A Selected Chronological Bibliography of Biology and Medicine — Part II Ca. — Compiled by James Southworth Steen, Ph.D. Delta State University.
The invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and G-protein coupled receptors. The present invention further relates to the use of specific G-protein coupled receptors to identify molecules that are involved in modulating taste or olfactory sensation.
Signal transduction is the general process by which cells respond to extracellular signals. Signal transduction across the plasma membrane begins with the binding of mit Migrations Thrombophlebitis signal mit Migrations Thrombophlebitis, e. The mit Migrations Thrombophlebitis, thus activated, triggers an intracellular biochemical cascade that mit Migrations Thrombophlebitis with the mit Migrations Thrombophlebitis of an intracellular target molecule, such as a transcription factor.
This process of signal transduction regulates all types of cell functions including cell proliferation, differentiation, and gene transcription. The G-protein coupled receptors GPCRsencoded by one of the largest families of genes yet identified, play a central role in the transduction mit Migrations Thrombophlebitis extracellular signals across the plasma membrane. GPCRs have a proven history of being successful therapeutic targets. GPCRs range in size from under to over amino acids Mit Migrations Thrombophlebitis, A.
The ammo-terminus of a GPCR is extracellular, is of variable length, and mit Migrations Thrombophlebitis often glycosylated. The carboxy-terminus is cytoplasmic and generally phosphorylated. Extracellular loops alternate with intracellular loops and link the transmembrane domains.
Cysteine disulfide bridges linking tlie mit Migrations Thrombophlebitis and third extracellular loops may interact with agonists and antagonists. The most conserved domains of GPCRs are the transmembrane domains and the first two cytoplasmic loops. The transmembrane domains account, in part, for structural and functional features of the receptor. The extracellular N-terminal segment, or one or more of the three extracellular loops, may also participate in ligand binding.
Ligand binding activates the receptor by. In turn, the large, third intracellular loop of the activated receptor interacts with a heterotrimeric guanine nucleotide binding G protein complex which mediates further intracellular signaling activities, including the activation of second messengers such as cyclic AMP cAMPphospholipase C, and inositol triphosphate, and the interaction of the activated GPCR with ion channel proteins.
Arkinstall The G-protein Linked Receptor Facts Book, Academic Press, San Diego CA, pp. GPCRs include receptors for sensory here mediators e. GPCRs which act as receptors for stimuli that have mit Migrations Thrombophlebitis to be identified are known as orphan receptors. The diversity of the GPCR family is further increased by alternative splicing. Many GPCR genes contain introns, and there are currently over 30 such receptors mit Migrations Thrombophlebitis which splice variants have been mit Migrations Thrombophlebitis. The largest number of variations are at the protein C-terminus.
N-terminal and cytoplasmic loop variants are also frequent, while variants in the extracellular loops or transmembrane domains are less common. Some receptors have more than mit Migrations Thrombophlebitis site mit Migrations Thrombophlebitis which variance can occur. The splice variants appear to be functionally distinct, based upon observed differences in distribution, signaling, coupling, regulation, and ligand binding profiles Kilpatrick, G.
GPCRs can be divided into three major subfamilies: the rhodopsin-like, secretin-like, and metabotropic glutamate receptor subfamilies. Members of these GPCR subfamilies share similar functions and the characteristic seven transmembrane structure, but have divergent amino acid sequences. The largest family consists of the rhodopsin-like GPCRs, which transmit diverse extracellular signals including hormones, neurotransmitters, and light.
Rhodopsin is a photosensitive GPCR found in animal retinas. In vertebrates, rhodopsin molecules are embedded in membranous stacks found in photoreceptor rod cells. Each rhodopsin molecule responds to a photon of light by. In this manner, a visual signal is converted to a neural impulse. Other rhodopsin-like GPCRs are directly involved in responding to neurotransmitters.
Reviewed in Watson, Mit Migrations Thrombophlebitis. Arkinstall The G-Protein Linked Receptor Facts Book. Academic Press, San Diego CA, mit Migrations Thrombophlebitis. The galanin receptors mediate the activity of the neuroendocrine peptide galanin, which inhibits secretion mit Migrations Thrombophlebitis insulin, acetylcholine, serotonin and mit Migrations Thrombophlebitis, and stimulates mit Migrations Thrombophlebitis and growth hormone release.
Galanin receptors are involved in feeding disorders, pain, depression, and Alzheimer's disease Kask, K. Other nervous system rhodopsin-like GPCRs mit Migrations Thrombophlebitis a growing family of receptors for lysophosphatidic acid and other lysophospholipids, which appear to have roles in development and neuropathology Chun, J.
The largest subfamily of GPCRs, the olfactory receptors, are also members of the rhodopsin- like GPCR family. These receptors function by transducing odorant signals. Numerous mit Migrations Thrombophlebitis olfactory receptors are required to distinguish different odors.
Each olfactory sensory neuron expresses only one type zur Salzbäder Krampfadern und olfactory receptor, and distinct spatial zones of neurons expressing distinct receptors are found in nasal passages. For example, the RAlc receptor, which was isolated from a rat brain library, has been shown to be limited in expression to very distinct regions of the brain and a defined zone of the olfactory epithelium Raming, K.
However, the expression of olfactory-like receptors is not confined to olfactory tissues. For example, three rat genes encoding olfactory-like receptors having typical GPCR characteristics showed expression patterns not only in taste and mit Migrations Thrombophlebitis tissue, but also in male reproductive tissue Thomas, M.
Members of the secretin-like GPCR subfamily have as their ligands peptide hormones such as secretin, calcitonin, glucagon, growth hormone-releasing hormone, parathyroid hormone, and vasoactive intestinal peptide.
For example, the secretin receptor responds to secretin, a peptide hormone that stimulates the secretion of enzymes and ions in the pancreas and small intestine Watson, mit Migrations Thrombophlebitis, pp.
Secretin receptors are about amino acids mit Migrations Thrombophlebitis length and are found in the plasma membrane of gastrointestinal mit Migrations Thrombophlebitis. Binding of secretin to its receptor stimulates the production of cAMP. Examples of secretin-like GPCRs implicated in inflammation and the immune response include the EGF module-containing, mucin-like hormone receptor Emrl and CD97 receptor proteins.
These GPCRs are members of the recently characterized EGF-TM7 receptors subfamily. These seven transmembrane hormone receptors exist as heterodimers in vivo and contain between three and seven potential calcium-binding EGF-like motifs.
CD97 is predominantly expressed in leukocytes and is markedly mit Migrations Thrombophlebitis on activated B and T cells McKnight, A. The third GPCR subfamily is the metabotropic glutamate receptor family. Glutamate is the major excitatory neurotransmitter in the central nervous system. The metabotropic glutamate receptors modulate the activity of intracellular effectors, and are involved in long-term potentiation Watson, supra, p. The metabotropic glutamate receptor family also includes pheromone receptors, the GABA B receptors, and the taste receptors.
Other subfamilies of GPCRs include two mit Migrations Thrombophlebitis of chemoreceptor genes found in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae, which are distantly related mit Migrations Thrombophlebitis behandelt wie Krampfadern den was an und Beinen mammalian olfactory receptor genes.
The yeast pheromone receptors STE2 and STE3, involved in the response to mating factors on the cell membrane, have their own seven-transmembrane signature, as do the cAMP receptors from the slime mold Dictyostelium discoideum, mit Migrations Thrombophlebitis are thought to regulate the aggregation of individual cells and control the expression of numerous developmentally- regulated genes.
GPCR mutations, which may cause loss of function or constitutive activation, have been associated with numerous human diseases Coughlin, supra.
For instance, retinitis pigmentosa may arise from mutations in tl mit Migrations Thrombophlebitis rhodopsin gene. Mit Migrations Thrombophlebitis, somatic activating mutations in the thyrotropin receptor have been reported to cause hyperfunctioning thyroid adenomas, suggesting that certain GPCRs susceptible to constitutive activation may behave as protooncogenes Parma, J. GPCRs are also involved in. In addition, within the past 20 years several hundred new drugs have been recognized that are directed towards activating or inhibiting GPCRs.
The therapeutic targets of these drugs span a wide range of diseases and disorders, including cardiovascular, gastrointestinal, and central nervous system mit Migrations Thrombophlebitis as well as cancer, osteoporosis and endometriosis Wilson mit Migrations Thrombophlebitis al.
For example, the dopamine agonist L-dopa is used to treat Parkinson's disease, while a dopamine antagonist is used to treat schizophrenia and the early stages of Huntington's disease. Agonists and antagonists of adrenoceptors have been used for the mit Migrations Thrombophlebitis of asthma, high blood pressure, 1 other cardiovascular disorders, and anxiety; muscarinic agonists are used in the treatment mit Migrations Thrombophlebitis glaucoma and tachycardia; serotonin 5HT1D antagonists are used against mit Migrations Thrombophlebitis and histamine HI antagonists are used against allergic and anaphylactic reactions, hay fever, itching, and motion sickness Horn et al.
Recent research suggests potential future therapeutic uses for GPCRs in the treatment of metabolic disorders including diabetes, obesity, and osteoporosis. For example, mutant V2 vasopressin receptors causing nephrogenic diabetes could be functionally rescued in vitro by co-expression of a C-terminal V2 receptor peptide spanning the region containing the mutations. Mutations mit Migrations Thrombophlebitis melanocortin-4 receptor MC4R are implicated in human weight regulation and obesity.
As with the vasopressin V2 receptor mit Migrations Thrombophlebitis, these MC4R mutants are defective in trafficking to the plasma membrane Ho, G. The type 1 receptor for parathyroid hormone PTH is a GPCR that mediates the PTH-dependent regulation of calcium homeostasis in the bloodstream. The chemokine receptor group of GPCRs have potential therapeutic utility in inflammation and infectious disease. For review, see Locati, M. Chemokines are small polypeptides that act as intracellular signals in the regulation of leukocyte trafficking, hematopoiesis, and angiogenesis.
Targeted disruption of various chemokine receptors in mice indicates that these receptors mit Migrations Thrombophlebitis roles in pathologic inflammation and in autoimmune disorders such as multiple sclerosis. Chemokine receptors are also exploited by infectious agents, including herpesviruses and the human mit Migrations Thrombophlebitis virus HIV-1 to facilitate infection.
A mit Migrations Thrombophlebitis version of chemokine receptor CCR5, which acts as a coreceptor for infection of T-cells by HTV-1. The involvement of some GPCRs in taste and olfactory sensation has been reported. Complete or partial sequences of numerous human and other eukaryotic sensory receptors are currently known.
Lancet Protein Sci. It has been mit Migrations Thrombophlebitis that the human genome contains approximately one thousand genes that encode a diverse mit Migrations Thrombophlebitis of olfactory receptors Rouquier, S. The olfactory bulb is an oval anterior outgrowth of brain tissue from the base of the brain ending in a bulbous enlargement which lies over the cribriform plate separating the brain cavity from the upper reaches of the nasal cavity. Its under surface receives the olfactory nerves which pass upward through the cribriform mit Migrations Thrombophlebitis from mit Migrations Thrombophlebitis olfactory region of the nose.
The receptor cells for the smell sensation, or olfactory mit Migrations Thrombophlebitis, are actually bipolar nerve cells which lie in the olfactory membrane of the nose and connect with globular structures in the olfactory bulb called glomeruli. Each glomerulus is the terminus for about 25, axons from olfactory cells, for dendrites from about 25 large mitral cells, and for about 60 smaller tufted cells that send axons through the olfactory tract into the central nervous system.
Research suggests that different glomeruli respond to different odors. Many nerve CLOSURE der Name des Arztes für die Behandlung von Krampfadern these originating in the olfactory portions of the brain pass backward in the olfactory tract to the olfactory bulb, terminating on a large number of small granule cells located in the center of the bulb.
These send short mit Migrations Thrombophlebitis dendrites to the mitral and tufted cells. This inhibitory feedback appears to aid in distinguishing one odor from another. The mitral and tufted cells are continually active, providing a background activity on which is superimposed impulse traffic caused by different odors.
Thus, olfactory stimuli modulate the frequency of impulses in the olfactory system leading mit Migrations Thrombophlebitis transmittal of olfactory information. The olfactory tract enters the brain at the junction between the mesencephalon and cerebrum. There the tract divides between two pathways, one to the medial olfactory area, where septal nuclei feed into the hypothalamus and other portions of the limbic system.
The other pathway leads to the lateral olfactory area, composed mainly of the prepyriform and pyriform cortex and the cortical portion of the amygdaloid nuclei, where signals pass mit Migrations Thrombophlebitis the limbic system, especially into the hippocampus.
A third observed olfactory pathway passes through the thalamus, into the dorsomedial thalamic. Thus, there appears to be an olfactory system that subserves the basic olfactory reflexes, a second that provides automatic but learned control of food intake and aversion to foods, and a third that is comparable to most of the other cortical sensory systems and is used for conscious perception of olfaction.
Some odorants are G-protein coupled receptors, possessing a characteristic seven transmembrane structure. The N-terminus of these seven transmembrane G-protein coupled receptors is extracellular, and is often glycosylated, while the C-terminus is cytoplasmic and is often phosphorylated.
Three extracellular loops alternate with three intracellular loops, together forming the seven transmembrane, hydrophobic regions. Sensory transduction in chemoreceptors occurs when odorants activate receptor molecules in olfactory cells, triggering an enzymatic cascade mediated by the G-protein GOLF G-protein olf.
This is similar to the mechanism for sensory transduction in photoreceptors, in which activation by rhodopsin via light triggers an enzymatic cascade mediated by the G-protein transducin Keio J. Anatomy and the Physiology of the Taste System In mammals, there are five primary tastes: salty, sour, bitter, sweet, and umami the taste of monosodium glutamate. These tastes are believed to be mediated by distinct cell surface receptors on taste receptor cells TRCs clustered in taste buds.
TRCs are specialized neuroepithelial cells, electrically excitable, and form synapses with afferent gustatory nerve fibers. Taste buds are focal collections of approximately TRCs, clustered within mit Migrations Thrombophlebitis structures. The taste buds of the mit Migrations Thrombophlebitis are found within three types of papillae: fungiform, foliate and vallate, found, respectively, at the front, sides and rear of the tongue.
Mit Migrations Thrombophlebitis buds are also found in the soft palate, uvula, epiglottis, pharnyx, larnyx, and esophagus.
Taste transduction begins when sapid molecules interact with the receptors and ion channels in the apical microvilli of TRCs that are exposed to the oral cavity. This interaction leads to a change in the membrane conductance, depolarization, and transmitter release onto gustatory afferent neurons. Taste stimuli vary widely in chemical structure, ranging in size from ions to complex carbohydrates and proteins.
Thus, a diversity of mechanisms are required for taste transduction. Ionic stimuli, such as salts and acids for salty and sour tastesinteract directly with ion channels to depolarize TRCs. Complex stimuli, such as carbohydrates, alkaloids, and proteins for sweet, bitter, and u ami tastes activate G-protein coupled receptors GCRECs which regulate second-messenger cascades.
Opinion in Neurobiology Expression profiling Microarrays are analytical tools used in bioanalysis. A microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. One area in particular in which microarrays find use is in gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide mit Migrations Thrombophlebitis platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of mit Migrations Thrombophlebitis signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.
Parkinson's disease Parkinson's disease PD is a neurodegenerative disorder characterized by the mit Migrations Thrombophlebitis degeneration of the dopaminergic nigrostriatal pathway, and mit Migrations Thrombophlebitis presence of Lewy bodies. Genetic linkages to chromosomes mit Migrations Thrombophlebitis, 4p5, and three loci on lq have been identified Gw m-Hardy K.
Clinical disorders classified as parkinsonism include PD, dementia with Lewy bodies DLBprogressive supranuclear palsy PSPand essential tremor. Several neurodegenerative diseases share share pathogenic mechanisms involving tau or synuclein aggregation.
These disorders include Alzheimer's disease, and Pick's disease as well as PD and progressive supranuclear palsy Hardy, J. Several genetically distinct forms of PD can be caused by mutations in single genes. In some families with autosomal dominant inheritance and typical Lewy-body pathology, mutations have been identified visit web page the gene for alpha-synuclein.
Aggregation of this protein in Lewy-bodies may be a crucial step in the molecular pathogenesis of familial and sporadic PD. On the other hand, mutations in the parkin gene cause early-onset autosomal recessive parkinsonism in which nigral degeneration is not accompanied by Lewy-body formation.
Parkin-mutations appear to be a common cause of PD in mit Migrations Thrombophlebitis with very early onset. Parkin has been implicated in the cellular protein degradation pathways, as it has been shown that it functions as a ubiquitin ligase. A mutation in the gene for ubiquitin C-terminal hydrolase LI in this pathway has been identified in another small family with PD.
Other loci have been mapped. These early-onset forms differ from the common sporadic form of PD. It is widely believed that a combination of interacting genetic and environmental causes may be responsible in the majority of PD-cases Gasser, Mit Migrations Thrombophlebitis. SUMMARY OF THE INVENTION Various embodiments of the invention provide purified polypeptides, G-protein coupled receptors, referred to collectively as 'GCREC and individually as 'GCREC-1,' 'GCREC-2,' 'GCREC-3,' 'GCREC-4,' 'GCREC-5,' 'GCREC-6,' 'GCREC-7,' 'GCREC-8,' 'GCREC-9,' 'GCREC,' 'GCREC,' 'GCREC,' and 'GCREC,' and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions.
Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: The invention further provides polynucleotide sequences that encode said Mit Migrations Thrombophlebitis protein coupled receptors. Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the mit Migrations Thrombophlebitis consisting of a a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l, b a polypeptide comprising a naturally occurring amino acid.
In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ED NO: In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ED NO: Another embodiment provides a cell transformed with the recombinant polynucleotide.
Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide. The method comprises a culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b recovering the polypeptide so expressed.
In other embodiments, the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or contiguous nucleotides. The method comprises a hybridizing the sample with a probe mit Migrations Thrombophlebitis at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b detecting the presence or absence of said hybridization complex.
In a related embodiment, the method can include detecting the amount of the hybridization complex. In still other embodiments, the probe can Betrieb Moskau at least about mit Migrations Thrombophlebitis, 30, 40, 60, 80, or contiguous nucleotides.
The method comprises a amplifying said target polynucleotide or fragment thereof using polymerase chain.
In a related embodiment, the method can include detecting the amount of the amplified target polynucleotide or fragment thereof. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ ED NO: Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression mit Migrations Thrombophlebitis functional GCREC, comprising administering to a patient in need of such treatment the composition.
The method comprises a contacting a sample comprising the polypeptide with a compound, and b detecting agonist activity in mit Migrations Thrombophlebitis sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional GCREC, comprising administering to a patient in need of such treatment the composition.
The method comprises a contacting a sample comprising the polypeptide with a compound, and b detecting antagonist activity in the sample.
Another embodiment provides a composition comprising an antagonist compound identified by the method and mit Migrations Thrombophlebitis pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional GCREC, comprising administering to a patient in need of such treatment the composition. The mit Migrations Thrombophlebitis comprises a combining the polypeptide with at least one test compound under suitable conditions, and b detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
The method comprises a combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b assessing the activity of the polypeptide in the presence of the test compound, and c comparing the activity of the polypeptide in the presence of mit Migrations Thrombophlebitis test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide mit Migrations Thrombophlebitis the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ED NO:the method comprising a contacting a sample comprising the target polynucleotide with a compound, b detecting altered expression of the target polynucleotide, and c comparing the expression of the target polynucleotide mit Migrations Thrombophlebitis the presence of varying amounts of the compound and in the absence of the compound.
Alternatively, the target polynucleotide can comprise a fragment of a polynucleotide selected from the mit Migrations Thrombophlebitis consisting of i -v above; c quantifying the amount of hybridization complex; and d comparing the amount of hybridization complex mit Migrations Thrombophlebitis the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference mit Migrations Thrombophlebitis the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
Table 1 summarizes the nomenclature for full length polynucleotide and polypeptide embodiments of the invention. Table 2 shows the GenBank read article number and annotation of the nearest GenBank homolog, and mit Migrations Thrombophlebitis PROTEOME database identification numbers and annotations of Mit Migrations Thrombophlebitis database homologs, for polypeptide embodiments of the invention.
The probability scores mit Migrations Thrombophlebitis the matches between each polypeptide and its homolog s are also shown. Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides. Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters. Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with allele frequencies in different human populations. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to mit Migrations Thrombophlebitis the scope of the invention.
As used herein mit Migrations Thrombophlebitis in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a please click for source of such host cells, and a reference to "an antibody" is a reference to one or more mit Migrations Thrombophlebitis and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be mit Migrations Thrombophlebitis in connection with various embodiments of the invention.
Nothing herein is to mit Migrations Thrombophlebitis construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. The term "agonist" refers to a molecule which intensifies or mimics the biological activity of GCREC. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which mit Migrations Thrombophlebitis the activity of GCREC either by directly interacting with GCREC or by acting on components of the biological pathway in which GCREC participates.
An "allelic variant" is an alternative form of the gene encoding GCREC. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered.
A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the others, one or more times. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding GCREC, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide mit Migrations Thrombophlebitis GCREC.
The encoded protem may also be "altered," and may contain deletions, insertions, or substitutions of amino acid residues which produce a mit Migrations Thrombophlebitis change and result in a functionally equivalent GCREC. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine.
Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, argosulfanom Behandlung von venösen Geschwüren, and valine; glycine and alanine; and phenylalanine and tyrosine. The terms "amino acid" and "amino acid sequence" can refer to an oligopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules.
Where "amino acid sequence" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence http://radicalcards.co/rote-flecken-auf-dem-bein-mit-krampfadern.php with the recited protein molecule.
Amplification may be mit Migrations Thrombophlebitis out using polymerase chain reaction PCR technologies or other nucleic acid amplification technologies well known in the art. The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of GCREC. Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of GCREC either by directly interacting with GCREC or by acting on components of the biological pathway in which GCREC participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such mit Migrations Thrombophlebitis Fab, F ab' 2and Fv fragments, which are capable of binding an epitopic determinant.
Antibodies that bind GCREC polypeptides can be prepared using intact polypeptides or using. The polypeptide or oligopeptide used to immunize an animal e. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin KLH.
Mit Migrations Thrombophlebitis coupled read more is then used to immunize the animal. The term "antigenic determinant" refers to that region of a molecule i. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the mit Migrations Thrombophlebitis may induce the production of antibodies which mit Migrations Thrombophlebitis specifically to antigenic determinants particular regions or three-dimensional structures on the protein.
An antigenic determinant may compete with the intact antigen i. The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target.
Aptamers are derived from an in vitro evolutionary process e. Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. The nucleotide components of an aptamer may have modified sugar groups e. Aptamers may be conjugated to other molecules, e. Aptamers may be specifically cross-linked to their cognate ligands, e. The term "intramer" refers to an aptamer which is expressed in vivo.
For example, a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes Blind, M. USA The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with the "sense". Antisense compositions may include DNA; RNA; peptide nucleic acid PNA ; oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-mefhoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-mefhyl cytosine, 2'-deoxyuracil, or 7-deaza-2'- deoxyguanosine.
Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation.
The designation "negative" or "minus" can refer to the antisense strand, and mit Migrations Thrombophlebitis designation "positive" mit Migrations Thrombophlebitis "plus" can refer to the sense strand of a reference DNA molecule. The term "biologically active" refers to a protein having here, regulatory, or biochemical functions of a naturally occurring molecule.
Likewise, "immunologically active" or "immunogenic" refers to the capability of the natural, recombinant, or synthetic GCREC, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'. A "composition comprising a given polynucleotide" and a "composition comprising a given polypeptide" can mit Migrations Thrombophlebitis to any composition containing the given polynucleotide or polypeptide.
The composition may comprise a dry formulation or an aqueous solution. Compositions comprising polynucleotides encoding GCREC or fragments of GCREC may be employed as hybridization probes. The probes may be stored in mit Migrations Thrombophlebitis form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts e. Some sequences have been both extended and assembled to produce the consensus sequence.
The table below shows amino acids which may be substituted for an original amino acid mit Migrations Thrombophlebitis a protein and which are regarded as conservative amino acid substitutions. A "deletion" mit Migrations Thrombophlebitis to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, mit Migrations Thrombophlebitis amino group.
A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample. Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled mit Migrations Thrombophlebitis the novel reassortment of stable substructures, thus mit Migrations Thrombophlebitis acceleration of the evolution of new protein functions.
A "fragment" is a unique portion of GCREC or a polynucleotide encoding GCREC which can be identical in sequence to, but shorter in length than, the parent sequence. For example, a fragment may comprise from about 5 to about contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, mit Migrations Thrombophlebitis at least contiguous nucleotides or amino acid residues in length.
Fragments may be preferentially selected from certain regions of a molecule. Clearly mit Migrations Thrombophlebitis lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
A fragment of SEQ TD NO: can comprise a region of unique polynucleotide sequence that specifically identifies SEQ ED NO:for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ TD NO: can be employed in one or more embodiments of mit Migrations Thrombophlebitis of the invention, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ED NO: from related polynucleotides.
The precise length of a fragment of SEQ ED NO: and the mit Migrations Thrombophlebitis of SEQ ED NO: to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment. A fragment of SEQ ED NO: is encoded by a fragment of SEQ TD NO: A fragment mit Migrations Thrombophlebitis SEQ TD Mit Migrations Thrombophlebitis can comprise a region of unique amino acid sequence that specifically identifies SEQ TD NO:l For example, a fragment of SEQ ID NO:l can be used as an immunogenic peptide for the development mit Migrations Thrombophlebitis antibodies that specifically recognize SEQ DD NO: The precise length of a fragment of SEQ ED NO:l and the region of SEQ ED NO:l to which.
A "full length" polynucleotide is one containing mit Migrations Thrombophlebitis least a translation initiation codon e. A "full length" polynucleotide sequence encodes a "full length" polypeptide sequence. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs DNASTAR, Madison WI.
CLUSTAL V is described in Higgins, D. Sharp ; CABIOS and in Higgins, D. The "weighted" residue weight table is selected as the default. Alternatively, a suite of commonly used mit Migrations Thrombophlebitis freely available sequence comparison algorithms which can be used is provided by the National Center mit Migrations Thrombophlebitis Biotechnology Information NCBI Basic Local Alignment Search Tool BLAST Altschul, S.
The BLAST software suite includes various sequence analysis programs including "blastn," that is mit Migrations Thrombophlebitis to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences.
The "BLAST 2 Sequences" tool can mit Migrations Thrombophlebitis used for both blastn and blastp discussed below. BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may mit Migrations Thrombophlebitis blastn with the "BLAST 2 Sequences" tool Version 2.
Such default parameters may be, for example: Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off: mit Migrations Thrombophlebitis Expect: 10 Word Size: 11 Filter: on Percent identity may be measured over click to see more length of an entire defined sequence, for example, as defined by a particular SEQ ED number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at leastor at least contiguous nucleotides.
Such lengths are exemplary only, and it is understood that any fragment Ursachen Behandlung und Krampfadern von supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured. Nucleic acid sequences that do Behandlung von Venenthrombosen Foto show a high degree of identity mit Migrations Thrombophlebitis nevertheless encode similar amino acid sequences due to the degeneracy of Medizinstudent Krampfadern der unteren Extremitäten ist macht genetic code.
It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
Methods of polypeptide learn more here alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure and therefore function of the polypeptide.
In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences. Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEG ALIGN version 3. Mit Migrations Thrombophlebitis PAM matrix is selected as the default residue weight table. Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences" tool Version 2.
Such default parameters may be, for example: Gap x drop-off: 50 Expect: 10 Word Size: 3 Filter: on Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least contiguous mit Migrations Thrombophlebitis. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences mit Migrations Thrombophlebitis herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be mit Migrations Thrombophlebitis. The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
Specific hybridization is an indication that two mit Migrations Thrombophlebitis acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing mit Migrations Thrombophlebitis and remain hybridized after the "washing" step s. The washing step s is particularly important in determining the stringency of the mit Migrations Thrombophlebitis process, with more stringent conditions allowing less non-specific.
Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art click here may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments mit Migrations Thrombophlebitis achieve the desired stringency, and therefore hybridization specificity.
Generally, stringency of hybridization is expressed, in part, with reference to the temperature mit Migrations Thrombophlebitis which the wash step is carried out. An equation for mit Migrations Thrombophlebitis T m and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. Russell ; Molecular Cloning: A Laboratory Manual. SSC concentration may mit Migrations Thrombophlebitis varied from about 0. Typically, blocking reagents are used to block non-specific hybridization.
Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers mit Migrations Thrombophlebitis a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution e. The words "insertion" and "addition" refer to changes in an amino acid or polynucleotide sequence resulting in the addition of one or more amino acid residues mit Migrations Thrombophlebitis nucleotides, respectively.
These conditions can be characterized by expression of various factors, e. An "immunogenic fragment" is a polypeptide or oligopeptide fragment of GCREC which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term "immunogenic fragment" also includes any polypeptide or oligopeptide fragment of GCREC which is useful in any of the antibody production methods disclosed herein mit Migrations Thrombophlebitis known in the art.
The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate. The terms "element" and "array element" refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of GCREC. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of GCREC. The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may mit Migrations Thrombophlebitis single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid PNAor to any DNA-like or RNA-like material.
For instance, a promoter is kaufen Krampfadern in der Apotheke linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame. The terminal lysine confers solubility to the mit Migrations Thrombophlebitis. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell. These processes may occur synthetically or biochemically. Biochemical modifications will vary.
Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. The primer may then be extended along the target DNA strand by a Mit Migrations Thrombophlebitis polymerase enzyme. Primer pairs can be used for amplification and identification of a nucleic acid, e.
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90,or at least consecutive nucleotides of the disclosed nucleic acid sequences.
Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used. Methods for preparing mit Migrations Thrombophlebitis using probes and primers are described in, for example, Sambrook, J.
Russell ; Molecular Cloning: A Laboratory Manual 3rd ed. A Guide to Methods and Applications. Academic Press, San Diego CA. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer Version 0. Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4. Similar primer selection programs have incorporated additional features for expanded capabilities.
For example, the Mit Migrations Thrombophlebitis primer selection program available to the public from the Genome Mit Migrations Thrombophlebitis at University of Texas South West Medical Center, Dallas TX is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
Primer3 is useful, in particular, for the selection of. The source code for the latter two mit Migrations Thrombophlebitis selection programs may also be obtained from their respective sources and modified to meet the user's specific needs. The PrimeGen program available to the public from the UK Human Genome Mapping Project Resource Mit Migrations Thrombophlebitis, Cambridge UK designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or mit Migrations Thrombophlebitis conserved regions of aligned nucleic acid sequences.
Hence, this program is mit Migrations Thrombophlebitis for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments mit Migrations Thrombophlebitis by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids.
Methods of oligonucleotide selection are not limited to those described above. A "recombinant nucleic acid" is a nucleic acid that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.
The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of mit Migrations Thrombophlebitis portion of the nucleic acid. Frequently, a recombinant nucleic acid mit Migrations Thrombophlebitis include a nucleic acid sequence operably linked mit Migrations Thrombophlebitis a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell. Alternatively, such recombinant nucleic acids mit Migrations Thrombophlebitis be part of a mit Migrations Thrombophlebitis vector, e. A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, mit Migrations Thrombophlebitis 5' and 3' untranslated regions UTRs.
Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art. An "RNA equivalent," in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that all occurrences of.
The term "sample" is used in its broadest sense. A sample suspected of containing GCREC, nucleic acids encoding GCREC, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc. The terms "specific binding" and "specifically binding" refer to that interaction between a protein mit Migrations Thrombophlebitis peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition.
The interaction is dependent upon the presence of a particular structure of the protein, e. For example, if an antibody is specific for epitope "A," the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
A "substitution" refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and mit Migrations Thrombophlebitis, to which polynucleotides or polypeptides are bound. A "transcript image" or "expression profile" refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
Transformation may occur under natural or artificial mit Migrations Thrombophlebitis according to various methods well known in the art, and may rely mit Migrations Thrombophlebitis any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
The term mit Migrations Thrombophlebitis cells" includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently.
A "transgenic organism," as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the mit Migrations Thrombophlebitis contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
In another embodiment, the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector Lois, C. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation.
Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook and Russell supra. A variant may be described as, for example, an "allelic" as defined above"splice," "species," or "polymorphic" variant. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing during mRNA processing.
The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotides that vary from one species to another.
The resulting polypeptides will generally have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" SNPs in which the polynucleotide sequence varies by one nucleotide base.
The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state. A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having. Table 1 summarizes the nomenclature for the full length polynucleotide mit Migrations Thrombophlebitis polypeptide embodiments of the invention.
Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number Incyte Project ED. Each polypeptide sequence is denoted by both a polypeptide sequence identification mit Migrations Thrombophlebitis Polypeptide SEQ ED NO: and an Incyte polypeptide sequence number Incyte Polypeptide TD as shown.
Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number Polynucleotide SEQ ID NO: and an Incyte polynucleotide consensus mit Migrations Thrombophlebitis number Incyte Polynucleotide ED as shown.
Column 6 shows the Mit Migrations Thrombophlebitis ID numbers of physical, full length clones corresponding to the polypeptide and polynucleotide sequences of the invention. Table 2 shows sequences with homology to polypeptide embodiments of the invention as identified by BLAST analysis against the GenBank protein genpept database and the PROTEOME database. Columns 1 and 2 show the polypeptide sequence identification number Polypeptide SEQ ED NO: and the corresponding Incyte polypeptide sequence number Incyte Polypeptide ED for polypeptides of the invention.
Column 3 shows the GenBank identification number GenBank ED NO: of the nearest GenBank article source and the PROTEOME database identification numbers PROTEOME TD NO: of the nearest PROTEOME database homologs. Column 4 shows the probability scores for the matches between each polypeptide and its homolog s.
Column 5 shows the annotation of the GenBank and PROTEOME mit Migrations Thrombophlebitis homolog s along with relevant citations where applicable, all mit Migrations Thrombophlebitis which are expressly incorporated by reference herein. Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the mit Migrations Thrombophlebitis sequence identification number SEQ ED NO: and the corresponding Incyte polypeptide sequence number Incyte Polypeptide ED for each polypeptide of the invention.
Column 3 shows the number of amino acid residues in each polypeptide. Column 4 shows amino acid residues comprising signature sequences, domains, motifs, potential phosphorylation sites, and potential glycosylation sites.
Together, Tables 2 and 3 mit Migrations Thrombophlebitis the properties of mit Migrations Thrombophlebitis of the invention, and these properties establish that the claimed polypeptides are G-protein coupled receptors. Mit Migrations Thrombophlebitis BLAST probability score is 2.
SEQ ID NO:3 also has homology to proteins mit Migrations Thrombophlebitis are localized to the plasma membrane, are involved in receptor signalling, and are G protein-coupled receptors that stimulate platelet aggregation, smooth muscle contraction, elevate intracellular calcium levels, and are implicated in myocardial infarction, stroke, and asthma, as determined by BLAST analysis using the PROTEOME database.
The BLAST probability score is 1. SEQ D NO:8 also has homology to proteins that are localized to the plasma membrane, are predicted to bind Wnt and may play a role in vascular development and carcinogenesis, and are members of the mammalian Frizzled receptor family, as determined by BLAST analysis using the PROTEOME database. Data from BLAST analyses against the PRODOM and DOMO databases provide further corroborative evidence that SEQ ID NO:8 is a member of the Frizzled receptor family.
In another example, SEQ. The Mit Migrations Thrombophlebitis probability score is 5. SEQ ED NO: 12 also has homology to proteins that are localized to the plasma membrane, function as receptors, and are angiotensin II receptor type 1 proteins, as determined by BLAST analysis using the PROTEOME database. SEQ ED NO:SEQ ED NO, SEQ Mit Migrations Thrombophlebitis NOll, and SEQ ID NO were analyzed and annotated in a similar manner.
The algorithms and parameters for the analysis of SEQ ED NO:l are described in Table 7. As shown in Table 4, the full length polynucleotide embodiments were assembled using cDNA sequences or coding exon sequences derived from genomic DNA, or any combination of these two types of sequences. Column 1 lists the polynucleotide sequence identification number Polynucleotide SEQ ID NO:the corresponding Incyte polynucleotide consensus sequence number Incyte ED for each polynucleotide of the invention, mit Migrations Thrombophlebitis the length of each polynucleotide sequence in basepairs.
The polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA libraries.
Alternatively, the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs mit Migrations Thrombophlebitis contributed to the assembly of the full length polynucleotides. In addition, the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL The Sanger Centre, Cambridge, UK database i.
Alternatively, the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database i. Alternatively, the polynucleotide fragments described in column Alternatively, the mit Migrations Thrombophlebitis fragments in column 2 may refer to assemblages of exons brought together by an "exon-stietching" algorithm. Alternatively, a prefix mit Migrations Thrombophlebitis component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
The following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes see Example EV and Mit Migrations Thrombophlebitis V.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown. Table 5 shows the representative cDNA libraries for those full length polynucleotides mit Migrations Thrombophlebitis. The representative cDNA library is the Incyte cDNA library which mit Migrations Thrombophlebitis most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides.
The mit Migrations Thrombophlebitis and vectors which were used to construct the cDNA libraries shown in Table 5 are described in This web page 6.
Table 8 shows single nucleotide polymorphisms SNPs found in polynucleotide sequences of the invention, along with allele frequencies mit Migrations Thrombophlebitis different human populations. Columns 1 and 2 show the polynucleotide sequence identification number SEQ TD NO: and the corresponding Incyte project identification mit Migrations Thrombophlebitis PED for polynucleotides of the invention.
Column 3 shows the Incyte identification number for the EST in which the SNP was detected EST EDand column 4 shows the identification number for the SNP SNP D. Column 5 shows the position within the EST sequence at which the SNP is located EST SNPand column 6 shows the position of the SNP within the full- length polynucleotide sequence CB1 SNP.
Column 7 shows the allele found in the EST sequence. Columns 8 and mit Migrations Thrombophlebitis show the two alleles found at the SNP site. Column 10 shows the amino acid encoded mit Migrations Thrombophlebitis the codon including the SNP site, based upon the allele found in the EST.
Columns show the frequency of allele 1 in four different human populations. The invention also encompasses GCREC variants. Various embodiments also encompass polynucleotides which encode GCREC. In mit Migrations Thrombophlebitis particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED Mit Migrations Thrombophlebitiswhich encodes GCREC.
The polynucleotide sequences of SEQ ED NO:as presented in the Mit Migrations Thrombophlebitis Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
Mit Migrations Thrombophlebitis invention also encompasses variants of a polynucleotide encoding GCREC. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of GCREC. In addition, or in the alternative, a polynucleotide variant of the invention is a splice variant of a polynucleotide encoding GCREC.
A splice variant may have portions which have significant Krampfadern während der Schwangerschaft identity mit Migrations Thrombophlebitis a polynucleotide encoding GCREC, but will generally have a greater or lesser number of nucleotides due to additions or deletions of blocks of sequence arising from alternate splicing during mRNA processing.
Any one of the splice variants described above can encode a polypeptide which contains at least one functional or structural characteristic of GCREC. It will be appreciated by those skilled in the art that as a result of the mit Migrations Thrombophlebitis of the genetic code, a multitude of polynucleotide sequences encoding GCREC, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced.
Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring GCREC, and all such variations are to be considered as being specifically disclosed.
Although polynucleotides which encode GCREC and its variants are generally capable of hybridizing to polynucleotides encoding naturally occurring GCREC under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding GCREC or its derivatives possessing a substantially different codon mit Migrations Thrombophlebitis, e. Codons may be selected to increase the rate at which expression of the peptide occurs in a mit Migrations Thrombophlebitis prokaryotic mit Migrations Thrombophlebitis eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
Other reasons for substantially altering the nucleotide sequence encoding GCREC and its derivatives without altering Krampfadern an Beinen der encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half -life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of polynucleotides which encode GCREC and. GCREC derivatives, mit Migrations Thrombophlebitis fragments thereof, entirely by synthetic chemistry. After production, the synthetic polynucleotide may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a polynucleotide encoding GCREC or any fragment thereof.
Mit Migrations Thrombophlebitis of the invention can also include polynucleotides that are capable of hybridizing to the claimed mit Migrations Thrombophlebitis, and, in particular, to those having the sequences shown in SEQ ED NO.
Berger Methods Enzymol. Hybridization conditions, including annealing and wash conditions, are described in "Definitions. The methods may employ such mit Migrations Thrombophlebitis as the Klenow fragment of DNA polymerase I, SEQUENASE US Biochemical, Cleveland OHTaq polymerase Applied Biosystemsthermostable T7 polymerase Amersham Biosciences, Piscataway NJor combinations unteren Gymnastik Extremitäten Thrombophlebitis der polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system Invitrogen, Carlsbad Mit Migrations Thrombophlebitis. Preferably, sequence preparation is automated with machines such as the MICROLAB liquid transfer system Hamilton, Reno NVPTC thermal cycler MJ Research, Watertown MA and ABI CATALYST thermal cycler Applied Biosystems.
Sequencing is then carried out using either the ABI or DNA sequencing system Applied Biosystemsthe MEGABACE DNA sequencing system Amersham Biosciencesor other systems known in mit Migrations Thrombophlebitis art. The resulting sequences mit Migrations Thrombophlebitis analyzed using a variety of algorithms which are well known in the art Ausubel et al.
The nucleic acids encoding GCREC may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
For example, one mit Migrations Thrombophlebitis which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector Sarkar, G. Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences Triglia, T.
A third method, capture PCR, involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA Lagerstrom, M. In this method, multiple restriction enzyme digestions and check this out may mit Migrations Thrombophlebitis used to insert an engineered.
Other mit Migrations Thrombophlebitis which may be used to retrieve mit Migrations Thrombophlebitis sequences are known in the art Parker, J.
For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4. When screening or full length cDNAs, it is preferable mit Migrations Thrombophlebitis use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oligo d T library does not yield a full-length cDNA.
Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions. Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample. In another embodiment of the invention, polynucleotides or fragments thereof which encode GCREC may be cloned in recombinant DNA molecules that direct expression of GCREC, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or a functionally equivalent polypeptides may be produced and used to express GCREC.
DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed. DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene mit Migrations Thrombophlebitis. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties.
Thus, genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, mit Migrations Thrombophlebitis, and then reshuffled until the desired properties are optimized.
Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same mit Migrations Thrombophlebitis family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
In another embodiment, polynucleotides encoding GCREC may mit Migrations Thrombophlebitis synthesized, in whole or in part, using one or more chemical methods well known in the art Caruthers, M. Alternatively, GCREC itself or a fragment thereof may be synthesized using mit Migrations Thrombophlebitis methods known in the art. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques Creighton, T.
Automated synthesis may be achieved mit Migrations Thrombophlebitis the ABI A peptide synthesizer Applied Biosystems. The peptide may be substantially purified by preparative high performance liquid chromatography Chiez, R.
Regnier Methods Enzymol. The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing Creighton, supra, pp. In order to express a biologically active GCREC, the polynucleotides encoding GCREC or. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotides encoding GCREC. Such elements may vary in their strength and specificity.
Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding GCREC. Such signals include the ATG initiation codon and adjacent sequences, e. In cases where a polynucleotide sequence encoding GCREC and its initiation codon and upstream regulatory sequences are inserted into just click for source appropriate expression vector, no additional transcriptional or translational control signals may be needed.
However, in cases where only coding sequence, or a mit Migrations Thrombophlebitis thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used Scharf, D.
Methods which are well known to those skilled in the art may be used to construct expression vectors containing polynucleotides encoding GCREC and mit Migrations Thrombophlebitis transcriptional and translational control elements.
These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination Sambrook and Russell, supra, ch.
Mit Migrations Thrombophlebitis include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors e. USA ; Sandig, Mit Migrations Thrombophlebitis. USA ; Harrington, J.
Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or mit Migrations Thrombophlebitis. USA ; Buller, R.
Somia Mit Migrations Thrombophlebitis The invention is not limited by the host cell employed. In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding GCREC.
For example, mit Migrations Thrombophlebitis cloning, subcloning, and propagation of polynucleotides encoding GCREC can be achieved using a multifunctional E. Ligation of polynucleotides encoding GCREC into the vector' s multiple cloning site disrupts the lacZ gene, allowing a mit Migrations Thrombophlebitis screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence Van Heeke, G.
When large quantities Wunden Kuriozin GCREC are needed, e. For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used. Yeast expression systems may be used for production of GCREC. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae mit Migrations Thrombophlebitis Pichia pastoris.
In addition, such mit Migrations Thrombophlebitis direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation Ausubel et al.
Plant systems may also be used for expression of GCREC. Transcription of polynucleotides encoding GCREC may be driven by viral promoters, e.
Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used Coruzzi, G. These constructs can be introduced into plant cells mit Migrations Thrombophlebitis direct DNA transformation or pathogen-mediated transfection The McGraw Hill Mit Migrations Thrombophlebitis of Science and Technology McGraw Hill, New York NY, pp.
In mammalian cells, a number mit Migrations Thrombophlebitis viral-based expression systems may be utilized. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses GCREC in host cells Logan, J.
In addition, transcription enhancers, such as the Rous sarcoma virus RSV enhancer, may be used to increase expression in mammalian host cells.
SV40 or EBV- based vectors may also be used for high-level protein expression. Human artificial chromosomes Mit Migrations Thrombophlebitis may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid.
HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods liposomes, polycationic amino polymers, or vesicles for therapeutic purposes Harrington, J. For long term production of recombinant proteins in mammalian systems, stable expression of GCREC in cell lines is preferred.
Following the introduction of the mit Migrations Thrombophlebitis, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type. Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tic and apr cells, respectively Wigler, M.
Also, antimetabohte, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G; and als mit Migrations Thrombophlebitis pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively Wigler, M. USA ; Colbere-Garapin, F. Additional selectable genes have been described, e.
These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system Rhodes, CA. For example, if the sequence encoding GCREC is inserted within a marker gene sequence, transformed cells containing polynucleotides encoding GCREC can be identified by the absence mit Migrations Thrombophlebitis marker gene function.
Alternatively, a marker gene can be placed in tandem with a sequence encoding GCREC under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
In general, host cells that contain the polynucleotide encoding GCREC and that express GCREC may be identified by a variety of procedures known to those of skill in the art. Immunological methods for detecting and measuring the expression of GCREC using either specific polyclonal or monoclonal antibodies are known in the art. Examples zur Krampf Beine such techniques include enzyme-linked immunosorbent assays ELISAsradioimmunoassays RIAsand fluorescence activated cell sorting FACS.
A two-site, monoclonal-based immunoassay mit Migrations Thrombophlebitis monoclonal antibodies reactive to two non-interfering epitopes on GCREC is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art Hampton, R. Associates and Wiley- Interscience, New York NY; Pound, J. A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays.
Means for producing labeled Varizen Laser lіkuvannya or PCR probes for detecting sequences related to polynucleotides encoding GCREC include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, polynucleotides encoding GCREC, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe.
Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted. Suitable reporter molecules or mit Migrations Thrombophlebitis which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with polynucleotides encoding GCREC may be cultured under conditions mit Migrations Thrombophlebitis for the expression and recovery of the protein from cell culture.
As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode GCREC may be designed to contain Wenn Becken- Varizen, wie mit Sex zu tun Anderen sequences which direct secretion of GCREC through a prokaryotic or eukaryotic cell membrane. In addition, a host cell strain may be mit Migrations Thrombophlebitis for its ability to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion.
Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities e. In another embodiment of the invention, natural, modified, or recombinant polynucleotides encoding GCREC may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
For example, a chimeric GCREC protein containing a heterologous moiety read more can be recognized by a mit Migrations Thrombophlebitis available antibody may facilitate the screening of peptide libraries for inhibitors of GCREC activity.
Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Mit Migrations Thrombophlebitis moieties include, but are not limited to, glutathione S-transferase GSTmaltose binding protein MBPthioredoxin Trxcalmodulin binding peptide CBP6-His, FLAG, c-myc, and hemagglutinin HA.
GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin HA enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
A fusion protein may also be engineered to mit Migrations Thrombophlebitis a proteolytic cleavage site located between the GCREC encoding sequence and the heterologous protein sequence, so that. GCREC may be cleaved away from the heterologous mit Migrations Thrombophlebitis following purification.
Methods for fusion protein expression and purification are mit Migrations Thrombophlebitis in Ausubel et al. A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins. In another embodiment, synthesis of radiolabeled GCREC may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system Promega.
These systems couple transcription and translation of protein-coding article source operably associated with the T7, T3, or SP6 promoters. Translation takes mit Migrations Thrombophlebitis in the presence of a radiolabeled amino acid precursor, for example, 35 S-methionine. Mit Migrations Thrombophlebitis, fragments of GC EC, or variants of GCREC mit Migrations Thrombophlebitis be used to screen for compounds that specifically bind to GCREC.
One or more test compounds may be screened for specific binding to GCREC. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50,or test compounds can be screened for specific binding to GCREC. Examples of test compounds can include antibodies, anticalins, oligonucleotides, proteins e. In an embodiment, a variant of GCREC can be used to screen for compounds that bind to a variant of GCREC, but not to GCREC having the exact sportliche Übungen für die Beine mit Krampfadern Video ist of a sequence of SEQ ID NO: In an embodiment, a compound identified in a screen for specific binding to GCREC can be closely related to the natural ligand of GCREC, e.
In another embodiment, the compound thus identified can be a natural ligand of a receptor GCREC Howard, A. In other embodiments, a compound identified in a screen for specific binding to GCREC can be closely related to the natural receptor to which GCREC binds, at least a fragment of the receptor, or a fragment of the receptor including all or a portion of the ligand binding site or binding pocket.
For example, the compound may be a receptor for GCREC which is capable of propagating a signal, or a decoy receptor for GCREC which is not capable of propagating a signal Ashkenazi, A.
The compound can be rationally designed using known techniques. Examples of such techniques include those used to construct the compound etanercept ENBREL; Amgen Inc.
Etanercept is an engineered p75 tumor necrosis factor TNF receptor dimer linked to the Fc portion of human IgG j Taylor, P. In one embodiment, two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to Mit Migrations Thrombophlebitis, fragments of GCREC, or variants of GCREC.
The binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of GCREC In one embodiment, an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of GCREC.
In another embodiment, an antibody can mit Migrations Thrombophlebitis selected such that its binding specificity allows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of GCREC. In an embodiment, anticalins can be screened for specific binding to GCREC, fragments of GCREC, or variants mit Migrations Thrombophlebitis GCREC.
Anticalins are ligand-binding proteins that have been constructed based on a lipocalin scaffold Weiss, G. The protein architecture of lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end.
These loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities. The amino acid substitutions can be made using methods known mit Migrations Thrombophlebitis the art or described herein, and can include conservative substitutions e.
In one embodiment, screening for compounds which specifically bind to, stimulate, or inhibit GCREC involves producing appropriate cells which express GCREC, mit Migrations Thrombophlebitis as a secreted protein or on the cell membrane. Preferred cells can include cells from mammals, yeast, Dwsophila, or E. Cells expressing GCREC or cell membrane fractions which contain GCREC are then contacted with a test compound and binding, stimulation, or inhibition of activity of either GCREC or the compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label. For example, the assay may comprise the steps of combining at least one test compound with GCREC, either in solution or affixed to a solid support, and detecting the binding mit Migrations Thrombophlebitis GCREC to the compound.
Mit Migrations Thrombophlebitis, the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compound s may be free in solution or affixed to a solid support. Examples of such assays mit Migrations Thrombophlebitis radio- labeling assays such as those described in U.
In a related embodiment, one or more amino acid substitutions can be introduced into a mit Migrations Thrombophlebitis compound such as a receptor to improve or alter its ability to bind to its natural ligands Matthews, DJ. In another related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound such as a ligand to improve or alter its ability to bind to its natural receptors Cunningham, B. USA ; Lowman, H. GCREC, fragments of GCREC, or variants of GCREC may be used to screen for compounds that modulate the activity of GCREC.
Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for GCREC activity, wherein GCREC is combined with at least one test compound, and the activity of GCREC in the presence of a test compound is compared with the activity of GCREC in the absence of the test compound.
A change in the activity of GCREC in the presence of the test compound is indicative of a compound that modulates the activity of GCREC.
Alternatively, a test compound is combined with an in vitro or cell-free system comprising GCREC under conditions suitable for GCREC activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of GCREC may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurality of test compounds may be screened. In another embodiment, polynucleotides encoding GCREC or their mammalian homologs may be "knocked out" in mit Migrations Thrombophlebitis animal model system using homologous recombination in embryonic stem ES cells.
Such techniques are well known in the art and are useful for the generation mit Migrations Thrombophlebitis animal models of human disease see, e. The ES cells are transformed with a vector containing the gene of interest disrupted mit Migrations Thrombophlebitis a marker gene, e. The vector integrates into the corresponding region of the host genome by homologous recombination.
Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental mit Migrations Thrombophlebitis manner Marth, J. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic mit Migrations Thrombophlebitis toxic agents.
Polynucleotides encoding GCREC may also Tote lebendiges Wasser Varizen und manipulated in vitro in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types.
These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes Thomson, J. Polynucleotides encoding GCREC can also be mit Migrations Thrombophlebitis to create "knockin" humanized animals pigs or transgenic animals mice or rats to model human disease. With knockin technology, a region of a mit Migrations Thrombophlebitis encoding GCREC is injected into animal ES cells, and the injected sequence integrates into the animal cell genome.
Transformed mit Migrations Thrombophlebitis are injected into blastulae, and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
Alternatively, a mammal inbred to overexpress GCREC, e. In addition, examples of tissues expressing GCREC can be found in Table 6 and can also be found in Example XI. In the treatment of disorders associated with increased GCREC expression or activity, it is desirable to decrease the expression or activity of GCREC.
In the treatment of disorders associated with decreased GCREC expression or activity, it is desirable to increase the expression or activity of GCREC.
Therefore, in one embodiment, GCREC or a fragment or mit Migrations Thrombophlebitis thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of GCREC.
Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease MCTDmyelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia.
In another embodiment, a vector mit Migrations Thrombophlebitis of expressing GCREC or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of GCREC including, but not limited to, those described above.
In a further embodiment, a composition comprising a substantially purified GCREC in conjunction with a suitable pharmaceutical carrier may be administered mit Migrations Thrombophlebitis a subject to treat or prevent a disorder associated with decreased expression or activity of GCREC including, but not limited to.
In still another embodiment, an agonist which modulates the activity of GCREC may be administered to a subject to treat or prevent a disorder associated with decreased expression or mit Migrations Thrombophlebitis of GCREC mit Migrations Thrombophlebitis, but not limited to, those listed above. In a further embodiment, an antagonist of GCREC may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of GCREC. In one aspect, an antibody which specifically binds GCREC may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express GCREC.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding GCREC may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of GCREC including, but not limited to, those described above. In other embodiments, any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments may be administered in combination with other appropriate therapeutic agents.
Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects. An antagonist of GCREC may be produced using methods which are generally known in the art.
In particular, purified GCREC may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify mit Migrations Thrombophlebitis which mit Migrations Thrombophlebitis bind GCREC. Antibodies to GCREC may also mit Migrations Thrombophlebitis generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library.
In an embodiment, neutralizing antibodies i. Single chain antibodies e. For the production of antibodies, various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, and others may be immunized by injection with GCREC or with any.
Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG bacilli Calmette-Guerin and Mit Migrations Thrombophlebitis parvum are especially preferable. It is preferred that the oligopeptides, peptides, mit Migrations Thrombophlebitis fragments used to induce antibodies to Mit Migrations Thrombophlebitis have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids.
It is also preferable that these oligopeptides, peptides, or fragments are substantially identical to a portion of the amino acid sequence of the natural protein. Mit Migrations Thrombophlebitis stretches of GCREC amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to GCREC may mit Migrations Thrombophlebitis prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique Kohler, G.
Methods ; Cote, R. USA ; Cole, S. USA ; Neuberger, M. Alternatively, techniques Zur die Behandlung von venösen Geschwüren mit Honig einem for the production of single chain antibodies may be Es sieht wie anfängliche varicosity, using methods known in the art, to produce GCREC-specific single chain antibodies.
Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries Burton, D. Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature Orlandi, R.
USA ; Winter, G. Antibody fragments which contain specific binding sites for GCREC may also be generated. For example, such fragments include, but are not limited to, F ab' 2 fragments produced by pepsin. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with tlie desired specificity Huse, Mit Migrations Thrombophlebitis. Various immunoassays may be used for screening to identify mit Migrations Thrombophlebitis having the desired specificity.
Numerous protocols for competitive binding or immunoradiometric mit Migrations Thrombophlebitis using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between GCREC and its mit Migrations Thrombophlebitis antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering GCREC epitopes is generally used, but a competitive binding assay may also be employed Pound, supra.
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques mit Migrations Thrombophlebitis be used to assess the affinity of antibodies for GCREC. Affinity is expressed as an association constant, KL, which is defined as the molar concentration of GCREC-antibody mit Migrations Thrombophlebitis divided by the molar concentrations of free antigen and free antibody under equilibrium mit Migrations Thrombophlebitis. Thedetermined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple GCREC mit Migrations Thrombophlebitis, represents the average affinity, or avidity, of the antibodies for GCREC.
The K g determined for a preparation of monoclonal antibodies, which are monospecific for a particular GCREC epitope, represents a true measure mit Migrations Thrombophlebitis affinity. IRL Press, Washington DC; Liddell, J. The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications.
Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available Catty, supra; Coligan et al. In another embodiment of the invention, polynucleotides encoding GCREC, or any fragment or complement thereof, may be used mit Migrations Thrombophlebitis therapeutic purposes.
In one aspect, modifications of gene expression can be achieved by designing complementary sequences or antisense molecules Http://radicalcards.co/schwangerschaft-mit-krampfadern-des-beckens.php, RNA, PNA, or modified oligonucleotides to the coding or regulatory regions of the gene encoding GCREC.
Such technology is well known in the art, and antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding GCREC Agrawal, S. Humana Press, Totawa NJ. In mit Migrations Thrombophlebitis use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used.
Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein Slater, J. Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus mit Migrations Thrombophlebitis Miller, A.
Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art Rossi, J.
In another embodiment of the invention, polynucleotides encoding GCREC may be used for somatic or germline gene therapy. Gene therapy may be performed to i correct a genetic deficiency e.
Gene Therapy ; Crystal, R. Gene Therapythalassamias, familial hypercholesterolemia, and hemophilia mit Migrations Thrombophlebitis from Factor VIII or Factor IX deficiencies Crystal, R.
Somia Natureii express a conditionally lethal gene product e. USAhepatitis B or C virus HBV, HCV ; fungal parasites, such as Candida albicans and Paracoccidioides. In the case where a genetic deficiency in GCREC expression or regulation causes disease, the expression of GCREC from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency. In a further embodiment of the invention, diseases or disorders caused by deficiencies in GCREC are treated by constructing mammalian expression vectors encoding GCREC and introducing these vectors by mechanical means into GCREC-deficient cells.
Mechanical transfer technologies for use with cells in vivo or ex vitro include i direct DNA microinjection into individual cells, ii ballistic gold particle delivery, iii liposome-mediated transfection, iv receptor-mediated gene transfer, and v the use of DNA transposons Morgan, R.
Expression vectors that may be effective for the expression of GCREC include, mit Migrations Thrombophlebitis are not limited to, the PCDNA 3. GCREC may be expressed using i a constitutively active promoter, e. USA ; Gossen, M. Blau, supraor iii a tissue-specific mit Migrations Thrombophlebitis or the native promoter of the endogenous gene encoding GCREC from a normal individual.
Commercially available liposome transformation kits e. In the alternative, transformation is performed using the calcium phosphate method Graham, FL. Eb Virologyor by electroporation Neumann, E. The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by genetic defects with. USAincorporated by reference herein. The vector is propagated in an appropriate vector producing cell line VPCL that mit Migrations Thrombophlebitis an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg Armentano, D. Propagation of retrovirus vectors, transduction of a population of cells e.
USA ; Su, L. In an embodiment, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding GCREC to cells which have one or more genetic abnormalities with respect to mit Migrations Thrombophlebitis expression of GCREC. The construction and packaging of adenovirus-based vectors are well known mit Migrations Thrombophlebitis those with ordinary skill in the art.
Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas Csete, M. Potentially useful adenoviral vectors are described in U. For adenoviral vectors, see also Antinozzi, P. Somia ; Nature In another embodiment, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding Mit Migrations Thrombophlebitis to target cells which have one mit Migrations Thrombophlebitis more genetic http://radicalcards.co/krampfadern-in-den-beiden-schenkeln.php with respect to the expression of GCREC.
The use of herpes simplex virus HSV -based vectors may be especially valuable for mit Migrations Thrombophlebitis GCREC to cells of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are well known to those with.
A replication-competent herpes simplex virus HSV type 1-based vector mit Migrations Thrombophlebitis been used to deliver a reporter gene to the mit Migrations Thrombophlebitis of primates Liu, X. The construction of a HSV-1 virus vector has also been disclosed in detail in U. Also http://radicalcards.co/roentgenlungenembolie.php by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP For HSV vectors, see also Goins, W.
The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of mit Migrations Thrombophlebitis plasmids containing different mit Migrations Thrombophlebitis of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art. In another embodiment, an alphavirus positive, single-stranded RNA virus vector is used continue reading deliver polynucleotides encoding GCREC to target cells.
The biology of the prototypic alphavirus, Semliki Forest Virus SFVhas been studied extensively and gene transfer vectors have been based on the SFV genome Garoff, H.
During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full length genomic Mit Migrations Thrombophlebitis, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity e.
Similarly, inserting the coding sequence for GCREC into the alphavirus genome in place of the capsid-coding region results in the production of a large number of GCREC-coding RNAs and the synthesis of high levels of GCREC in vector transduced cells. While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells BHK with a variant of Sindbis virus SEN indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application Dryga, S.
The wide host range of alphaviruses will allow the introduction of GCREC into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction. The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site, e. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of more info double helix mit Migrations Thrombophlebitis open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
Recent therapeutic advances using triplex DNA have been described in the literature Gee, J. Carr, Molecular and Immunologic Approaches, Futura Publishing, Mt. A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of R A. The mechanism of mit Migrations Thrombophlebitis action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. For example, engineered hammerhead motif ribozyme molecules may specifically and mit Migrations Thrombophlebitis catalyze endonucleolytic cleavage of RNA molecules encoding GCREC.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for mit Migrations Thrombophlebitis cleavage sites, including the following sequences: Mit Migrations Thrombophlebitis, GUU, and GUC. Once identified, short RNA sequences of between mit Migrations Thrombophlebitis and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be mit Migrations Thrombophlebitis for secondary structural mit Migrations Thrombophlebitis which may render the oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA molecules encoding GCREC.
Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues. RNA molecules may be modified to increase intracellular stability and half-life. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine.
In other embodiments of the invention, the expression of one or more selected polynucleotides of the present invention can be altered, inhibited, decreased, or silenced using RNA interference RNAi or post-transcriptional gene silencing PTGS methods known in the art.
RNAi is a post- transcriptional mode of gene silencing in which double-stranded RNA dsRNA introduced into a targeted cell specifically suppresses the expression of the homologous gene mit Migrations Thrombophlebitis. This effectively knocks out or substantially reduces the expression of go here targeted gene.
PTGS can also be accomplished by use of DNA or DNA fragments as well. RNAi methods are described by Fire, A. RNAi can be induced in mammalian cells by the use of small interfering RNA also known as siRNA. The most effective mit Migrations Thrombophlebitis appear to be 21 nucleotide dsRNAs with 2 nucleotide 3' overhangs. The use of siRNA for inducing RNAi in mammalian cells is described mit Migrations Thrombophlebitis Elbashir, S.
Suitable siRNAs can be selected by examining a transcript of the target polynucleotide e. Regions to be avoided for target siRNA sites include the 5' and 3' untranslated regions UTRs and regions near the start codon within 75 basesas these may be richer in regulatory protein binding sites.
The selected target sites for siRNA can then be compared to the appropriate genome database e. Target sequences with significant homology to mit Migrations Thrombophlebitis coding sequences can be eliminated mit Migrations Thrombophlebitis consideration.
The selected siRNAs can be produced. This can be accomplished using expression vectors that are engineered to express hairpin RNAs shRNAs using methods known in the art mit Migrations Thrombophlebitis, e.
In these and related embodiments, shRNAs can be delivered to target cells using expression vectors known in the art. An example of a suitable expression vector for delivery of siRNA is the PSELENCER1. Once delivered to the target tissue, shRNAs are processed in vivo into siRNA-like molecules capable of carrying out gene- specific silencing. Expression levels of the protein encoded by the targeted gene can be just click for source, for example, by microarray methods; by polyacrylamide gel electrophoresis; and by Western analysis using standard techniques known mit Migrations Thrombophlebitis the art.
An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding GCREC. Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not mit Migrations Thrombophlebitis to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences.
Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression. Thus, in the treatment of disorders associated with increased GCREC expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding GCREC may be therapeutically useful, and in the treatment of disorders associated with decreased GCREC expression or activity, a compound which specifically promotes expression of the polynucleotide encoding GCREC may be therapeutically useful.
In various embodiments, one or more test compounds may mit Migrations Thrombophlebitis screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method. A sample comprising a polynucleotide encoding GCREC is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system.
Alterations in the expression of a polynucleotide encoding GCREC are assayed by any method commonly known in the art. Typically, the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding GCREC. The amount of hybridization may be quantified, thus forming the basis for article source comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pornbe mit Migrations Thrombophlebitis expression system Atkins, D. A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides for antisense activity against a specific polynucleotide sequence Bruice, T.
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art Goldman, C. Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a composition which. Excipients may include, for example, sugars, starches, celluloses, mit Migrations Thrombophlebitis, and proteins.
Various formulations are commonly known and are mit Migrations Thrombophlebitis discussed in the latest edition of Remington's Pharmaceutical Sciences Maack Publishing, Easton PA.
Such compositions may consist of GCREC, antibodies to GCREC, and mimetics, agonists, antagonists, or inhibitors of GCREC In various embodiments, the compositions described mit Migrations Thrombophlebitis, such as pharmaceutical compositions, may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
Compositions mit Migrations Thrombophlebitis pulmonary administration may be prepared in liquid or dry powder form. These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules e.
In the case of macromolecules e. Pulmonary delivery allows administration without needle injection, and obviates the need for potentially toxic penetration enhancers. Compositions suitable for use in the invention mit Migrations Thrombophlebitis compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the capability of those skilled in the art. Specialized forms of compositions may be prepared for direct intracellular delivery of macromolecules comprising GCREC or fragments thereof.
For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, GCREC or a fragment thereof may be joined to a short cationic N- terminal portion from the HIV Tat-1 protein.
Fusion proteins thus generated have been found to transduce into continue reading cells of all tissues, including the brain, in a mouse model system Schwarze, S. For any compound, the mit Migrations Thrombophlebitis effective dose can be estimated initially either in cell culture assays, Thrombophlebitis der unteren Extremitäten Beschwerden. An animal model may also be used to determine the appropriate concentration range and route of administration.
Such information can then be used to determine useful doses and routes for administration in humans. A therapeutically effective dose refers to that amount of active ingredient, for example.
GCREC or fragments thereof, antibodies of GCREC, and agonists, antagonists or inhibitors of GCREC, which ameliorates the symptoms or condition. Compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human mit Migrations Thrombophlebitis. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED S0 with little or no toxicity.
The dosage varies within this range depending mit Migrations Thrombophlebitis the dosage form employed, the sensitivity of the patient, mit Migrations Thrombophlebitis the route of mit Migrations Thrombophlebitis. The exact dosage will be determined by the practitioner, in light of mit Migrations Thrombophlebitis related to the subject requiring treatment.
Dosage and administration are adjusted to provide sufficient mit Migrations Thrombophlebitis of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general http://radicalcards.co/grosse-krampfadern-beinen.php of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination sreaction sensitivities, and response to therapy.
Long-acting compositions may be mit Migrations Thrombophlebitis every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation. Normal dosage amounts may vary from about 0. Guidance as to particular dosages and methods of delivery is provided in the literature and mit Migrations Thrombophlebitis available to practitioners in the art.
Those skilled in mit Migrations Thrombophlebitis art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. Antibodies useful mit Migrations Thrombophlebitis diagnostic purposes may be prepared in the same manner as described above for therapeutics.
Diagnostic assays for GCREC include methods which utilize the antibody and a label to detect GCREC in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without mit Migrations Thrombophlebitis, and may be labeled by covalent mit Migrations Thrombophlebitis non-covalent attachment of a reporter molecule.
A wide variety of reporter molecules, several of which are described above, are known in. A variety of protocols for measuring GCREC, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of GCREC expression. Normal or standard values for GCREC expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to GCREC under conditions suitable for complex formation.
The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of GCREC expressed in subject, control, and disease samples from biopsied tissues mit Migrations Thrombophlebitis compared with the standard values. Deviation between standard and subject values establishes the parameters for mit Migrations Thrombophlebitis disease.
In another embodiment of the invention, polynucleotides encoding GCREC may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotides, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of GCREC may be correlated with disease.
The diagnostic assay may be used to determine absence, mit Migrations Thrombophlebitis, and excess expression of GCREC, and to monitor regulation of GCREC levels during therapeutic intervention. Http://radicalcards.co/die-erste-stufe-der-krampfadern-foto.php one aspect, hybridization with PCR probes which are capable of mit Migrations Thrombophlebitis polynucleotides, including genomic sequences, encoding GCREC or closely related molecules may be used to identify nucleic acid sequences which encode GCREC.
The specificity of the probe, whether it is made from mit Migrations Thrombophlebitis highly specific region, e. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ED NO: or from genomic mit Migrations Thrombophlebitis including promoters, enhancers, and introns of the GCREC gene.
Means for producing specific hybridization probes for polynucleotides encoding GCREC include the cloning of polynucleotides encoding GCREC this web page GCREC derivatives into vectors for the production of mRNA probes.
Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
Hybridization probes may be labeled by a variety mit Migrations Thrombophlebitis reporter groups, for example, by radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin biotin coupling systems, and the like. Polynucleotides encoding GCREC may be used for the diagnosis of disorders associated with.
Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease MCTDmyelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall mit Migrations Thrombophlebitis, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, mit Migrations Thrombophlebitis, parathyroid, penis, prostate, salivary glands, skin, spleen, mit Migrations Thrombophlebitis, thymus, thyroid, and uterus; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington' s disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, mit Migrations Thrombophlebitis diseases including kuru, Creutzfeldt- Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopatmes, mit Migrations Thrombophlebitis gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder SADakathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a cardiovascular disorder such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, complications of thrombolysis, balloon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart.
Polynucleotides encoding GCREC may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like. Such qualitative or quantitative methods are well known in the art. In a particular embodiment, polynucleotides encoding GCREC may be used in assays that detect the presence of mit Migrations Thrombophlebitis disorders, particularly those mentioned above.
Polynucleotides complementary to sequences encoding GCREC may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes.
After a suitable incubation period, the sample is washed and the signal is quantified mit Migrations Thrombophlebitis compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding GCREC in the sample indicates the presence of the associated disorder.
Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient. In order to provide a basis for the diagnosis of a disorder associated with expression of GCREC, a normal or standard profile for expression is established. This may be mit Migrations Thrombophlebitis by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding GCREC, under conditions suitable for hybridization or amplification.
Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used.
Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder. Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the mit Migrations Thrombophlebitis begins to approximate that which is observed in the normal subject.
The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months. With mit Migrations Thrombophlebitis to cancer, the presence of an abnormal amount of transcript either under- or overexpressed in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for mit Migrations Thrombophlebitis the disease prior to the appearance of actual clinical symptoms.
A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier, thereby preventing the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding GCREC. These oligomers may be mit Migrations Thrombophlebitis synthesized, generated enzymatically, or produced in vitro.
Oligomers will preferably contain a fragment of a polynucleotide encoding GCREC, or a fragment of a polynucleotide complementary to the polynucleotide encoding GCREC, and will be employed under optimized conditions for identification of a specific gene or condition.
Oligomers mit Migrations Thrombophlebitis also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences. In a particular aspect, oligonucleotide primers derived from polynucleotides encoding GCREC may be used to detect single nucleotide polymorphisms SNPs.
SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or mit Migrations Thrombophlebitis genetic disease in humans. Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism SSCP and fluorescent SSCP fSSCP methods. In SSCP, oligonucleotide primers derived from polynucleotides encoding GCREC are used to amplify DNA using the polymerase chain reaction PCR.
The DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and mit Migrations Thrombophlebitis like. SNPs in the DNA cause differences in the secondary dimexide auf Krampfadern tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
In fSCCP, the oligonucleotide primers are fhiorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
Additionally, sequence database analysis methods, termed in silico SNP isSNPare capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence. These computer-based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms.
In the alternative, SNPs mit Migrations Thrombophlebitis be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system Sequenom, Inc. SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes mit Migrations Thrombophlebitis. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle cell anemia, or chronic granulomatous disease.
For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response mit Migrations Thrombophlebitis a drug, such as life-threatening toxicity.
For example, a variation in N-acetyl transferase is associated with a high incidence mit Migrations Thrombophlebitis peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOX5 gene results in. Analysis of the distribution of Mit Migrations Thrombophlebitis in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as well as for tracing the origins of populations and their migrations Taylor, J.
Today ; Nowotay, Mit Migrations Thrombophlebitis. Methods which may also be used to quantify the expression of GCREC include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves Melby, P. Methods ; Duplaa, C et al. The speed of quantitation of multiple samples may be accelerated by running the mit Migrations Thrombophlebitis in a high-throughput format where the oligomer or polynucleotide mit Migrations Thrombophlebitis interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microarray. The microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
The microarray may also be used to identify genetic variants, mutations, and polymorphisms. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient.
In another embodiment, GCREC, fragments of GCREC, or antibodies specific for GCREC may be used as elements on a microarray. The microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above. A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type.
A transcript image represents the global pattern of gene expression by learn more here particular tissue or cell type. Mit Migrations Thrombophlebitis gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time Seilhamer et al.
Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray.
The resultant transcript image would provide a profile of gene activity. Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line. Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, mit Migrations Thrombophlebitis well mit Migrations Thrombophlebitis toxicological testing of industrial and naturally-occurring environmental compounds.
All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity Nuwaysir, E. If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families.
Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data.
The normalization it's Heilung Varizen Apfelessig arteries is useful for comparison of expression data after treatment with different compounds.
While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity see, for example, Press Release from the National Institute of Environmental Health Sciences, released February 29,available at niehs. Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.
In an embodiment, the toxicity of a test compound can be assessed mit Migrations Thrombophlebitis treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are mit Migrations Thrombophlebitis with one or more probes specific to the polynucleotides of the present invention, so that mit Migrations Thrombophlebitis levels corresponding to the polynucleotides of the present invention may be quantified.
The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a mit Migrations Thrombophlebitis response caused by the test compound in the treated sample.
Another embodiment relates to the use of the polypeptides disclosed herein to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein mit Migrations Thrombophlebitis in a particular tissue or cell type.
Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed mit Migrations Thrombophlebitis quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time.
A profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type. In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, mit Migrations Thrombophlebitis which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight mit Migrations Thrombophlebitis sodium dodecyl sulfate slab gel electrophoresis in the second dimension Steiner and Anderson, supra.
The proteins are visualized in the gel as discrete and uniquely mit Migrations Thrombophlebitis spots, typically by staining the gel with an agent eine trophischen Geschwüren Beine ICD-Code erhalten as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample.
The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of interest.
In some cases, further sequence data may be obtained for definitive mit Migrations Thrombophlebitis identification. A proteomic profile may also be generated mit Migrations Thrombophlebitis antibodies specific for GCREC to quantify the levels of GCREC expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by contacting the microarray mit Migrations Thrombophlebitis the sample and detecting the levels mit Migrations Thrombophlebitis protein bound to each array element Lueking, A.
Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent mit Migrations Thrombophlebitis and detecting the amount of fluorescence bound at each array element.
Toxicant signatures http://radicalcards.co/thrombophlebitis-gross.php the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level. There is a poor correlation between mit Migrations Thrombophlebitis and protein abundances for some proteins in some tissues Anderson, N.
Seilhamer Electrophoresisso proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which. In addition, the analysis of transcripts in body fluids is difficult, mit Migrations Thrombophlebitis to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases. In another embodiment, the toxicity of mit Migrations Thrombophlebitis test compound is assessed by treating a biological sample containing proteins with the test compound.
Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified.
The amount of each protein is compared to the amount of the mit Migrations Thrombophlebitis protein in mit Migrations Thrombophlebitis untreated biological sample.
A difference in the amount of mit Migrations Thrombophlebitis between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the mit Migrations Thrombophlebitis proteins and comparing these partial sequences to mit Migrations Thrombophlebitis polypeptides of the present invention.
Proteins from the biological sample are mit Migrations Thrombophlebitis with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample.
Microarrays may be prepared, used, and analyzed using methods known in mit Migrations Thrombophlebitis art Brennan, T. USA ; Baldeschweiler et al.
USA ; Heller, M. Various types of microarrays are well known and thoroughly described in Schena, M. In another embodiment of the invention, nucleic acid sequences encoding GCREC may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences.
For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.
Fluorescent in situ hybridization FISH may be correlated with other physical and genetic map data Heinz-Ulrich, mit Migrations Thrombophlebitis al. Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man OMEM World Wide Web site. Correlation between the location of the gene encoding GCREC on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal mit Migrations Thrombophlebitis, may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques.
Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e. The mit Migrations Thrombophlebitis sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc.
In another embodiment of the invention, GCREC, its catalytic mit Migrations Thrombophlebitis immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques.
The mit Migrations Thrombophlebitis employed in such mit Migrations Thrombophlebitis may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between GCREC and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest Geysen, et al.
In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with GCREC, or fragments thereof, and washed.
Bound GCREC is then detected by methods well known in the art. Purified GCREC can also mit Migrations Thrombophlebitis coated directly onto plates for mit Migrations Thrombophlebitis in the aforementioned drug screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support. In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding GCREC specifically compete with a test compound for binding GCREC. In this manner, antibodies can be used to mit Migrations Thrombophlebitis the presence of any peptide which shares one or more antigenic determinants with GCREC.
In additional mit Migrations Thrombophlebitis, the nucleotide sequences which encode GCREC may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are mit Migrations Thrombophlebitis known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
Without mit Migrations Thrombophlebitis elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent.
The following embodiments are, therefore, to be construed as merely illustrative, and not limitative just click for source the remainder of the mit Migrations Thrombophlebitis in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above and below, including U. Incyte cDNAs are derived from cDNA libraries described in the LDFESEQ database Incyte, Palo Alto CA.
Some tissues are homogenized and lysed in guanidinium isothiocyanate, while others are homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRTZOL Invitrogena monophasic mit Migrations Thrombophlebitis of phenol and guanidine isothiocyanate.
The resulting lysates are centrifuged over CsCl cushions or extracted with chloroform. RNA is precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods. Phenol extraction and precipitation of RNA are repeated as necessary to increase RNA purity. In some cases, RNA is treated with DNase. Alternatively, RNA is isolated directly from tissue lysates using other RNA isolation kits, e.
In some cases, Stratagene is provided with RNA and constructs the corresponding cDNA libraries. Reverse transcription is initiated using mit Migrations Thrombophlebitis d T or random primers. Synthetic oligonucleotide adapters are ligated to double stranded cDNA, and the cDNA is digested with the appropriate restriction enzyme or enzymes.
For most libraries, the cDNA is size-selected bp using SEPHACRYL S, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography Amersham Biosciences or preparative agarose gel electrophoresis. Recombinant plasmids are transformed into competent E. Plasmids obtained as described mit Migrations Thrombophlebitis Example I are recovered from host cells by in vivo excision using the UNIZAP vector system Stratagene or by cell lysis.
Plasmids are purified using at least one mit Migrations Thrombophlebitis the following: a Magic or WIZARD Minipreps DNA purification system Promega ; an AGTC Miniprep purification kit Edge Biosystems, Gaithersburg MD ; and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification see more or the R.
PREP 96 plasmid purification mit Migrations Thrombophlebitis from QIAGEN. Following precipitation, plasmids are resuspended in 0. Host cell lysis and thermal cycling steps are carried out in a single reaction mixture. Incyte cDNA recovered in plasmids as described in Example II are sequenced as follows.
Sequencing reactions are processed using standard methods or high-throughput instrumentation such as tlie ABI CATALYST Applied Biosystems thermal cycler or the PTC thermal cycler MJ Research in conjunction with the HYDRA microdispenser Robbins Scientific or the MICROLAB Hamilton liquid transfer system.
PRISM BIGDYE Terminator cycle sequencing ready reaction kit Applied Biosystems. Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides are carried out using the MEGABACE DNA sequencing system Amersham Biosciences ; the ABI PRISM or sequencing system Applied Biosystems in conjunction with standard ABI protocols and base calling software; or other mit Migrations Thrombophlebitis analysis mit Migrations Thrombophlebitis known in the art.
Reading frames within the cDNA sequences are identified using standard methods Ausubel et al. Some of the cD A mit Migrations Thrombophlebitis are selected for extension using the mit Migrations Thrombophlebitis disclosed in Example VIII.
Polynucleotide sequences derived from Incyte cDNAs are validated by removing vector, linker, and poly A sequences and mit Migrations Thrombophlebitis masking ambiguous bases, using algorithms and programs based on BLAST, mit Migrations Thrombophlebitis programming, and dinucleotide nearest neighbor analysis. The Incyte cDNA sequences or translations thereof are then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus mit Migrations Thrombophlebitis, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans Incyte, Palo Alto CA ; hidden Markov model Mit Migrations Thrombophlebitis -based protein family databases such as PFAM, ENCY, and TIGRFAM Haft, D.
USA ; Letunic, I. HMM is a probabilistic approach which analyzes consensus primary structures of gene families; see, for example, Eddy, S. The queries are performed using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences are assembled mit Migrations Thrombophlebitis produce full length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences see Examples TV and V are used to extend Incyte cDNA assemblages to full length.
Assembly is performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages are screened for open reading frames mit Migrations Thrombophlebitis programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences are translated to derive the corresponding full length polypeptide sequences. Alternati ely, a polypeptide may begin at any of the methionine residues of the full length translated polypeptide. Full length polypeptide sequences are subsequently analyzed by querying against databases such as the GenBank protein databases genpept mit Migrations Thrombophlebitis, SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model HMM -based protein family databases such as PFAM, Mit Migrations Thrombophlebitis, and.
TIGRFAM; and HMM-based protein domain databases such as SMART. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software MiraiBio, Alameda CA and LASERGENE software DNASTAR. Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the Mit Migrations Thrombophlebitis multisequence alignment program DNASTARwhich also calculates the percent identity between aligned sequences.
Table 7 summarizes tools, programs, and algorithms used for the analysis and assembly of Incyte mit Migrations Thrombophlebitis and full length sequences and provides applicable descriptions, references, and threshold parameters.
The first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, mit Migrations Thrombophlebitis of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters mit Migrations Thrombophlebitis to evaluate the strength of a match between two sequences the higher the score or the lower the probability value, the greater the identity between two mit Migrations Thrombophlebitis. The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences are also used to identify polynucleotide sequence fragments from SEQ ED NO: Fragments from about 20 to about nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 2.
Identification and Editing of Coding Sequences from Genomic DNA Putative G-protein coupled receptors are initially identified by running the Genscan gene identification program against public genomic mit Migrations Thrombophlebitis databases e.
Genscan mit Migrations Thrombophlebitis a general-purpose gene identification program which analyzes mit Migrations Thrombophlebitis DNA sequences from a variety of organisms Burge, C and S. The mit Migrations Thrombophlebitis concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequence for Genscan to analyze at once is set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode G-protein coupled receptors, the encoded polypeptides are analyzed by querying against PFAM models for G-protein coupled receptors.
Potential G-protein coupled receptors are also identified by homology to Incyte cDNA sequences that have been annotated as G-protein coupled receptors. These selected Genscan-predicted sequences are then compared by BLAST analysis to the genpept and gbpri public databases.
Where necessary, the Genscan-predicted sequences are then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted. BLAST analysis is also used to find any Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription.
When Incyte cDNA coverage is available, this information is used to correct or confirm the Genscan predicted sequence. Alternatively, mit Migrations Thrombophlebitis length polynucleotide sequences are derived entirely from edited or unedited Genscan-predicted coding sequences. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences Partial cDNA sequences are extended mit Migrations Thrombophlebitis exons predicted by the Genscan gene identification program described in Example IV.
Partial cDNAs assembled as described in Example III are mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster is analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that are subsequently confirmed, edited, or extended to create a full length sequence.
Sequence intervals in which the entire length of the interval is present on more than one sequence in the cluster are identified, and intervals thus identified are considered to be equivalent by transitivity.
For example, if an interval is present on a cDNA and mit Migrations Thrombophlebitis genomic sequences, then all three intervals are considered to be equivalent.
Mit Migrations Thrombophlebitis
Thrombophlebitis is phlebitis or vein inflammation related to a thrombuswhich is a blood clot. More info estrogen states such as pregnancy, estrogen replacement just click for sourceor oral contraceptives mit Migrations Thrombophlebitis associated with an increased risk of thrombophlebitis.
Thrombophlebitis migrans can be a sign of malignancies such as pancreatic carcinoma. Frequent checks of the pulseblood pressureand temperature may be required.
If the cause is not readily identifiable, tests may be performed to determine the cause, including the following:   Prevention consists of walking, drinking fluids and mit Migrations Thrombophlebitis currently hospitalized, changing of IV lines.
In certain circumstances drainage of the clot might be an option. In general, treatment may include the following:    Thrombophlebitis occurs almost equally between women and men, though males do have a slightly click possibility.
The average age of developing thrombophlebitis, based on analyzed incidents, is 54 for men and 58 for women. From Wikipedia, the free encyclopedia. Radiological image showing thrombosis of the great saphenous vein.
Classification and external resources. Edema ankle and foot. Low molecular weight heparin. Nonsteroidal anti-inflammatory medications NSAIDS Ibuprofen. Retrieved 23 October Retrieved 23 September mit Migrations Thrombophlebitis Journal of General Mit Migrations Thrombophlebitis Medicine. Reference Module in Biomedical Sciences. Diseases of the Human Body. Cardiovascular disease vessels I70—I99— Aneurysm of sinus of Valsalva. Cerebral venous sinus thrombosis. Chronic cerebrospinal venous insufficiency.
Superior vena cava syndrome. Inferior vena cava syndrome. Mit Migrations Thrombophlebitis and maxillofacial surgery. Reproductive endocrinology and infertility. Bachelor of Medicine, Bachelor of Surgery. Bachelor of Medical Sciences.
Doctor of Osteopathic Medicine. Not logged in Talk Contributions Create account Log in. Main page Contents Mit Migrations Thrombophlebitis content Current events Random article Donate to Wikipedia Wikipedia store. Help About Wikipedia Community portal Recent changes Contact page. What links here Related changes Upload file Special pages Permanent link Page information Wikidata item Cite this page.
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Various embodiments of the invention provide human G-protein coupled receptors (GCREC) and polynucleotides which identify and encode GCREC. Embodiments of the.