whereafter the product is dried. Preferably, the lipid phase is solid or semi-solid at ambient temperature and preferably is present at the surface or at the surface portion of one or both sides of the sheet. In particular said sheet is made of a non-woven material, more in particular a non-woven material made by the spunlace or the hydro-entanglement procedure. In a further aspect, this invention relates to products that comprise a porous or absorbent sheet whereto a waxy lipid phase has been applied. Or, in a different aspect, this invention relates to products that comprise a porous or absorbent sheet whereto a waxy lipid phase has been applied and which products are dry or essentially dry. Dry refers to the situation where the water content is very low, i.e. lower than 1% and essentially dry means that the product contains limited amounts of water, e.g. less than 10% of the total weight of the product, preferably less than 8%, more preferably less than 5%, still more preferably less than 2%. The waxy lipid phase in particular comprises one or more triglycerides, more in particular a C 12-24 fatty acid triglycerides, or further in particular a C 16-20 fatty acid triglyceride; or the lipid phase contains C 12 -C 50 -fatty alcohols, in particular C 12 -C 24 -fatty alcohols; or the lipid phase comprises a component selected from dialkyl(ene) ethers, dialkyl(ene) carbonates, dicarboxylic acids or hydroxy fatty alcohols; or mixtures thereof. The lipid phase preferably has a low water content, in particular lower than 10%. The lipid phase preferably contains an active ingredient. In a preferred embodiment, the lipid phase is present at the surface or at the surface portion of one or both sides of the sheet. In another aspect there is provided a method of manufacturing a product as described herein, said method comprising applying to the sheet a lipid phase and an aqueous phase, either subsequently or simultaneously. In a preferred method of manufacturing, said sheet is first coated with a lipid phase which is of oily nature and subsequently sprayed or impregnated with an aqueous phase which is of aqueous nature. In still a further aspect there is provided the use of a product as described herein as a cleansing tool, in particular in personal care applications. In another aspect the invention concerns the use of a product as described herein as an applicator of active substances. In still another aspect the invention provides the use of a product as described herein as a combined cleanser and applicator of active substances. detailed-description description="Detailed Description" end="lead"? |
Modulators of ceramidase and methods of used based thereon |
The present invention relates to compounds which can be used as inhibitors of mitochondrial ceramidase, in particular human mitochondrial ceramidase. The invention also relates to methods of designing and making the compounds, as well as methods screening for compounds that inhibit mitochondrial ceramidase. The invention also relates to the use of the compounds as a regulator of the level of ceramide by inhibiting ceramidase activity. The invention also relates to methods for the prevention and treatment of diseases associated with cell overproliferation and sphingolipid signal transduction including cancer, cardiovascular diseases, and inflammation. |
1. A compound having the formula: wherein A is —CH2CH2—, trans —CH═CH—, or —C≡C—; B is —CH2—, —CH(OH)—, —CH(alkyl)—, —C(═O)—, —C(═NOH)—, or —C(═N—NH2)—; X is O, S, or NH; Y is NH, O, C═O, CHR4, CH2C═O, or CH2CHR4; R1 is CH3, CH2OH, CH2SH, CH2—NH2, CH2N3, CH2—NH—OH, CH═N—OH, CH═N—NH2, C(═O)H, C(═O)CH3, C(═O)CF3, C(═O)NH2, CH2R5, C(OH)R5, or C(═O)R5; R3 is H, CH3, OH, NH2, NH2.HCl, NHC(═O)NH(CH2)LN(CH3)2, NHCONH(CH2)LN(CH3)2.HCl or C(═O)R5; R4 is H, OH, OR5, or C(═O)R5; R5 is a five-membered monocyclic heterocycle; six-membered monocyclic heterocycle; five- and five-membered bicyclic heterocycle; six- and six-membered bicyclic heterocycle; five- and six-membered bicyclic heterocycle; five-, five-, and five-membered tricylic heterocycle; six-, six-, and six-membered tricylic heterocycle; five-, five-, and six-membered tricylic heterocycle; five-, six-, and six-membered tricylic heterocycle; six-, five-, and six-membered tricylic heterocycle; five-, six-, and five-membered tricylic heterocycle; R6-phenyl; or R6-condensed aromatic ring system; R6 is H, Cl, OH, CH3, NO2, NH2, N(CH3)2, or NHC(═O)(CH2)kCH3; L is an integer from 2 to10; k is an integer from 5 to16; n is an integer from 4 to 20; m is an integer from 0 to 26; with the proviso that the compound is not (i) (2S, 3R, 4E), (2S, 3R, 4E), (2R, 3R, 4E), or (2R, 3R, 4E) ceramide wherein R1 is CH2OH; B is —CH(OH)—; X is O; Y is CHR4; A is trans —CH═CH—; R3 is CH3, H, or OH; R4 is H or OH; n is 12; and m is an integer from 0 to 24; and (ii) (2S, 3R, 4E) 3-keto ceramide wherein R1 is CH2OH; B is —C(═O)—; X is O; Y is CHR4; A is trans —CH═CH—; R3 is CH3 or OH; R4 is H or OH; n is 12; and m is 13. 2. A compound having the formula: wherein A is —CH2CH2—, trans —CH═CH—, or —C≡C—; B is —CH2—, —CH(OH, —CH(alkyl)—, —C(═O)—, —C(═NOH)—, or —C(═N—NH2)—; R1 is CH3, CH2OH, CH2SH, CH2—NH2, CH2N3, CH2—NH—OH, CH═N—OH, CH═N—NH2, C(═O)H, C(═O)CH3, C(═O)CF3, C(═O)NH2, CH2R5, C(OH)R5, or C(═O)R5; R3 is H, CH3, OH, NH2, NH2.HCl, NHC(═O)NH(CH2)LN(CH3)2, NHCONH(CH2)LN(CH3)2.HCl or C(═O)R5; R5 is a five-membered monocyclic heterocycle; six-membered monocyclic heterocycle; five- and five-membered bicyclic heterocycle; six- and six-membered bicyclic heterocycle; five- and six-membered bicyclic heterocycle; five-, five-, and five-membered tricylic heterocycle; six-, six-, and six-membered tricylic heterocycle; five-, five-, and six-membered tricylic heterocycle; five-, six-, and six-membered tricylic heterocycle; six-, five-, and six-membered tricylic heterocycle; five-, six-, and five-membered tricylic heterocycle; R6-phenyl; or Rr-condensed aromatic ring system; R6 is H, Cl, OH, CH3, NO2, NH2, N(CH3)2, or NHC(═O)(CH2)kCH3; L is an integer from 2 to10; k is an integer from 5 to16; n is an integer from 4 to 20; m is an integer from 0 to 26; with the proviso that the compound is not (i) (2S, 3R, 4E), (2S, 3R, 4E), (2R, 3R, 4E), or (2R, 3R, 4E) sphingosine wherein R1 is CH2OH; B is —CH(OH)—; A is trans —CH═CH—; R3 is H; n is 12; and m is 0; (ii) (2S, 3R, 4E)-N-methyl-sphingosine wherein R1 is CH2OH; B is —CH(OH)—; A is trans —CH═CH—; R3 is CH3; n is 12; and m is 0; and (iii) (2S, 3R, 4E) 1-O-methyl-sphinogosine wherein R1 is CH2OH; B is —CH(OH)—; A is trans —CH═CH—; R3 is H; n is 12; and m is 0. 3. A compound selected from the group consisting of D-erythro-C16-urea-ceramide, cis-D-erythro-sphingosine, D-erythro-dihydrosphingosine, 1-O-methyl-D-erythro-sphingosine, 3-keto-sphingosine, 3-keto-dihydrosphingosine, 3-keto-dehydrosphingosine, N-stearyl-D-erythro-sphingosine, N,N-dimethyl-sphingosine, C16-urea-ceramide, and C6-urea-ceramide. 4-12. (Canceled) 13. A compound having the formula of wherein A is —CH2CH2—, trans —CH═CH—, or —C≡C—; B is —CH2—, —CH(OH)—, —CH(alkyl)—, —C(═O)—, —C(═NOH)—, or —C(═N—NH2)—; X is O, S, or NH; Y is NH, O, C═O, CHR4, CH2C═O, or CH2CHR4; R1 is a phosphate group; R3 is H, CH3, OH, NH2, NH2.HCl, NHC(═O)NH(CH2)LN(CH3)2, NHCONH(CH2)LN(CH3)2.HCl or C(═O)R5; R4 is H, OH, OR5, or C(═O)R5; R5 is a five-membered monocyclic heterocycle; six-membered monocyclic heterocycle; five- and five-membered bicyclic heterocycle; six- and six-membered bicyclic heterocycle; five- and six-membered bicyclic heterocycle; five-, five-, and five-membered tricylic heterocycle; six-, six-, and six-membered tricylic heterocycle; five-, five-, and six-membered tricylic heterocycle; five-, six-, and six-membered tricylic heterocycle; six-, five-, and six-membered tricylic heterocycle; five-, six-, and five-membered tricylic heterocycle; R6-phenyl; or R6-condensed aromatic ring system; R6 is H, Cl, OH, CH3, NO2, NH2, N(CH3)2, or NHC(═O)(CH2)kCH3; L is an integer from 2 to10; k is an integer from 5 to16; n is an integer from 4 to 20; and m is an integer from 0 to 26; with the proviso that the compound is not (2S, 3R, 4E) ceramide wherein R1 is CH2OH; B is —CH(OH)—; X is O; Y is CHR4; A is trans —CH═CH—; R3 is CH3, H, or OH; R4 is H or OH; n is an integer from 12 to16; and m is an integer from 0 to 24. 14-16. (Canceled) 17. A method for increasing mitochondrial ceramidase activity comprising contacting a composition comprising mitochondrial ceramidase activity with the compound of claim 13. 18. (Canceled) 19. A method for treating or preventing a disorder in a subject characterized by deficient cell proliferation or growth, or in which cell proliferation is desired, said method comprising administering the compound of claim 13 to the subject. 20. (Canceled) 21. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the compound of claim 13. 22-36. (Canceled) 37. A method for increasing mitochondrial ceramidase activity comprising contacting a composition comprising mitochondrial ceramidase activity with the compound of claim 1. 38. A method for treating or preventing a disorder in a subject characterized by deficient cell proliferation or growth, or in which cell proliferation is desired, said method comprising administering the compound of claim 1 to the subject. 39. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the compound of claim 1. 40. A method for increasing mitochondrial ceramidase activity comprising contacting a composition comprising mitochondrial ceramidase activity with the compound of claim 2. 41. A method for treating or preventing a disorder in a subject characterized by deficient cell proliferation or growth, or in which cell proliferation is desired, said method comprising administering the compound of claim 2 to the subject. 42. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the compound of claim 2. 43. A method for increasing mitochondrial ceramidase activity comprising contacting a composition comprising mitochondrial ceramidase activity with the compound of claim 3. 44. A method for treating or preventing a disorder in a subject characterized by deficient cell proliferation or growth, or in which cell proliferation is desired, said method comprising administering the compound of claim 3 to the subject. 45. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the compound of claim 3. |
<SOH> 2. BACKGROUND <EOH>Ceramide is a potent signal transducer that affects cell growth, differentiation and death (Hannun, Y. A. (1996) Science 274, 1855-1859; Obeid, L. M., Linardic, C. M., Karolak, L. A., and Hannun, Y. A. (1993) Science 259, 1769-1771; Perry, D. K. and Hannun, Y. A., (1998) Biochim Biophys Acta 436, 233-243). It occupies a central position in sphingolipid metabolism. As an acceptor of carbohydrates, phosphorylcholine and phosphate, it serves as precursor of the various complex sphingolipids. Alternatively, the enzymatic breakdown of these sphingolipids releases ceramide which may consequently be hydrolyzed into fatty acid and sphingosine; the latter exerting bioeffector functions on its own as well as acting as a precursor of sphingosine phosphate, another signal mediator and regulator of various cell functions. A controlled level of ceramide, therefore, reflects an intricate balance between the catabolic and anabolic pathways of ceramide. Multiple enzymes are directly involved in regulating intracellular ceramide concentration. These include ceramide-generating enzymes such as ceramide synthase, cerebrosidase, sphingomyelinase and ceramide-consuming enzymes such as cerebroside synthase, sphingomyelin synthase, ceramide kinase and ceramidase (Luberto, C. and Hannun, Y. A., (1999) Lipids 34, 5-11). Ceramidases are enzymes that hydrolyze ceramides at the amide bond linking the sphingosine moiety to the fatty acids. In that sense they provide a target site for regulating ceramide-sphingosine inter-conversion (Hassler, D. F. and Bell, R. M., (1993) Adv. Lipid Res. 49-57). At least three different types of ceramidases have been reported. A lysosomal acid ceramidase, the defect of which underlies the human disorder Farber's disease (Sugita, M., Dulaney, J. T. and Moser, H. W., (1972) Science 178, 1100-1102), was purified from human urine (Koch, J., Gartner, S. Li. C-M., Quinten, L. E., Bernardo, K., Levarn, O., Schnabel, D., Desnick, R. J., Schuchman, E. H., and Sandhoff, K., (1996) J. Biol. Chem. 271, 33110-33115), and the cDNA encoding the enzyme was also cloned from mouse brain and human fibroblasts (Koch, J., Gartner, S. Li. C-M., Quinten, L. E., Bernardo, K., Levarn, O., Schnabel, D., Desnick, R. J., Schuchman, E. H., and Sandhoff, K., (1996) J. Biol. Chem. 271, 33110-33115; Li, C.-M., Hong, S. B., Kopal, G., He, X., Linke, T., Hou, W. S., Koch, J., Gatt, S., Sandhoff, K., and Shuchman, E. H., (1998) Genomics 50, 267-274). Alkaline ceramidases, CDase-I and CDase-II, were purified from guinea pig skin (Yada, Y., Higuch, K., and Imokawa, G., (1995) J. Biol. Chem. 270, 12677-12684) and from gram-negative bacterium Pseudomonas aeruginosa (Okino, N., Tani, M., Imayama, S., and Ito, M., (1998) J. Biol. Chem. 273, 14368-14373; Okino, N., Ichinose, S., Omori, A., Imayama, S., Nakamura, T., and Ito, M., (1999) J. Biol. Chem. 274, 36616-36622). A non-lysosomal, ceramidase with a neutral to alkaline pH optimum was also purified to homogeneity from rat brain (El-Bawab, S., Bielawska, A., and Hannun, Y. A., (1999) J. Biol. Chem. 274, 27948-27955) and cloned from mouse (Tani, M., Okino, N., Mori, K., Tanigawa, T., Izu, H., and Ito, M., (2000) J. Biol. Chem. 235, 11229-11234) and human (El-Bawab, S., Roddy, P., Qian, T., Bielawska, A., Lemasters, J. J., and Hannun, Y. A., (2000) J. Biol. Chem. 275, 21508-21513). The human form was found to localize to mitochondria (El-Bawab, S., Roddy, P., Qian, T., Bielawska, A., Lemasters, J. J., and Hannun, Y. A., (2000) J. Biol. Chem. 275, 21508-21513). Citation of references hereinabove shall not be construed as an admission that such references are prior art to the present invention. |
<SOH> 3. SUMMARY OF THE INVENTION <EOH>The present invention provides mitochondrial ceramidase inhibitors. The invention is based on the observation that the interaction of mitochondrial ceramidase with its ligand occurs in a high affinity-low specificity manner, which is in contrast to catalysis which is highly specific for D-erythro-ceramide (D-e-Cer) but occurs with a lower affinity. The compounds of the invention are structurally related to of ceramide or sphingosine. In one embodiment of the invention, the compounds of the invention are designed according to modifications of key structural elements in ceramide and sphingosine, including stereochemistry, the primary and secondary hydroxyl groups, the trans double bond in the sphingosine backbone, and the amide bond. In general, the compounds of the invention interfere with one or more of the following structure of the ceramide or sphingosine: the primary and secondary hydroxyl groups, the C4-C5 double bond, the trans configuration of the C4-C5 double bond, or the NH-protons from either the amide of ceramide or the amine of sphingosine. In specific embodiments, the mitochondrial ceramidase inhibitors of the invention are 1) all stereoisomers of D-erythro-ceramide (D-e-Cer) with an IC 50 (the concentration of an inhibitor at which ceramidase activity is inhibited by 50% of the level observed in the absence of the inhibitor) of the range 0.01-0.8 mole %, preferably an IC 50 of 0.11, 0.21 and 0.26 mole %, for the L-threo, D-threo and L-erythro isomers respectively; 2) all stereoisomers of sphingosine with IC 50 ranging from 0.01-0.8 mole %, preferably an IC 50 of 0.04 to 0.14 mole %, for the N-alkyl-D-erythro-sphingosine (most preferably for N-Me-Sph, IC 50 0.13 mole %); and 3) D-erythro-urea-C 16 -ceramide (most preferably for C 16 -urea-Cer IC 50 0.33 mole %). In preferred embodiments, the compounds are potent competitive inhibitors: urea-ceramide (C 16 -urea-Cer) and ceramine (C 18 -ceramine). In various preferred embodiments, the invention encompasses potent mitochondrial ceramidase inhibitors, such as but not limited to, D-erythro-sphinganine with an IC 50 of the range 0.01-0.8 mole % (more preferably for D-e-dh-Sph, IC 50 0.20 mole %), D-erythro-dehydro sphingosine with an IC 50 of the range 0.01-0.8 mole % (more preferably for D-e-deh-Sph, IC 50 0.25 mole %), (2S)-3-keto-sphinganine with an IC 50 of the range 0.01-0.8 mole % (more preferably for 3-keto-dh-Sph, IC 50 0.34 mole %), (2S) 3-keto-ceramide with an IC 50 of the range 0.01-0.8 mole % (more preferably for 3-keto-C 16 -Cer, IC 50 0.60 mole %). In other embodiments, the invention encompasses weaker mitochondrial ceramidase inhibitors, such as but not limited to, 1-O-Methyl-D-erythro-sphingosine (1-O-Me-Sph), cis-D-erythro-sphingosine (cis-D-e-Sph), (2S)-3-keto-sphingosine (3-keto-Sph), (2S)-3-keto-dehyrosphingosine (3-keto-deh-Sph), and N,N-dimethyl-D-erythro-sphingosine (N,N-diMe-Sph). In yet another embodiment, the invention provides the use of ceramide-1-phosphate (Cer-1-P) and sphingosine-1-phosphate (Sph-1-P) to stimulate mitochondrial ceramidase. The present invention provides method of designing and screening for compounds that inhibit mitochondrial ceramidase. Methods of making the compounds that inhibits mitochondrial ceramidase are also provided. The present invention encompasses methods, pharmaceutical compositions, and dosage forms for the treatment and prevention of disorders that are ameliorated by the inhibition of mitochondrial ceramidase in mammals, including humans. Examples of such disorders include, but are not limited to, various cancers, hyperproliferative diseases, cardiovascular diseases, and inflammation. The methods of the invention comprise administering to a patient in need of such treatment or prevention a therapeutically or prophylactically effective amount of a compound of the invention, or a pharmaceutically acceptable salt,-or solvate thereof. Pharmaceutical compositions of the invention comprise a therapeutically or prophylactically effective amount of a mitochondrial ceramidase inhibitor. Preferred compounds are those that are active in decreasing cell survival and viability (e.g., which can be demonstrated in in vitro assays or in breast cancer cell line assays, or can be identified using in vitro assays, animal models, or cell culture assays). Pharmaceutical compositions of the invention can further comprise other anticancer or anti-inflammatory drug substances. The present invention provides compounds for increasing mitochondrial ceramidase activity having the Formula V and VI as shown below. In a more preferred embodiment, such compounds include ceramide 1-phosphate and sphingosine 1-phosphate. The invention also provides methods of treatment of disorders involving deficient cell proliferation or growth, or in which cell proliferation is otherwise desired (e.g., degenerative disorders, growth deficiencies, lesions, physical trauma) by administering compounds that activates mitochondrial ceramidase (e.g., ceramide-1-phosphate (Cer-1-P) and sphingosine-1-phosphate (Sph-1-P). Activating ceramide function can also be done to grow larger animals and plants, e.g., those used as food or material sources. The present invention further relates to a method of synthesizing cis-D-erythro-sphingosine, which comprises regioselective catalytic hydrogenation of N-Boc-4.5-dehydro-D-erythro-sphingosine using Raney® 2800 nickel catalyst performed in ethanol solution, in the presence of pyridine; deprotecting of the formed 9:1 mixture of cis/trans isomers of N-Boc-D-erythro-sphingosine using chlorotrimethylsilane in methanol, and final separation of the formed cis/trans-D-erythro-sphingoid bases using silica gel column chromatography and chloroform-methano1-concentrated ammonium hydroxide (5:1:0.05 v/v/v/) as an eluent system. The present invention also further relates to a method of synthesizing of varied chains urea analogs of ceramides, which comprises regioselective condensation of sphingosine base with an alkyl isocyanate, performed in an inert solvent system at room temperature. |
Data recording medium, data recording method and device, and encode method and device |
A recording method for converting m-bit data into n-bit (where n>m) data whose run length is restricted and recording the converted data on a recording medium, the recording method comprising the step of selecting first n-bit data according to an immediately preceded n-bit data, first n-bit data immediately followed thereby, and second n-bit data immediately followed thereby so that the cumulative value of DC components per unit time becomes small. |
1. A recording method for converting m-bit data into an n-bit (where n>m) data symbol whose run length is restricted and placing a connection bit after the m-bit data symbol so that the cumulative value of DC components per unit time becomes small, the recording method comprising the steps of: selecting a first connection bit to be added to an immediately preceded data symbol from a plurality of connection bits according to at least one first connection bit that can be added between the immediately preceded data symbol and a first data symbol immediately followed thereby and according to at least one second connection bit that can be added between the first data symbol and at least one second data symbol immediately followed thereby; adding the selected first connection bit to the immediately preceded data symbol so as to generate record data; and recording the generated record data on a recording medium. 2. The recording method as set forth in claim 1, wherein the selecting step comprises the steps of: selecting at least one first connection bit that can be added between the immediately preceded data symbol and the first data symbol immediately followed thereby; selecting at least one second connection bit that can be added between the first data symbol and at least one second data symbol immediately followed thereby; and selecting the first connection bit from the selected first connection bit and the selected second connection bit so that the cumulative value of the DC components becomes small. 3. The recording method as set forth in claim 2, wherein the first connection bit selecting step is performed by selecting the first connection bit so that when the selected first connection bit and the selected second connection bit are combined, the cumulative value of the DC components becomes small. 4. A recording method for converting m-bit data into an n-bit (where n>m) data symbol whose run length is restricted and adding a connection bit after the m-bit data symbol so that the cumulative value of DC components per unit time becomes small, the recording method comprising the steps of: when an immediately preceded data symbol is a special data symbol, selecting a first connection bit to be added to the immediately preceded data symbol from a plurality of connection bits according to at least one first connection bit that can be added between the immediately preceded data symbol and a first data symbol immediately followed thereby and according to at least one second connection bit that can be added between the first data symbol and at least one second data symbol immediately followed thereby; adding the selected first connection bit to the immediately preceded data symbol so as to generate record data; and recording the generated record data on a recording medium. 5. The recording method as set forth in claim 4, wherein the selecting step comprises the steps of: selecting at least one first connection bit that can be added between the immediately preceded data symbol and the first data symbol immediately followed thereby; selecting at least one second connection bit that can be added between the first data symbol and at least one second data symbol immediately followed thereby; and selecting the first connection bit from the selected first connection bit and the selected second connection bit so that the cumulative value of the DC components becomes small. 6. The recording method as set forth in claim 5, wherein the first connection bit selecting step is performed by selecting the first connection bit so that when the selected first connection bit and the selected second connection bit are combined, the cumulative value of the DC components becomes small. 7. The recording method as set forth in claim 4, wherein when the first data symbol is selected according to the immediately preceded data symbol and the first data symbol, with the special data symbol, the second connection bit added between the first data symbol and the second data symbol is unconditionally selected. 8. The recording method as set forth in claim 4, wherein the m-bit data is modulated according to the 8-14 modulating system. 9. A recording method for converting m-bit data into n-bit (where n>m) data whose run length is restricted and recording the converted data on a recording medium, the recording method comprising the step of: selecting first n-bit data according to an immediately preceded n-bit data, first n-bit data immediately followed thereby, and second n-bit data immediately followed thereby so that the cumulative value of DC components per unit time becomes small. 10. The recording method as set forth in claim 9, wherein the n-bit data is composed of an n1-bit data symbol and n2-bit (=n−n1) connection bits immediately followed thereby and selected from a plurality of connection bits so that the cumulative value of DC components per unit time becomes small, and wherein the recording method further comprises the step of: selecting a first connection bit to be added to the immediately preceded data symbol according to at least one connection bit that can be added between the immediately preceded data symbol and the first data symbol and at least one second connection bit that can be added between the first data symbol and the second data symbol. 11. The recording method as set forth in claim 10, wherein the selecting step comprises the steps of: selecting at least one first connection bit that can be added between the immediately preceded data symbol and the first data symbol immediately followed thereby; selecting at least one second connection bit that can be added between the first data symbol and the second data symbol; and selecting the first connection bit from the selected first connection bit and the second connection bit so that the cumulative value of the DC components becomes small. 12. The recording method as set forth in claim 11, wherein the first connection bit selecting step is performed by selecting the first connection bit so that when the selected first connection bit and the selected second connection bit are combined, the cumulative value of the DC components becomes small. 13. The recording method as set forth in claim 9, wherein the m-bit data is modulated according to the 8-14 modulating system. 14. The recording method as set forth in claim 9, wherein the m-bit data is modulated according to the 8-16 modulating system. 15. The recording method as set forth in claim 9, wherein the n-bit data is composed of an n1-bit data symbol and n2-bit (=n−n1) connection bits immediately followed thereby and selected from a plurality of connection bits so that the cumulative value of DC components per unit time becomes small, and wherein the recording method further comprises the step of: when the immediately preceded data symbol is a special data symbol, selecting a first connection bit to be added to the immediately preceded data symbol according to at least one connection bit that can be added between the immediately preceded data symbol and the first data symbol and at least one second connection bit that can be added between the first data symbol and the second data symbol. 16. The recording method as set forth in claim 15, wherein the selecting step comprises the steps of: selecting at least one first connection bit that can be added between the immediately preceded data symbol and the first data symbol immediately followed thereby; selecting at least one second connection bit that can be added between the first data symbol and the second data symbol; and selecting the first connection bit from the selected first connection bit and the second connection bit so that the cumulative value of the DC components becomes small. 17. The recording method as set forth in claim 16, wherein the first connection bit selecting step is performed by selecting the first connection bit so that when the selected first connection bit and the selected second connection bit are combined, the cumulative value of the DC components becomes small. 18. The recording method as set forth in claim 15, wherein when the first data symbol is selected according to the immediately preceded data symbol and the first data symbol, with the special data symbol, the second connection bit added between the first data symbol and the second data symbol is unconditionally selected. 19. A recording apparatus, comprising: an encoding process portion for performing an encoding process for input data; a converting portion for converting m-bit data that is output from the encoding processing portion into n-bit (where n>m) data whose run length is restricted by selecting first n-bit data according to an immediately preceded n-bit data, first n-bit data immediately followed thereby, and second n-bit data immediately followed thereby so that the cumulative value of DC components per unit time becomes small; and a recording portion for recording data that is output from the converting portion on a recording medium. 20. The recording apparatus as set forth in claim 19, wherein the n-bit data is composed of an n1-bit data symbol and n2-bit (=n−n1) connection bits immediately followed thereby and selected from a plurality of connection bits so that the cumulative value of DC components per unit time becomes small, and wherein the converting portion is configured to select a first connection bit to be added to the immediately preceded data symbol according to at least one connection bit that can be added between the immediately preceded data symbol and the first data symbol and at least one second connection bit that can be added between the first data symbol and the second data symbol. 21. The recording apparatus as set forth in claim 20, wherein the converting portion is configured to select at least one first connection bit that can be added between the immediately preceded data symbol and the first data symbol immediately followed thereby, select at least one second connection bit that can be added between the first data symbol and the second data symbol, and select the first connection bit from the selected first connection bit and the second connection bit so that the cumulative value of the DC components becomes small. 22. The recording apparatus as set forth in claim 21, wherein the converting portion is configured to select the first connection bit so that when the selected first connection bit and the selected second connection bit are combined, the cumulative value of the DC components becomes small. 23. The recording apparatus as set forth in claim 19, wherein the m-bit data is modulated according to the 8-14 modulating system. 24. The recording apparatus as set forth in claim 19, wherein the m-bit data is modulated according to the 8-16 modulating system. 25. The recording apparatus as set forth in claim 19, wherein the n-bit data is composed of an n1-bit data symbol and n2-bit (=n−n1) connection bits immediately followed thereby and selected from a plurality of connection bits so that the cumulative value of DC components per unit time becomes small, and wherein the converting portion is configured to select a first connection bit to be added to the immediately preceded data symbol according to at least one connection bit that can be added between the immediately preceded data symbol and the first data symbol and at least one second connection bit that can be added between the first data symbol and the second data symbol when the immediately preceded data symbol is a special data symbol. 26. The recording apparatus as set forth in claim 25, wherein the converting portion is configured to select at least one first connection bit that can be added between the immediately preceded data symbol and the first data symbol immediately followed thereby, select at least one second connection bit that can be added between the first data symbol and the second data symbol, and select the first connection bit from the selected first connection bit and the second connection bit so that the cumulative value of the DC components becomes small. 27. The recording apparatus as set forth in claim 26, wherein the converting portion is configured to select the first connection bit so that when the selected first connection bit and the selected second connection bit are combined, the cumulative value of the DC components becomes small. 28. The recording apparatus as set forth in claim 25, wherein when the first data symbol is selected according to the immediately preceded data symbol and the first data symbol, with the special data symbol, the second connection bit added between the first data symbol and the second data symbol is unconditionally selected. 29. A recording medium on which when m-bit data is converted into n-bit (where n>m) data whose run length is restricted, first n-bit data is selected according to an immediately preceded n-bit data, first n-bit data immediately followed thereby, and second n-bit data immediately followed thereby so that the cumulative value of DC components per unit time becomes small and the selected first n-bit data is recorded after the immediately preceded n-bit data. 30. The recording medium as set forth in claim 29, wherein the n-bit data is composed of an n1-bit data symbol and n2-bit (=n−n1) connection bits immediately followed thereby and selected from a plurality of connection bits so that the cumulative value of DC components per unit time becomes small, and wherein a first connection bit to be added to the immediately preceded data symbol is selected according to at least one connection bit that can be added between the immediately preceded data symbol and the first data symbol and at least one second connection bit that can be added between the first data symbol and the second data symbol and recorded on the recording medium. 31. The recording medium as set forth in claim 30, wherein at least one first connection bit that can be added between the immediately preceded data symbol and the first data symbol immediately followed thereby is selected, wherein at least one second connection bit that can be added between the first data symbol and the second data symbol is selected, wherein the first connection bit is selected from the selected first connection bit and the second connection bit so that the cumulative value of the DC components becomes small, and wherein the resultantly selected first connection bit is recorded on the recording medium. 32. The recording medium as set forth in claim 31, wherein the first connection bit is selected so that when the selected first connection bit and the selected second connection bit are combined, the cumulative value of the DC components becomes small, and wherein the resultantly selected first connection bit is recorded on the recording medium. 33. The recording medium as set forth in claim 29, wherein the m-bit data is modulated according to the 8-14 modulating system and recorded on the recording medium. 34. The recording medium as set forth in claim 29, wherein the m-bit data is modulated according to the 8-16 modulating system and recorded on the recording medium. 35. The recording medium as set forth in claim 29, wherein the n-bit data is composed of an n1-bit data symbol and n2-bit (=n−n1) connection bits immediately followed thereby and selected from a plurality of connection bits so that the cumulative value of DC components per unit time becomes small, and wherein when the immediately preceded data symbol is a special data symbol, a first connection bit to be added to the immediately preceded data symbol is selected according to at least one connection bit that can be added between the immediately preceded data symbol and the first data symbol and at least one second connection bit that can be added between the first data symbol and the second data symbol and recorded on the recording medium. 36. The recording medium as set forth in claim 35, wherein at least one first connection bit that can be added between the immediately preceded data symbol and the first data symbol immediately followed thereby is selected, wherein at least one second connection bit that can be added between the first data symbol and the second data symbol is selected, and wherein the first connection bit is selected from the selected first connection bit and the second connection bit so that the cumulative value of the DC components becomes small, and wherein the resultantly selected first connection bit is recorded on the recording medium,. 37. The recording medium as set forth in claim 36, wherein the first connection bit is selected so that when the selected first connection bit and the selected second connection bit are combined, the cumulative value of the DC components becomes small, and wherein the resultantly selected first connection bit is recorded on the record medium. 38. The recording medium as set forth in claim 35, wherein when the first data symbol is selected according to the immediately preceded data symbol and the first data symbol, with the special data symbol, the second connection bit added between the first data symbol and the second data symbol is unconditionally selected. 39. A data converting method, comprising the step of: when m-bit data is converted into n-bit (where n>m) data whose run length is restricted, selecting first n-bit data according to an immediately preceded n-bit data, first n-bit data immediately followed thereby, and second n-bit data immediately followed thereby so that the cumulative value of DC components per unit time becomes small. 40. The data converting method as set forth in claim 39, wherein the n-bit data is composed of an n1-bit data symbol and n2-bit (=n−n1) connection bits immediately followed thereby and selected from a plurality of connection bits so that the cumulative value of DC components per unit time becomes small, and wherein the data converting method further comprises the step of: selecting a first connection bit to be added to the immediately preceded data symbol according to at least one connection bit that can be added between the immediately preceded data symbol and the first data symbol and at least one second connection bit that can be added between the first data symbol and the second data symbol. 41. The data converting method as set forth in claim 40, wherein the selecting step comprises the steps of: selecting at least one first connection bit that can be added between the immediately preceded data symbol and the first data symbol immediately followed thereby; selecting at least one second connection bit that can be added between the first data symbol and the second data symbol; and selecting the first connection bit from the selected first connection bit and the second connection bit so that the cumulative value of the DC components becomes small. 42. The data converting method as set forth in claim 41, wherein the first connection bit selecting step is performed by selecting the first connection bit so that when the selected first connection bit and the selected second connection bit are combined, the cumulative value of the DC components becomes small. 43. The data converting method as set forth in claim 39, wherein the m-bit data is modulated according to the 8-14 modulating system. 44. The data converting method as set forth in claim 39, wherein the m-bit data is modulated according to the 8-16 modulating system. 45. The data converting method as set forth in claim 39, wherein the n-bit data is composed of an n1-bit data symbol and n2-bit (=n−n1) connection bits immediately followed thereby and selected from a plurality of connection bits so that the cumulative value of DC components per unit time becomes small, and wherein the data converting method further comprises the step of: when the immediately preceded data symbol is a special data symbol, selecting a first connection bit to be added to the immediately preceded data symbol according to at least one connection bit that can be added between the immediately preceded data symbol and the first data symbol and at least one second connection bit that can be added between the first data symbol and the second data symbol. 46. The data converting method as set forth in claim 45, wherein the selecting step comprises the steps of: selecting at least one first connection bit that can be added between the immediately preceded data symbol and the first data symbol immediately followed thereby; selecting at least one second connection bit that can be added between the first data symbol and the second data symbol; and selecting the first connection bit from the selected first connection bit and the second connection bit so that the cumulative value of the DC components becomes small. 47. The data converting method as set forth in claim 46, wherein the first connection bit selecting step is performed by selecting the first connection bit so that when the selected first connection bit and the selected second connection bit are combined, the cumulative value of the DC components becomes small. 48. The data converting method as set forth in claim 35, wherein when the first data symbol is selected according to the immediately preceded data symbol and the first data symbol, with the special data symbol, the second connection bit added between the first data symbol and the second data symbol is unconditionally selected. |
<SOH> BACKGROUND ART <EOH>Since optical discs such as a CD (Compact Disc) and a CD-ROM (Compact Disc Read Only Memory) are easy to handle and are produced at relatively low cost, they have been widely used as recording mediums for storing data. In recent years, a CD-R (Compact Disc Recordable) disc, on which data can be recorded once, and a CD-RW (Compact Disc ReWritable) disc, on which data can be rewritten, have come out. Thus, data can be easily recorded on such recordable optical discs. As a result, optical discs that accord with the CD standard such as a CD-DA (Compact Disc Digital Audio) disc, a CD-ROM disc, a CD-R disc, and a CD-RW disc have become the mainstream of data recording mediums. In addition, in recent years, audio data is compressed according to the MP3 (MPEG1 Audio Layer-3) and the ATRAC (Adaptive TRansform Acoustic Coding) 3 and recorded on the CD-R disc, the CD-RW disc, and so forth. However, as a CD-R disc and a CD-RW disc have come out, data recoded on a CD disc can be easily copied to these discs. As a result, a problem about copyright protection has arisen. Thus, when content data is recorded to a CD disc, it is necessary to take measures to protect content data. As a conventional copy protection technology for protecting a copy of data from a CD-ROM disc to a CD-R disc or a CD-RW disc, a method for physically deforming a disc with for example wobble pits has been proposed. In the physically deforming method, however, if an original disc is a CD-R disc or a CD-RW disc, the copy protection cannot be performed. In addition, to protect a copying operation, a method for encrypting content data has been proposed. However, even if content data has been encrypted, a disc on which the same data as an original disc is recorded can be produced. To protect content data recorded on a CD disc that accords with the CD standard, there is a method for determining whether the disc is an original CD or a CD whose data has been copied from an original CD (this CD is referred to as copied CD). When the disc is an original CD, a copying operation thereof can be permitted. When the disc is a disc whose data has been copied from an original disc (this disc is referred to as copied disc), a further copying operation thereof can be prohibited. To determine whether the disc is an original disc or a copied disc, a method for placing a defect on a disc in a master disc production stage, detecting the defect from the disc during a reproduction, and determining that the disc is an original disc based on the detected defect has been proposed. In this method, however, an original disc may contain such a defect. In addition, depending on the type of a defect, it may be copied as it is. Thus, content data of an original disc cannot be prevented from being copied to a CD-R disc. Therefore, an object of the present invention is to provide a data recording medium, a data recording method, and a data recording apparatus that contribute to copy protection without need to physically deform a medium and intentionally place a defect thereon. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a block diagram showing an example of the structure of a mastering apparatus according to an embodiment of the present invention. FIG. 2A and FIG. 2B are schematic diagrams showing an example of a special pattern recorded on a disc. FIG. 3 is a schematic diagram describing the format of an EFM frame of a CD. FIG. 4 is a schematic diagram describing a sub code block of a CD. FIG. 5 is a schematic diagram describing Q channel of a sub code. FIG. 6A and FIG. 6B are schematic diagrams describing data of a sub code. FIG. 7 is a schematic diagram showing an EFM conversion table. FIG. 8 is a schematic diagram showing an EFM conversion table. FIG. 9 is a schematic diagram showing an EFM conversion table. FIG. 10 is a schematic diagram showing an EFM conversion table. FIG. 11 is a schematic diagram showing an EFM conversion table. FIG. 12 is a schematic diagram showing an EFM conversion table. FIG. 13A to FIG. 13D are schematic diagrams describing a method for selecting merging bits. FIG. 14A , FIG. 14B , and FIG. 14C are schematic diagrams showing an example of a special pattern of which DSV does not converge. FIG. 15A , FIG. 15B , and FIG. 15C are schematic diagrams showing another example of a special pattern of which DSV does not converge. FIG. 16A , FIG. 16B , and FIG. 16C are schematic diagrams showing a further example of a special pattern of which DSV does not converge. FIG. 17A to FIG. 17E are schematic diagrams showing an example of a special pattern of which DSV does not converge in the EFM modulation when DSV is controlled according to only the relation of an immediately preceded code symbol and a code symbol immediately followed thereby. FIG. 18 is a schematic diagram describing an example of a special pattern of which DSV does not converge in the EFM modulation when DSV is controlled according to only the relation of an immediately preceded code symbol and a code symbol immediately followed thereby. FIG. 19 is a flow chart describing a merging bit selecting process performed by a modulator according to the present invention. FIG. 20A and FIG. 20B are schematic diagrams describing the merging bit selecting process performed by the modulator according to the present invention. FIG. 21 is a block diagram showing an example of a CD reproducing apparatus. FIG. 22 is a flow chart describing a merging bit selecting process performed by a conventional modulator. FIG. 23A and FIG. 23B are schematic diagrams describing the merging bit selecting process performed by the conventional modulator. FIG. 24 is a block diagram describing a flow of a disc copying process. FIG. 25 is a block diagram showing an outline of a reproducing process portion when a disc copying process is performed. FIG. 26 is a block diagram showing an outline of a recording process portion when a disc copying process is performed. detailed-description description="Detailed Description" end="lead"? |
Products comprising a sheet and a lipid and aqueous phase |
This invention concerns products for cleansing and other applications that comprise a sheet of absorbent material, in particular of non-woven material, such as a wipe, to which are applied a lipid and an aqueous phase. The invention further concerns the manufacture and use of such products. |
1. A product comprising a porous or absorbent sheet whereto an aqueous and a lipid phase have been applied. 2. A product according to claim 1 wherein the melting point or melting range of the lipid phase is above or equal to 25° C. 3. A product according to any of claims 2 wherein the melting point or melting range of the lipid phase is in the range of 32 to 40° C. 4. A product according to claims 1 wherein the lipid phase comprises mono-, di- or triglycerides. 5. A product according to claim 4 wherein the lipid phase comprises mono-, di- or triglycerides derived from or present in natural oils. 6. A product according to claim 4 wherein the lipid phase comprises fatty acid mono-, di- or triglycerides wherein the fatty acids contain from 12 to 24, preferably from 16 to 20 carbon atoms. 7. A product according to claim 4 wherein the lipid phase comprises triglycerides selected from glyceryl stearate, glyceryl oleate, glyceryl laurate, glyceryl myristate, cocoglycerides, or hydrogenated palm oil glycerides, hydrogenated castor oil, or hydrogenated rapeseed oil. 8. A product according to claims 4 wherein the lipid phase comprises mono-, di- or triglycerides in an amount of at least 50%, preferably at least 70%, more preferably at least 90%, w/w of the total amount of components making up the lipid phase. 9. A product according to claims 1 wherein the lipid phase contains fatty alcohols. 10. A product according to claim 9 wherein the lipid phase contains C12-C50-fatty alcohols, in particular the C12-C24-fatty alcohols. 11. A product according to claim 10 wherein the fatty alcohols are selected from myristyl alcohol, 1-pentadecanol, cetyl alcohol, lauryl alcohol, oleyl alcohol, palmityl alcohol, 1-heptadecanol, stearyl alcohol, cetearyl alcohol, 1-nonadecanol, arachidyl alcohol, 1-heneicosanol, behenyl alcohol, brassidyl alcohol, lignoceryl alcohol, ceryl alcohol or myricyl alcohol and C16/C18-Guerbet alcohols. 12. A product according to claims 9 wherein the fatty alcohols are present in the lipid phase, in an amount relative to the total weight amount of the lipid phase, which is in the range of 1-40%, preferably 1-30% (w/w), more preferably of 1-20% (w/w), still more preferably from 1-10% (w/w). 13. A product according to claims 1 wherein the lipid phase contains fatty acids. 14. A product according to claim 13 wherein the fatty acids are C14-C40-fatty acids or in particular are C16-C30-fatty acids. 15. A product according to claim 13 wherein the fatty acids are selected from myristic-, pentadecanoic-, palmitic-, margaric-, stearic-, nonadecanoic-, arachic-, behenic-, lignoceric-, cerotic-, melissic-, erucaic-, elaeostearic-, oleic-, linoleic-, lauric acid and hydroxy-substituted fatty acids. 16. A product according to claims 13 wherein the total amount of the fatty acids present in the lipid phase, relative to the total weight amount of the lipid phase, is in the range of 1-30% (w/w), preferably of 1-20% (w/w), more preferably from 1-10% (w/w). 17. A product according to claims 1 wherein the lipid phase contains one or more of components (a), (b), (c), (d), (e) or (f) as defined hereafter: (a) at least 1-50% (w/w), in particular at least 1-10% of an oily or waxy component (b) 0.1-5% (w/w) of at least one active ingredient (c) 1-10% (w/w) of at least one oil (d) 0.1-10% (w/w) of at least one emulsifier (e) 5-90% (w/w) of further waxy components (f) 0-5% (w/w) water. 18. A product according to claim 17 wherein the lipid phase contains all components (a)-(f). 19. A product according to claim 17 wherein component (a) is an oily or waxy component selected from C14-C30-dialkyl ethers, C14-C30-dialkyl carbonates, C4-C34-dicarbonic acids or C12-C30-hydroxyfatty alcohols or mixtures thereof. 20. A product according to claims 1 wherein the lipid phase contains dialkyl(ene) ethers or -carbonates, dicarboxylic acids or hydroxy fatty alcohols, or a combination thereof. 21. A product according to claims 1 wherein the lipid or the aqueous phase contains one or more active substances. 22. A product according to claim 21 wherein the active substance(s) is or are anti-microbials, e.g. anti-bacterials and antifungals, anti-inflammatory agents, anti-irritating, anti-itching, anti-perspirant agents. 23. A product according to claim 1 wherein the lipid or the aqueous phase contains at least one moisturizer, deodorant, skin caring ingredient, plant extract, vitamin, perfume oil, dye, sunscreen filter, hydrotrope or self-tanning agent. 24. A product according to claim 1 wherein the lipid or the aqueous phase contains at least one emulsifier. 25. A product according to claim 1 wherein the lipid phase contains at least one superfatting agent, thickener, cationic polymer, aniomic polymer, zwitterionic polymer, amphoteric polymer, consistency agent, anti-oxidant. 26. A product according to claim 1 wherein the lipid or the aqueous phase contains an insect repellent, a sunscreen filter, a powder or a peeling agent. 27. A product according to claim 1 which is a wipe. 28. A product according to claim 27 wherein the wipe is a non-woven wipe. 29. A method of manufacturing a product as claimed in claim 1 said method comprising contacting the sheet with a lipid phase and with an aqueous phase, either subsequently or simultaneously. 30. A method according to claim 29 wherein a lipid phase having a melting point or a melting range of above room temperature is first applied to the surface of the sheet and subsequently the aqueous phase is applied. 31. A method according to claim 29 wherein the the aqueous phase by spraying, drippling, immersing or running through a bath, and the lipid phase is applied by spraying, contacting, printing or a direct contact process where there is a direct contact between the sheet and an application head having slit nozzles. 32. A method of cleansing comprising using the product as claimed in claim 1 as a combined cleanser and sheet of active substances. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Wipe products have become an important product category that has found a wide variety of applications for adults and babies. Examples include face or body cleansing wipes, wipes for skin treatment, and skin conditioning wipes. Over the last couple of decades so-called wet wipes have become successful as products particularly suited for these applications. These products typically are manufactured by impregnating sheets made of non-woven fabric with a suitable lotion. Recent innovations in the wipes area included improvements in the fabric, in the impregnating liquid as well as in product presentation. Initially, wet wipe products were made of traditional non-woven materials based on paper making technology (pulp based products). These products were well accepted but deficient in softness of the fabric material. The introduction of the ‘spunlace’ non-woven technology offered products that, compared to traditional paper based products, were superior in terms of softness. This is mainly due to (i) the use of long soft fibres (most frequently rayon and PET/PP or a mixture of these fibres) in the spunlace process and (ii) the fact that during the spunlace process no binder is added to the fabric. Another innovation was the introduction of the so-called ‘Pop-up’ technology that offered advantages as regards the dispensing of individual wipes. In addition to the above lotions have been developed which offered skincare benefits in addition to the basic cleansing properties of the wipe. One approach was the introduction of lotions that were based on oil-in-water emulsions which delivered useful properties such as superior mildness, moisturisation, protection and skin smoothness, when compared to simple aqueous cleansing formulations. Another approach encompassed the incorporation of active skincare ingredients, e.g. chamomile, into simple aqueous formulations, thereby delivering useful properties such as soothing. Current wet wipe products still are based on these approaches in that they are impregnated with either aqueous lotions or with oil in water emulsions. However, these approaches have several limitations. Firstly, only a small portion of the lotion (usually about 15%) is released from the wipes during use. Thus a large quantity of the relatively expensive lotion is not delivered to the skin providing no benefit to the consumer and is wasted when the product is discharged after use. This also prevents the use of expensive but more effective ingredients. Secondly, from a formulation point there is an apparent contradiction in the optimization of cleansing performance and skincare benefits in one single lotion, since ingredients which are effective in cleansing usually are not compatible with efficient skin care agents. Another important factor in cleansing is the fact that a number of soils are water-compatible and therefore more easily removed by water-based formulations, whereas others are lipid-compatible and therefore adequately removed by lipid or oil based formulations. A complete and effective removal of soils therefore requires the presence in or on a wipe of as well water and oil-based components. This is in particularly required in wipes for personal cleansing and in particular in wipes used for babies and infants. In the latter instance wet wipes are used for cleansing the perianal region when changing diapers. Inadequate cleaning not only results in personal discomfort but also gives rise to diaper rash and other infection related phenomena. It has been shown that the most effective way of preventing diaper rash is to cleanse the skin thoroughly and to remove the microorganisms that have been identified as causative. The source of these microorganisms is often the fecal deposits that can remain on a baby's skin while wearing the diaper. Because fecal deposits consist of both water-soluble and oil-soluble matter, however, complete removal of fecal deposits from the diaper area requires both water-based and oil-based cleansing agents. Thus, it is an object of this invention to provide a mechanism for cleansing babies' skin in order both to remove waste deposits and to reduce the number of microorganisms available to cause infection. WO 96/14835 discloses dry tissues to which a water-in-lipid emulsion has been applied and WO 99/25923 concerns a process and an apparatus for selectively coating a wipe with a water-in-lipid emulsion. WO 99/01536 discloses wipes wherein the carrier comprises two regions of different basis weight being applied with an emulsion comprised of a solidified lipid phase, a polar phase dispersed therein and an emulsifier. Other prior art in this field is WO 95/35411, WO 95/35412, WO 95/16824, WO 97/30216, DE 33 09 530 and the publication of R. E. Mathis in Nonwovens World 1999, pp. 59-65. It is an object of this invention to offer a cleansing article and in particular a wet wipe product that allows to independently optimize the cleansing and skincare attributes of the product and at the same time improves the delivery of skincare actives onto the skin during use. It is a further object of this invention to provide products that have an improved release of the active ingredient(s) onto the skin during use. It is still a further object of the present invention to provide a product for use as a cleansing tool that effectively and completely removes oil and water compatible soils. Another object of this invention is to provide products for cleansing and other applications that allow convenient and quick application, are easy to carry, as well as an easier and more evenly distribution of the ingredients in or on the product. They moreover should be convenient for application on babies and children. These objects are attained by the products according to the present invention, which comprise a sheet that contains an aqueous and a lipid phase. Whereas traditional wet wipes have been based on impregnation of a fabric with one phase, the products of this invention concern the application of two distinctly different phases onto a sheet. Both phases differ in terms of physical properties and may be applied on various parts or portions of the sheet. This approach allows combined optimal cleansing performance and superior skincare properties. |
<SOH> SUMMARY OF THE INVENTION <EOH>This invention relates to products that comprise a porous or absorbent sheet whereto a lipid and an aqueous phase have been applied. Preferably, the lipid phase is solid or semi-solid at ambient temperature and preferably is present at the surface or at the surface portion of one or both sides of the sheet. In particular said sheet is made of a non-woven material, more in particular a non-woven material made by the spunlace or the hydro-entanglement procedure. In a further aspect there is provided a method of manufacturing a product as described herein, said method comprising applying to the sheet a lipid phase and an aqueous phase, either subsequently or simultaneously. In a preferred method of manufacturing, said sheet is first coated with a lipid phase and subsequently sprayed or impregnated with an aqueous phase. In still a further aspect there is provided the use of a product as described herein as a cleansing tool, in particular in personal care applications. In another aspect the invention concerns the use of a product as described herein as an applicator of active substances. In still another aspect the invention provides the use of a product as described herein as a combined cleanser and applicator of active substances. detailed-description description="Detailed Description" end="lead"? |
Respiratory therapy device for keeping free the natural respiratory tract of a human body and the use thereof in order to prevent the sound of snoring |
In order to treat snoring and obstructive sleep apnea, the inner respiratory tract is subjected to atmospheric pressure and the outer surrounding area of the respiratory tract is subjected to artificial low-pressure, the difference between the inner atmospheric pressure and the outer low-pressure remaining constant. The respiratory therapy device consists of a pressure stable hollow body which surrounds the human body while keeping free the natural respiratory opening. Set hollow body forms the low-pressure chamber (7) is connected to a suction pump (3) by a suction tube (2). The invention method and respiratory therapy device make it possible to breathe in a free atmosphere. |
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