WO2000026362A2 - Proteins of the stomatin family and their use as target proteins for pain therapy - Google Patents
Proteins of the stomatin family and their use as target proteins for pain therapy Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- Proteins of the stomatin family and their use as target proteins for pain therapy are also known.
- the invention relates to proteins of the stomatin family and their use as target proteins for pain therapy.
- Red blood cells contain a lot of proteins, both inside the cell and in the cell envelope (cell membrane); one of the main membrane proteins is stomatin. It is absent in patients suffering from overhydrated stomatocytosis. It has long been known that in these patients the red blood cells have an increased permeability of their cell membranes to sodium and potassium. Because of this, it has been postulated that stomatin regulates an - unidentified - ion channel in the cell membrane. Research on stomatin therefore focuses on the function in erythrocytes. The human stomatin cDNA was cloned independently in 1991/92 by three groups (Stewart et al. (1992) Blood 79: 1593-1601).
- mec-2 is involved in the conversion of a mechanical stimulus into a receptor potential (mechanotransduction). This enables the worm to perceive a touch stimulus (Huang et al, (1995) Nature 378: 292-295).
- mec-2 connects an ion channel to the cytoskeleton, thus opening the ion channel through a mechanical stimulus.
- Mec-2 has a high sequence homology to stomatin (66% in the central 260 amino acids).
- the object of the invention is to identify and isolate further members of the stomatin family and to develop medical applications for this protein family. This object is achieved according to the claims.
- the invention relates to stomatin and new proteins of the stomatin family, in particular N-stomatin, comprising 287 amino acids and its fragments, mutants and polymorphisms, its coding cDNA with a length of 1847 nucleotides and their fragments, mutants and polymorphisms and the use as target proteins for pain therapy . Furthermore, it relates to the use for the development of pain therapeutics, analgesics / anesthetics, for the construction of genes, for the development of pharmaceutically relevant substances and for diagnostic kits.
- the invention is based on our own knowledge that stomatin has an important function in mechanotransduction in mammals. Stomatin occurs in the nerve cells that are responsible for the perception of touch and pain stimuli. These cells are called sensory neurons. It was found that the sensitivity to mechanical stimuli in mice that lack stomatin (knockout mice) is severely impaired.
- an in vitro skin-nerve preparation was used, which makes it possible to irritate the skin with defined stimuli and to measure the response of the nerves in the form of action potentials.
- a neuron responds to a weak stimulus with few action potentials, stronger stimuli trigger higher frequencies of action potentials. This transmits the strength of the stimulus to the brain.
- AM receptors A-Mechnanonociceptors
- Stomatin RO mice the AM receptors (A-Mechnanonociceptors), which are responsible for the perception of pain caused by strong mechanical stimulation of the skin, are clearly disturbed in their function in the Stomatin RO mice. They are unable to respond to stronger touch stimuli with higher frequencies of action potentials. Pain perception is disturbed in mice that lack stomatin.
- stomatin is an important target for pain therapy: if the function of stomatin is disturbed, the pain sensation is reduced.
- An essential part of the invention is that a protein analogous to stomatin has been found and isolated.
- the complete coding sequence was cloned using RACE (Rapid Amplification of cDNA Ends).
- This cDNA has a length of 1847 base pairs (sequence II).
- the largest open reading frame contains 864 base pairs and codes for a protein of 287 amino acids in length (sequence I).
- Stomatin and the analog protein called N-stomatin are 67% identical. In situ hybridizations were used to perform expression analysis.
- N-stomatin Since the RNA for N-stomatin is formed by all (with a few exceptions) neurons of the nervous system, N-stomatin stands for neuronal stomatin. It was also found that N-stomatin is most strongly expressed in the nervous system but to a lesser extent in various other parts of the body. Because of its high homology to stomatin, N-stomatin also has a function in mechanotransduction and pain sensation and can therefore also be used as a target protein for pain therapy according to the invention.
- N-stomatin Since stomatin is particularly involved in the warning of strong mechanical stimuli, it is likely that N-stomatin has a function in the perception of weak touch stimuli. With almost all forms of chronic pain or other diseases, even a light touch is felt to be painful. This symptom of touch-induced pain is called allodynia. Since N-stomatin is likely to be involved in the perception of light touches, this protein according to the invention is the target protein for therapeutic agents for the treatment of allodynia.
- both stomatin and N-stomatin are important for the effect of volatile anesthetics on the body.
- the mode of action of volatile anesthetics at the molecular level has so far hardly been investigated. It is only known that they act on the neurons of the central nervous system. Such a function can be assumed especially for N-stomatin, since it is expressed by all neurons of the central nervous system (Stomatin, however, only by a few).
- mice were anesthetized with CO 2 and killed by decapitation.
- the skin of the right hind leg was extracted together with the saphenous nerve, an afferent nerve that innervates this area of the skin.
- the skin was in one Organ chamber with hairy side down. This chamber was permanently filled with 32 ° C warm SIF buffer (synthetic interstitial fluid; 2mM CaCl 2 , 5.5mM glucose, 10mM Hepes, 3.5mM KC1, 0.7mM MgS0 4 , 123mM NaCl, 1.5mM NaH 2 P0 4 , 9.5mM Na Gluconate, 7.5mM sucrose).
- SIF buffer synthetic interstitial fluid
- the nerve was guided into a lead chamber, which is connected to the organ chamber, and split into finer bundles of axons using fine forceps. For extracellular derivation, these were placed in succession on a silver electrode. If you stimulate the receptive field of one of the neurons whose axon is currently touching the lead electrode, you can determine the properties of a single sensory neuron. First, the receptive field was stimulated electrically and the delay until an action potential was triggered was measured. The line speed was calculated from this time delay and the distance between the receptive field and the lead electrode. The receptive field was then stimulated with von Frey hair in order to determine the activation threshold. Finally, the stimulus response function was measured. For this purpose, the receptive field of the neuron was stimulated with permanent stimuli of various strengths by placing the rounded end of a fine metal rod on the receptive field for 10 seconds with the help of a micrometer screw.
- the specific primers tcacgcagggaaacacaat and tcagctggaggcgatgag were used for them.
- the first PCR was carried out with the Oligo dT Anchor Primer and the specific primer agctgagacggcgctgtagattc.
- the PCR anchor primer and the specific primer gtagacgactccatccacttg were used for the second ('nested') PCR.
- the 550 bp fragment resulting from this second PCR was cloned and sequenced and assembled with the EST clone. Based on this sequence, a second RACE experiment was carried out.
- the specific primer CTC GGG TGA ATC CAT CTC ATT was used in the first PCR.
- the sequence for the second specific primer was: CAG ACG TTT GTC AAA TCT CGA.
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Abstract
Description
Proteine der Stomatinfamilie und ihre Verwendung als Zielproteine für die SchmerztherapieProteins of the stomatin family and their use as target proteins for pain therapy
Beschreibungdescription
Die Erfindung betrifft Proteine der Stomatinfamilie und ihre Verwendung als Zielproteine für die Schmerztherapie.The invention relates to proteins of the stomatin family and their use as target proteins for pain therapy.
Anwendungsgebiete der Erfindung sind die Medizin und die pharmazeutische Industrie.Areas of application of the invention are medicine and the pharmaceutical industry.
Rote Blutkörperchen (Erythrocyten) enthalten sehr viele Proteine, sowohl im Zellinneren als auch in der Zellhülle (Zellmembran); eines der Haupt-Membranproteine ist Stomatin. Es fehlt bei Patienten, die an überhydrierter Stomatocytose leiden. Seit längerem ist bekannt, daß bei diesen Patienten die roten Blutkörperchen eine vermehrte Permeabilität ihrer Zellmembranen für Natrium und Kalium aufweisen. Aufgrund dieser Tatsache wurde postuliert, dass Stomatin einen - nicht identifizierten - Ionenkanal in der Zellmembran reguliert. Die Forschung an Stomatin konzentriert sich also auf die Funktion in Erythrocyten. Die cDNA für humanes Stomatin wurde 1991/92 von drei Gruppen unabhängig voneinander kloniert (Stewart et al. (1992) Blood 79: 1593-1601). Später wurde auch die cDNA aus Maus veröffentlicht (Gallagher et al. (1995) J. Biol. Chem. 44: 26358-26363). Zusätzlich wurden einige Angaben über die biochemischen Eigenschaften dieses Proteins veröffentlicht. Stomatin ist ein Membranprotein, das wie eine Haarnadel in die Zellmembran inseriert ist; N- und C-Terminus befinden sich auf der cytoplasmatischen Seite. Stomatin bildet Oligomere mit sich selbst. Neueste Forschungsergebnisse, die in diesem Jahr veröffentlicht wurden, zeigen, daß Stomatin entgegen den Vermutungen doch nicht an der Entstehung von Stomatocytose beteiligt ist (Zhu et al, 1999, Blood 3: 2404-2410, Innes et al. 1999, Am. J. Hematol 60: 72-74) . In dem Wurm Caenorhabditis elegans wurde 1995 nachgewiesen, dass mec-2 an der Umsetzung eines mechanischen Stimulus in ein Rezeptorpotential (Mechanotransduktion) beteiligt ist. Hierdurch wird der Wurm befähigt, einen Berührungsreiz wahrzunehmen (Huang et al, (1995) Nature 378: 292-295). Dem Modell nach verbindet mec-2 einen Ionenkanal mit dem Cytoskelett und bewirkt so die Öffnung des Ionenkanals durch einen mechanischen Reiz. Mec-2 weist eine hohe Sequenzhomologie zu Stomatin auf (66% in den zentralen 260 Aminosäuren) .Red blood cells (erythrocytes) contain a lot of proteins, both inside the cell and in the cell envelope (cell membrane); one of the main membrane proteins is stomatin. It is absent in patients suffering from overhydrated stomatocytosis. It has long been known that in these patients the red blood cells have an increased permeability of their cell membranes to sodium and potassium. Because of this, it has been postulated that stomatin regulates an - unidentified - ion channel in the cell membrane. Research on stomatin therefore focuses on the function in erythrocytes. The human stomatin cDNA was cloned independently in 1991/92 by three groups (Stewart et al. (1992) Blood 79: 1593-1601). The mouse cDNA was also later published (Gallagher et al. (1995) J. Biol. Chem. 44: 26358-26363). In addition, some information about the biochemical properties of this protein has been published. Stomatin is a membrane protein that is inserted into the cell membrane like a hairpin; The N and C terminus are on the cytoplasmic side. Stomatin forms oligomers with itself. Recent research results published this year show that contrary to what is suspected, stomatin is not involved in the development of stomatocytosis (Zhu et al, 1999, Blood 3: 2404-2410, Innes et al 1999, Am. J. Hematol 60: 72-74). In 1995 the worm Caenorhabditis elegans showed that mec-2 is involved in the conversion of a mechanical stimulus into a receptor potential (mechanotransduction). This enables the worm to perceive a touch stimulus (Huang et al, (1995) Nature 378: 292-295). According to the model, mec-2 connects an ion channel to the cytoskeleton, thus opening the ion channel through a mechanical stimulus. Mec-2 has a high sequence homology to stomatin (66% in the central 260 amino acids).
Der Erfindung liegt die Aufgabe zugrunde, weitere Mitglieder der Stomatinfamilie zu identifizieren und zu isolieren sowie medizinische Anwendungsmöglichkeiten für diese Proteinfamilie zu entwickeln. Diese Aufgabe wird gemäß den Ansprüchen gelöst.The object of the invention is to identify and isolate further members of the stomatin family and to develop medical applications for this protein family. This object is achieved according to the claims.
Die Erfindung betrifft Stomatin und neue Proteine der Stomatinfamilie, insbesondere N-Stomatin, umfassend 287 Aminosäuren und seine Fragmente, Mutanten und Polymorphismen, seine kodierende cDNA mit einer Länge von 1847 Nukleotiden und deren Fragmente, Mutanten und Polymorphismen sowie die Verwendung als Zielproteine für die Schmerztherapie. Ferner betrifft sie die Verwendung zur Entwicklung von Schmerztherapeutika, Analgetika/Anästhetika, zum Aufbau von Genen, zur Entwicklung pharmazeutisch relevanter Substanzen und für diagnostische Kits.The invention relates to stomatin and new proteins of the stomatin family, in particular N-stomatin, comprising 287 amino acids and its fragments, mutants and polymorphisms, its coding cDNA with a length of 1847 nucleotides and their fragments, mutants and polymorphisms and the use as target proteins for pain therapy . Furthermore, it relates to the use for the development of pain therapeutics, analgesics / anesthetics, for the construction of genes, for the development of pharmaceutically relevant substances and for diagnostic kits.
Der Erfindung liegen die eigenen Erkenntnisse zugrunde, daß Stomatin eine wichtige Funktion in der Mechanotransduktion bei Säugetieren erfüllt. Stomatin kommt in den Nervenzellen vor, die für die Wahrnehmung von Berührungs- und Schmerzreizen verantwortlich sind. Diese Zellen nennt man sensorische Neurone. So wurde gefunden, daß die Sensivität für mechanische Reize in Mäusen, denen Stomatin fehlt (knockout Mäuse) stark beeinträchtigt ist. Für die Erarbeitung der Erfindung wurde eine in vitro Haut- Nerv-Präparation verwendet, die es erlaubt, die Haut mit definierten Stimuli zu reizen und die Antwort der Nerven in Form von Aktionspotentialen zu messen. Dabei antwortet ein Neuron auf einen schwachen Reiz mit wenigen Aktionspotentialen, stärkere Reize lösen höhere Frequenzen von Aktionspotentialen aus. Dadurch wird die Stärke des Reizes an das Gehirn weitergeleitet. Besonders die AM- Rezeptoren (A-Mechnanonociceptoren) , die verantwortlich sind für die Wahrnehmung von Schmerzen, die durch starke mechanische Stimulation der Haut hervorgerufen werden, sind in ihrer Funktion in den Stomatin RO Mäusen deutlich gestört. Sie sind nicht in der Lage, auf stärkere Berührungsreize mit höheren Frequenzen von Aktionspotentialen zu antworten. Bei Mäusen, denen Stomatin fehlt, ist also die Schmerzwahrnehmung gestört.The invention is based on our own knowledge that stomatin has an important function in mechanotransduction in mammals. Stomatin occurs in the nerve cells that are responsible for the perception of touch and pain stimuli. These cells are called sensory neurons. It was found that the sensitivity to mechanical stimuli in mice that lack stomatin (knockout mice) is severely impaired. For the development of the invention, an in vitro skin-nerve preparation was used, which makes it possible to irritate the skin with defined stimuli and to measure the response of the nerves in the form of action potentials. A neuron responds to a weak stimulus with few action potentials, stronger stimuli trigger higher frequencies of action potentials. This transmits the strength of the stimulus to the brain. In particular, the AM receptors (A-Mechnanonociceptors), which are responsible for the perception of pain caused by strong mechanical stimulation of the skin, are clearly disturbed in their function in the Stomatin RO mice. They are unable to respond to stronger touch stimuli with higher frequencies of action potentials. Pain perception is disturbed in mice that lack stomatin.
Gemäß der Erfindung ist Stomatin ein wichtiger Angriffspunkt für eine Schmerztherapie: wenn die Funktion von Stomatin gestört wird, wird das Schmerzempfinden gesenkt.According to the invention, stomatin is an important target for pain therapy: if the function of stomatin is disturbed, the pain sensation is reduced.
Ein wesentlicher Teil der Erfindung besteht darin, daß ein dem Stomatin analoges Protein gefunden und isoliert wurde. Ausgehend von einem EST-Klon aus einer Klonbank wurde mit Hilfe von RACE (Rapid Amplification of cDNA Ends) die vollständige codierende Sequenz kloniert. Diese cDNA hat eine Länge von 1847 Basenpaaren (Sequenz II). Das grösste offene Leseraster enthält 864 Basenpaare und kodiert für ein Protein von 287 Aminosäuren Länge (Sequenz I). Stomatin und das analoge Protein, genannt N-Stomatin, sind zu 67% identisch. Es wurden In Situ Hybridisierungen benutzt, um eine Expressionsanalyse durchzuführen. Da die RNA für N-Stomatin von allen (von wenigen Ausnahmen abgesehen) Neuronen des Nervensystems gebildet wird, steht N-Stomatin für neuronales Stomatin. Zudem wurde festgestellt, daß N-Stomatin am stärksten im Nervensystem aber in geringem Maße auch in verschiedenen anderen Teilen des Körpers exprimiert wird. Aufgrund seiner hohen Homologie zu Stomatin hat N-Stomatin ebenfalls eine Funktion in der Mechanotransduktion und Schmerzempfindung und ist somit gemäß der Erfindung auch als Zielprotein für eine Schmerztherapie einsetzbar.An essential part of the invention is that a protein analogous to stomatin has been found and isolated. Starting from an EST clone from a clone bank, the complete coding sequence was cloned using RACE (Rapid Amplification of cDNA Ends). This cDNA has a length of 1847 base pairs (sequence II). The largest open reading frame contains 864 base pairs and codes for a protein of 287 amino acids in length (sequence I). Stomatin and the analog protein called N-stomatin are 67% identical. In situ hybridizations were used to perform expression analysis. Since the RNA for N-stomatin is formed by all (with a few exceptions) neurons of the nervous system, N-stomatin stands for neuronal stomatin. It was also found that N-stomatin is most strongly expressed in the nervous system but to a lesser extent in various other parts of the body. Because of its high homology to stomatin, N-stomatin also has a function in mechanotransduction and pain sensation and can therefore also be used as a target protein for pain therapy according to the invention.
Da Stomatin besonders an der Warneh ung starker mechanischer Reize beteiligt ist, ist es wahrscheinlich, daß N-Stomatin eine Funktion bei der Wahrnehnung schwacher Berührungsreize besitzt. Bei fast allen Formen von chronischen Schmerzen oder auch anderen Krankheiten kommt es vor, daß schon eine leichte Berührung als schmerzhaft empfunden wird. Dieses Symptom des berührungs-induzierten Schmerzes wird als Allodynia bezeichnet. Da N-Stomatin wahrscheinlich an der Wahrnehmung leichter Berührungen beteiligt ist, ist dieses Protein gemäß der Erfindung Zielprotein für Therapeutika zur Behandlung von Allodynia.Since stomatin is particularly involved in the warning of strong mechanical stimuli, it is likely that N-stomatin has a function in the perception of weak touch stimuli. With almost all forms of chronic pain or other diseases, even a light touch is felt to be painful. This symptom of touch-induced pain is called allodynia. Since N-stomatin is likely to be involved in the perception of light touches, this protein according to the invention is the target protein for therapeutic agents for the treatment of allodynia.
Weiterhin sind sowohl Stomatin als auch N-Stomatin für die Wirkung volatiler Anästhetika auf den Körper von Bedeutung. Die Wirkungsweise volatiler Anästhetika auf molekularer Ebene ist zur Zeit noch kaum untersucht. Bekannt ist lediglich, daß sie auf die Neurone des zentralen Nervensystems wirken. Besonders für N-Stomatin kann somit eine solche Funktion angenommen werden, da es von allen Neuronen des zentralen Nervensystems exprimiert wird (Stomatin dagegen nur von einigen) .Furthermore, both stomatin and N-stomatin are important for the effect of volatile anesthetics on the body. The mode of action of volatile anesthetics at the molecular level has so far hardly been investigated. It is only known that they act on the neurons of the central nervous system. Such a function can be assumed especially for N-stomatin, since it is expressed by all neurons of the central nervous system (Stomatin, however, only by a few).
Die Erfindung soll nachfolgend durch Ausführungsbeispiele näher erläutert werden.The invention will be explained in more detail below by means of exemplary embodiments.
Physiologische Charakterisierung der sensorischen NeuronePhysiological characterization of sensory neurons
Mäuse wurden mit C02 betäubt und durch Dekapitieren getötet. Die Haut des rechten Hinterbeines wurde zusammen mit dem Nervus Saphenus, ein afferenter Nerv, der dieses Hautgebiet innerviert, herauspräpariert. Die Haut wurde in einer Organkammer mit der haarigen Seite nach unten aufgespannt. Diese Kammer wurde permanent mit 32 °C warmen SIF-Puffer (Synthetische Interstielle Flüssigkeit; 2mM CaCl2, 5.5mM Glucose, lOmM Hepes , 3.5mM KC1 , 0.7mM MgS04 , 123mM NaCl , 1.5mM NaH2P04, 9.5mM Na-Gluconat, 7.5mM Saccharose) durchspült. Der Nerv wurde in eine Ableitungskammer geführt, die mit der Organkammer verbunden ist, und mit feinen Pinzetten in feinere Axonbündel aufgespalten. Zur extrazellulären Ableitung wurden diese nacheinander an eine Silber-Elektrode gelegt. Wenn man das rezeptive Feld eines der Neurone stimuliert, dessen Axon gerade an der Ableitungselektrode anliegt, so kann man die Eigenschaften eines einzelnen sensorischen Neurons ermitteln. Zunächst wurde das rezeptive Feld elektrisch stimuliert und die Verzögerung bis zum Auslösen eines Aktionspotentials gemessen. Aus dieser zeitlichen Verzögerung und des Abstandes zwischen rezeptivem Feld und der Ableitungselektrode wurde die Leitungsgeschwindigkeit errechnet. Sodann wurde das rezeptive Feld mit von Frey-Haaren gereizt, um die Aktivierungsschwelle zu ermitteln. Schließlich wurde die Reiz-Antwort-Funktion gemessen. Hierzu wurde das rezeptive Feld des Neurons mit dauerfhaften Reizen verschienener Stärke stimuliert, indem mit Hilfe einer Mikrometerschraube das abgerundete Ende eines feinen Metallstabes für jeweils 10 Sekunden auf das rezeptive Feld abgesetzt wurde.Mice were anesthetized with CO 2 and killed by decapitation. The skin of the right hind leg was extracted together with the saphenous nerve, an afferent nerve that innervates this area of the skin. The skin was in one Organ chamber with hairy side down. This chamber was permanently filled with 32 ° C warm SIF buffer (synthetic interstitial fluid; 2mM CaCl 2 , 5.5mM glucose, 10mM Hepes, 3.5mM KC1, 0.7mM MgS0 4 , 123mM NaCl, 1.5mM NaH 2 P0 4 , 9.5mM Na Gluconate, 7.5mM sucrose). The nerve was guided into a lead chamber, which is connected to the organ chamber, and split into finer bundles of axons using fine forceps. For extracellular derivation, these were placed in succession on a silver electrode. If you stimulate the receptive field of one of the neurons whose axon is currently touching the lead electrode, you can determine the properties of a single sensory neuron. First, the receptive field was stimulated electrically and the delay until an action potential was triggered was measured. The line speed was calculated from this time delay and the distance between the receptive field and the lead electrode. The receptive field was then stimulated with von Frey hair in order to determine the activation threshold. Finally, the stimulus response function was measured. For this purpose, the receptive field of the neuron was stimulated with permanent stimuli of various strengths by placing the rounded end of a fine metal rod on the receptive field for 10 seconds with the help of a micrometer screw.
Insgesamt wurden ca. 200 Fasern von 15 verschiedenen Tieren charakterisiert. Da der Nervus Saphenus nur ca. 500 Axone enthält, wurden also 20% aller sensorischen Neurone, die dieses Hautstück innervieren, charakterisiert (Abbildung 1). Dies läßt somit gesicherte Aussagen über Veränderungen in den knock out Mäusen gegenüber den Kontroll-Mäusen zu.A total of approximately 200 fibers from 15 different animals were characterized. Since the saphenous nerve contains only about 500 axons, 20% of all sensory neurons that innervate this piece of skin have been characterized (Figure 1). This allows reliable statements about changes in the knock out mice compared to the control mice.
Klonierung von N-StomatinCloning of N-stomatin
Eine Blast-Recherche der EST-Datenbank von NCBI mit der kodierenden Sequenz von murinem Stomatin zeigte hohe Homologien zu einem EST-Klon (W54879), der aus dem Gehirn der Ratte isoliert wurde. Die enthaltene Sequenz wies ca. 70% Homologie zu dem 3 '-Ende von Stomatin auf. Mit Hilfe von 5'- RACE (Rapid Amplification of cDNA Ends) wurde das fehlende 5 '-Ende kloniert. Hierzu wurde der 5'-RACE-Kit (Cat.# 1734792) von Boehringer Mannheim verwendet und die darin angegebenen Protokolle befolgt. Für die RT-Reaktion wurde als Ausgangsmaterial mRNA aus sensorischen Neuronen verwendet. Für sie wurden die spezifischen Primer tcacgcagggaaacacaat und tcagctggaggcgatgag verwendet. Die erste PCR erfolgte mit dem Oligo dT Anchor Primer und dem spezifischen Primer agctgagacggcgctgtagattc. Für die zweite ('nested') PCR wurden der PCR Anchor Primer und der spezifische Primer gtagacgactccatccacttg verwendet. Das 550 bp-Fragment, das aus dieser zweiten PCR hervorging, wurde kloniert und sequenziert und mit dem EST-Klon zusammengesetzt. Ausgehend von dieser Sequenz wurde ein zweites RACE-Experiment durchgeführt. Hierbei wurde in der ersten PCR der spezifische Primer CTC GGG TGA ATC CAT CTC ATT verwendet.A blast search of NCBI's EST database with the coding sequence of murine stomatin showed high homologies to an EST clone (W54879) which emerged from the brain of the Rat was isolated. The sequence contained approximately 70% homology to the 3 'end of stomatin. The missing 5 'end was cloned using 5'-RACE (Rapid Amplification of cDNA Ends). For this purpose, the 5'-RACE kit (Cat. # 1734792) from Boehringer Mannheim was used and the protocols specified therein followed. For the RT reaction, mRNA from sensory neurons was used as the starting material. The specific primers tcacgcagggaaacacaat and tcagctggaggcgatgag were used for them. The first PCR was carried out with the Oligo dT Anchor Primer and the specific primer agctgagacggcgctgtagattc. The PCR anchor primer and the specific primer gtagacgactccatccacttg were used for the second ('nested') PCR. The 550 bp fragment resulting from this second PCR was cloned and sequenced and assembled with the EST clone. Based on this sequence, a second RACE experiment was carried out. The specific primer CTC GGG TGA ATC CAT CTC ATT was used in the first PCR.
Die Sequenz für den zweiten spezifischen Primer lautete: CAG ACG TTT GTC AAA TCT CGA. Das mit Hilfe der zweiten RACE gefundene PCR-Produkt hatte eine Größe von 130 bp. Setzt man die Sequenzen aus den beiden RACE-Experimenten mit der des EST-Klons zusammen, so erhält man die angegebene Sequenz von N-Stomatin (Abb. 2). The sequence for the second specific primer was: CAG ACG TTT GTC AAA TCT CGA. The PCR product found with the help of the second RACE had a size of 130 bp. If the sequences from the two RACE experiments are combined with that of the EST clone, the sequence given is obtained from N-stomatin (Fig. 2).
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Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19850015.7 | 1998-10-30 | ||
| DE1998150015 DE19850015A1 (en) | 1998-10-30 | 1998-10-30 | Proteins of the stomatin family and their use as target proteins for ore therapy |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO2000026362A2 true WO2000026362A2 (en) | 2000-05-11 |
| WO2000026362A3 WO2000026362A3 (en) | 2000-06-22 |
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ID=7886124
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/DE1999/003434 Ceased WO2000026362A2 (en) | 1998-10-30 | 1999-10-28 | Proteins of the stomatin family and their use as target proteins for pain therapy |
Country Status (2)
| Country | Link |
|---|---|
| DE (1) | DE19850015A1 (en) |
| WO (1) | WO2000026362A2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004028555A1 (en) * | 2002-09-29 | 2004-04-08 | Yeda Research And Development Co. Ltd | Resistin binding proteins, their preparation and use |
| WO2004040299A3 (en) * | 2002-10-30 | 2004-07-15 | Max Delbrueck Centrum | Method for identifying compounds which inhibit mechanotransduction in neurons |
| US7820876B2 (en) | 2000-09-04 | 2010-10-26 | Institut Pasteur | Mouse mutant for expression of the alpha6 subunit of the nicotinic acetylcholine receptor |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5763589A (en) * | 1997-01-09 | 1998-06-09 | Incyte Pharmaceuticals, Inc. | Human membrane protein |
| WO1999025825A2 (en) * | 1997-11-13 | 1999-05-27 | Genset | EXTENDED cDNAs FOR SECRETED PROTEINS |
-
1998
- 1998-10-30 DE DE1998150015 patent/DE19850015A1/en not_active Withdrawn
-
1999
- 1999-10-28 WO PCT/DE1999/003434 patent/WO2000026362A2/en not_active Ceased
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7820876B2 (en) | 2000-09-04 | 2010-10-26 | Institut Pasteur | Mouse mutant for expression of the alpha6 subunit of the nicotinic acetylcholine receptor |
| WO2004028555A1 (en) * | 2002-09-29 | 2004-04-08 | Yeda Research And Development Co. Ltd | Resistin binding proteins, their preparation and use |
| WO2004040299A3 (en) * | 2002-10-30 | 2004-07-15 | Max Delbrueck Centrum | Method for identifying compounds which inhibit mechanotransduction in neurons |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2000026362A3 (en) | 2000-06-22 |
| DE19850015A1 (en) | 2000-05-04 |
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