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Methods for determining cellular response to stimuliRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay, Involving A Micro-organism Or Cell Membrane Bound Antigen Or Cell Membrane Bound Receptor Or Cell Membrane Bound Antibody Or Microbial Lysate, Animal CellMethods for determining cellular response to stimuli description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20080050756, Methods for determining cellular response to stimuli. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATION [0001] This patent document is a continuation of U.S. application Ser. No. 11/313,418, filed on Dec. 21, 2005, which claims the benefit of priority of U.S. application Ser. No. 60/639,152, filed Dec. 22, 2004, and PCT application number PCT/US2005/41946 filed on Nov. 17, 2005, which applications are herein incorporated by reference. BACKGROUND OF THE INVENTION [0002] Taste transduction is one of the most sophisticated forms of chemotransduction in animals. Gustatory signaling is found throughout the animal kingdom, from simple metazoans to the most complex vertebrates. Its main purpose is to provide a reliable signaling response to non-volatile ligands. Humans typically distinguish several perceptual taste qualities or modalities: sweet, sour, salty, bitter and umami. Each of these modalities is thought to be mediated by distinct signaling pathways mediated by receptors or channels, leading to receptor cell depolarization, generation of a receptor or action potential, and release of neurotransmitter at gustatory afferent neuron synapses. [0003] Taste transduction in animals is mediated by specialized neuroepithelial cells, referred to as taste receptor cells. These cells are organized into groups of about 40 to 100 cells to form taste buds. Taste buds contain precursor cells, support cells, and taste receptor cells. Receptor cells are innervated at their base by afferent nerve endings that transmit information to the taste centers of the cortex through synapses in the brain stem and thalamus. Taste buds are distributed into different papillae in the tongue epithelium. Circumvallate papillae, found at the very back of the tongue, contain hundreds to thousands of taste buds. By contrast, foliate papillae, localized to the posterior lateral edge of the tongue, contain dozens to hundreds of taste buds. Further, fungiform papillae, located on the anterior two-thirds of the tongue, contain only a single or few taste buds, depending upon the species. Taste cells are also found in the palate and other tissues, such as the esophagus and the stomach. [0004] Taste buds are ovoid structures and are primarily embedded within the epithelium of the tongue. It is believed that taste transduction is initiated at the apical portion of a taste bud at the taste pore, where microvilli of the taste receptor cells make contact with the outside environment. Various taste stimulants cause either depolarization (i.e., a reduction in membrane potential) or hyperpolarization (i.e., an increase in membrane potential) of taste cells and regulate neurotransmitter release from the cells at chemical synapses with afferent nerve fibers. The primary gustatory sensory fibers, which receive the chemical signals from the sensory cells, enter the base of each taste bud. Inter-cellular connections between taste cells in the same bud may also modulate the signals transmitted to the afferent nerve fibers. Molecules that elicit specific taste sensations are often referred to as "tastants." Although much is known about the psychophysics and physiology of taste cell function, very little is known about the molecules and pathways that mediate its sensory signaling response. [0005] In general, each taste modality is associated with particular types of receptor proteins expressed in some of the cells that form each taste bud. Genes encoding taste receptor proteins for sweet, bitter, umami and salty taste substances have been cloned from a variety of species, including humans. The nature of the coupling of stimulus-receptor interaction to a cellular response in the receptor cells has also been defined for some receptors. Some of these receptors have been used to develop bioassays for use in identifying potential taste enhancers, blockers and modifiers. Although these "chip" based systems have the potential for high throughput screening of large numbers of compounds, they do not incorporate the normal cellular components of the taste signaling pathways that are required for normal receptor-response coupling. Consequently, these assays are best at providing initial information about binding of potential stimuli with a particular receptor. They do not, however, provide information about a subsequent cellular response, if any, to the test substance. [0006] An alternative approach is to express cloned taste receptors in heterologous cells, typically a mammalian cell line such as human embryonic kidney cells.(HEK293), and to measure changes in intracellular calcium induced by taste stimuli. This approach requires coupling between stimulus-receptor interaction and a cellular pathway leading to an increase in calcium, and it permits measurements in many cells at once. The normal cellular organization of the taste receptor unit, the taste bud, however, is lost along with any processing of taste information occurring between cells within the taste bud. This limitation is particularly important in light of recent results suggesting that sweet and bitter receptors are localized in taste cells that do not directly communicate with afferent nerve fibers, but rather communicate with adjacent taste bud cells that are innervated. SUMMARY OF THE INVENTION [0007] Over the years substantial efforts have been directed to the development of various agents that interact with taste receptors to mimic or block natural taste stimulants. Examples of agents that have been developed to mimic sweet tastes are saccharin, monellin, and the thaumatins. Many taste-mimicking or taste-blocking agents developed to-date are not suitable as food additives, however, because they are not economical, are high in calories, or are carcinogenic. Development of new agents that mimic or block the basic tastes has been limited by a lack of knowledge of the taste cell biology involved in the transduction of taste modalities. Thus, there is a continuing need for new products and methods involved in or affect taste transduction. [0008] The present invention provides a method for determining the functional cellular response of a taste cell or taste cells contained in a section of taste-bud containing lingual epithelium (i.e., taste sensory cells in taste bud-containing intact epithelial tissue) to one or more stimuli. In the present invention, one contacts tissue such as taste tissue (such as taste bud-containing epithelial tissues or taste papillae), from an animal, with one or more stimuli, and quantitatively determines the magnitude of at least one cellular signaling event initiated by the stimulus/stimuli. Multiple data values may be collected, such as at differing concentrations of stimulus/stimuli and/or at different time points. The term "isolated taste bud-containing intact epithelial tissue" refers to a tissue sample isolated from an animal, where the tissue has been removed such that the tissue, and the cells contained in the tissue, retains its integrity. For example, the isolated intact taste tissue may be an intact taste bud in an intact lingual epithelial tissue sample that includes precursor cells, support cells, and taste receptor cells such that the polarization of the epithelium is retained. Taste cells have an apical surface and a basal surface. In certain embodiments of the present invention, the stimuli contact the apical surface of the taste cell or cells, but does not contact basal surface of the cell or cells. [0009] The invention provides methods of testing different taste stimuli, e.g., activators, inhibitors, stimulators, enhancers, agonisis, and antagonists of taste cells and tissues. As used herein, the term "taste cells" include neuroepithelial cells that are organized into groups to form taste buds of the tongue in structures known as papillae, e.g., foliate, fungiform, and circumvallate papillae (see, e.g., Roper et al. (1989)). Taste cells also include cells of the palate and other tissues that may contain taste cells, such as the esophagus, the stomach, the gastrointestinal tract or other internal organs such as the liver or pancreas. The taste cells may be taken from a biological sample. Such samples include, but are not limited to, tissue isolated from humans, mice, rats, and pigs. In addition to fresh tissue, biological samples may include sections of tissues such as frozen sections taken for histological purposes. A biological sample is typically obtained from a eukaryotic organism, such as an insect, protozoa, bird, fish, reptile, or mammal (e.g., a rat, mouse, cow, dog, pig, rabbit, chimpanzee, or human). Tissues include tongue tissue and isolated taste buds. [0010] A "functional cellular response" in the context of the present invention includes one or more cellular changes in response to any parameter that is indirectly or directly under the influence of the test stimulus, e.g., a functional, physical or chemical effect of the stimulus on the taste cell, cells, or tissue. A functional cellular response includes ligand binding, changes in ion flux, membrane potential, current flow, transcription, signal transduction, receptor-ligand interactions, messenger concentrations (including, but not limited to, cyclic AMP (cAMP), inositol trisphosphate (IP.sub.3), or intracellular Ca.sup.++ or other positive and/or negative ions including, but not limited to, chloride, sodium, or protons), in vitro, in vivo, and ex vivo and also includes other physiologic effects, such as increases or decreases of neurotransmitter or hormone release. [0011] As used herein, "determining a functional cellular response," means assaying for an increase or decrease in a parameter in or on a cell that is indirectly or directly under the influence of the test stimulus, e.g., functional, physical and chemical effects. Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in optical or spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties, patch clamping, voltage-sensitive dyes, whole cell currents, radioisotope efflux (or influx), inducible markers; tissue culture cell expression; transcriptional activation; ligand binding assays; voltage, membrane potential and conductance changes; ion flux assays; changes in intracellular second messengers such as cAMP and inositol triphosphate (IP3); changes in intracellular calcium levels; neurotransmitter release, and the like. "Inhibitors," "activators," and "modulators" are used to refer to inhibitory, activating, or modulating molecules identified using in vitro and in vivo assays for taste transduction, e.g., ligands, agonists, antagonists, and their homologs and mimetics. Inhibitors are compounds that, e.g., bind to a cell or cell component (e.g., receptor), partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate taste transduction, e.g., antagonists. Activators are compounds that, e.g., bind to a cell or cell component (e.g., receptor), stimulate, increase, activate, facilitate, enhance activation, sensitize or up regulate taste transduction, e.g., agonists. Modulators include compounds that alter the interaction of a polypeptide with receptors or extracellular proteins that bind activators or inhibitor, such as kinases. Modulators include genetically modified, naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Such assays for inhibitors and activators include, e.g., applying putative modulator compounds, in the presence or absence of tastants, and then determining the functional effects on taste transduction. Samples or assays comprising a cell in intact taste tissue but that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of modulation. Positive control samples (e.g., a tastant without added modulators) are assigned a relative activity value of 100%. Samples treated with an inhibitor, activator, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition, activation or modulation. Control samples (untreated with an inhibitor, activator, or modulator) are assigned a relative activity value of 100%. Inhibition is achieved when the activity value relative to the control is about 80%, optionally 50%, or 25, or even 0%. Activation is achieved when the activity value relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000%, or higher. [0012] The present invention provides a method for simulating a taste, comprising ascertaining the extent to which a cell in intact taste tissue interacts with a tastant. Interaction of a tastant with a cell in intact taste tissue can be determined using any of the assays described herein. The tastant can be combined with other tastants to form a mixture. If desired, one or more of the plurality of the compounds can be combined covalently. [0013] The present invention also provides a method wherein one or more control tastants are tested against one or more test tastants, to ascertain the extent to which a sensory cell or group of sensory cells in taste bud-containing lingual epithelial tissue interacts with each control tastants, thereby generating a stimulation profile for each control tastants. These stimulation profiles may then be stored in a relational database on a data storage medium. The method may further comprise providing a desired stimulation profile for a taste; comparing the desired stimulation profile to the relational database; and ascertaining one or more combinations of control tastants that most closely match the desired stimulation profile. The method may further comprise combining control tastants in one or more of the ascertained combinations to simulate the taste. [0014] The invention also provides methods of screening for modulators, e.g., activators, inhibitors, stimulators, enhancers, agonists, and antagonists, of tastants. Such modulators of taste transduction are useful for pharmacological, chemical, and genetic modulation of taste signaling pathways. These methods of screening can be used to identify high affinity agonists and antagonists of taste cell activity. These modulatory compounds can then be used in the food and pharmaceutical industries to customize taste, e.g., to modulate the tastes of foods, beverages, or drugs. [0015] An "ingestible substance" is a food, beverage, or other comestible, or orally administered products or compositions. A "flavor" herein refers to the perception of taste and/or smell in a subject, which include sweet, sour, salty, bitter, umami, and others. The subject may be a human or an animal. A "flavoring agent" herein refers to a compound or a biologically acceptable salt thereof that induces a flavor or taste in an animal or a human. A "flavor modifier" herein refers to a compound or biologically acceptable salt thereof that modulates, including enhancing or potentiating, and inducing, the tastes and/or smell of a natural or synthetic flavoring agent in an animal or a human. A "flavor enhancer" herein refers to a compound or biologically acceptable salt thereof that enhances the tastes or smell of a natural or synthetic flavoring agent. [0016] "Savory flavor" herein refers to the savory "umami" taste typically induced by MSG (mono sodium glutamate) in an animal or a human. "Savory flavoring agent" or "savory compound" herein refers to a compound or biologically acceptable salt thereof that elicits a detectable savory flavor in a subject, e.g., MSG (mono sodium glutamate). A "savory flavor modifier" herein refers to a compound or biologically acceptable salt thereof that modulates, including enhancing or potentiating, inducing, and blocking, the savory taste of a natural or synthetic savory flavoring agents, e.g., monosodium glutamate (MSG) in an animal or a human. A "savory flavor enhancer" herein refers to a compound or biologically acceptable salt thereof that enhances or potentiates the savory taste of a natural or synthetic savory flavoring agents, e.g., monosodium glutamate (MSG) in an animal or a human. [0017] A "savory flavoring agent amount" herein refers to an amount of a compound that is sufficient to induce savory taste in a comestible or medicinal product or composition, or a precursor thereof. A fairly broad range of a savory flavoring agent amount can be from about 0.001 parts per million (ppm) to 100 ppm, or a narrow range from about 0.1 ppm to about 10 ppm. Alternative ranges of savory flavoring agent amounts can be from about 0.01 ppm to about 30 ppm, from about 0.05 ppm to about 15 ppm, from about 0.1 ppm to about 5 ppm, or from about 0.1 ppm to about 3 ppm. [0018] A "savory flavor modulating amount" herein refers to an amount of a compound that is sufficient to alter (either increase or decrease) savory taste in a comestible or medicinal product or composition, or a precursor thereof, sufficiently to be perceived by a human subject. A fairly broad range of a savory flavor modulating amount can be from about 0.001 ppm to 100 ppm, or a narrow range from about 0.1 ppm to about 10 ppm. Alternative ranges of savory flavor modulating amounts can be from about 0.01 ppm to about 30 ppm, from about 0.05 ppm to about 15 ppm, from about 0.1 ppm to about 5 ppm, or from about 0.1 ppm to about 3 ppm. [0019] A "savory flavor enhancing amount" herein refers to an amount of a compound that is sufficient to enhance the taste of a natural or synthetic flavoring agents, e.g., monosodium glutamate (MSG) in a comestible or medicinal product or composition. A fairly broad range of a savory flavor enhancing amount can be from about 0.001 ppm to 100 ppm, or a narrow range from about 0.1 ppm to about 10 ppm. Alternative ranges of savory flavor enhancing amounts can be from about 0.01 ppm to about 30 ppm, from about 0.05 ppm to about 15 ppm, from about 0.1 ppm to about 5 ppm, or from about 0.1 ppm to about 3 ppm. [0020] Similar definitions are applicable to the other taste modalities of sweet, sour, salty, and bitter. [0021] The terms "isolated," "purified" or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. Continue reading about Methods for determining cellular response to stimuli... Full patent description for Methods for determining cellular response to stimuli Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods for determining cellular response to stimuli patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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