Patent application title: Method for regulating neurite growth
Inventors:
David Lovejoy (Stouffville, CA)
Arij Al Chawaf (North York, CA)
IPC8 Class: AA61K3800FI
USPC Class:
514 12
Class name: Designated organic active ingredient containing (doai) peptide containing (e.g., protein, peptones, fibrinogen, etc.) doai 25 or more peptide repeating units in known peptide chain structure
Publication date: 2008-12-25
Patent application number: 20080318854
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Patent application title: Method for regulating neurite growth
Inventors:
David Lovejoy
Arij Al Chawaf
Agents:
McCarthy Tetrault LLP
Assignees:
Origin: TORONTO, ON CA
IPC8 Class: AA61K3800FI
USPC Class:
514 12
Abstract:
This invention relates to a method of inhibiting neuronal cell death,
including protecting neronal cells from cell death and the effects of
stress, such as high or low pH, comprising administering to the cells an
effective amount of Teneurin C-terminal Associated Peptide (TCAP). The
invention provides the use of TCAP to prevent and/or treat a number of
brain conditions, such as hypoxiaischemia and brain alkalosis or various
brain or spinal cord injuries due to physical or physiological stresse.
In one aspect the invention provides a use of TCAP to increase
β-tubulin, β-actin levels in neuronal cells and/or to increase
fasciculation among neuronal cells, in culture or in tissue. In another
aspect, the invention provides a method of treating various pH induced
neuronal conditions.Claims:
1. A method of treating a neuronal condition that would benefit from
induction of neurite growth comprising administering to a patient in need
thereof an effective amount of TCAP, pharmaceutical acceptable salt or
ester thereof or obvious chemical equivalent thereof.
2. The method of claim 1, wherein the neuronal condition is physiological trauma.
3. The method of claim 2, wherein the physiological trauma is selected from the group consisting of: hypoxia, injury, infection, cytokine deprivation, carcinogenic agents and cancer.
4. The method of claim 2, wherein the physiological trauma is a result of neurodegenerative disease.
5. The method of claim 4, wherein the neurodegenerative disease is selected from the group consisting of: Alzheimer's, Parkinson's, Huntington's, Multiple Sclerosis and brain ischemia.
6. The method of claim 2, wherein the physiological traumas is selected from the group consisting of: hypothermia, hypoxia, acute ischemia, hypoxia-ischemia, respiratory alkalosis, metabolic alkalosis and brain alkalosis.
7. The method of claim 2, wherein the physiological trauma is traumatic injury to the brain or spinal cord.
8. The method of claim 7, wherein cell death is a result of secondary energy failure post the physiological trauma.
9. A method for increasing fasciculation in neuronal cell cultures or tissue comprising administering to the cells or tissue an effective amount of TCAP, pharmaceutical acceptable salt or ester thereof or obvious chemical equivalent thereof.
10. A method for increasing β-tubulin and/or β-actin levels in neuronal cells comprising administering to the cells an effective amount of TCAP, pharmaceutical acceptable salt or ester thereof or obvious chemical equivalent thereof.
Description:
RELATED APPLICATIONS
[0001]This application is a continuation-in-part of U.S. application Ser. No. 10/510,959, filed Aug. 10, 2005, entitled "Teneurin C-Terminal Associated Peptides (TCAP) and Uses Thereof" which was a national phase entry of PCT/CA2003/00622 filed May 2,2003, which was a non-provisional of U.S. provisional patent application No. U.S. 60/376,879, filed May 2,2002, and a non-provisional of U.S. provisional patent application No. U.S. 60/377,231, filed May 3, 2002, and a non-provisional of U.S. provisional patent application No. U.S. 60/424,016, filed Nov. 6, 2002. This application also claims priority from U.S. provisional patent application No., U.S. 60/73,309, filed Feb. 15, 2006, entitled "A Method for Inhibiting Neuronal Cell Death". This application also claims priority from U.S. provisional patent application No., US 60/783,321, filed Mar. 21, 2005, entitled "Method for Regulating Neurite Growth". All of these references are incorporated in their entirety by reference.
FIELD OF THE INVENTION
[0002]This invention relates to a method for regulating neurite growth. In another aspect, it relates to a method for inhibiting neuronal cell death. In another aspect, it further relates to the neuroprotective effects of teneurin C-terminal associate peptides (TCAP) and to methods and uses of TCAP as a neuroprotective agent and/or to inhibit neuronal cell death and to regulate neurite growth. It further relates to the use of TCAP to induce neuronal growth, increase β-tubulin and β-actin levels in neuronal cells and induce fasciculation of neuronal cells, cultures or tissue, such as primary embryonic hippocampal cultures.
BACKGROUND OF THE INVENTION
[0003]The teneurins are a family of four vertebrate type II transmembrane proteins preferentially expressed in the central nervous system (Baumgartner et al., 1994). The teneurins are about 2800 amino acids long and possess a short membrane spanning region. The extracellular face consists of a number of structurally distinct domains suggesting that the protein may possess a number of distinct functions (Minet and Chiquet-Ehrismann, 2000; Minet et al., 1999; Oohashi et al., 1999). The gene was originally discovered in Drosophila as a pair rule gene and was named tenascin-major (Ten-M) or Odz (Baumgartner et al., 1994; Levine et al., 1994). It is expressed in the Drosophila nervous system and targeted disruption of the genes leads to embryonic lethality (Baumgartner et al., 1994). In immortalized mouse cells, expression of the teneurin protein led to increased neurite outgrowth (Rubin et al., 1999).
[0004]The extracellular C-terminal region of each teneurin is characterized by a 40 or 41 amino acid sequence flanked by enzymatic cleavage sites, which predicts the presence of an amidated cleaved peptide (Qian et al., 2004; Wang et al., 2005). A synthetic version of this peptide was named teneurin C-terminus associated peptide (TCAP) and is active in vivo and in vitro. The mouse TCAP from teneurin- 1 (TCAP-1) can modulate cAMP concentrations and proliferation in mouse hypothalamic cell lines as well as regulate the teneurin protein in a dose dependent manner (Wang et al, 2004). Intracerebroventricular injection of TCAP-1 into rats can induce changes in the acoustic startle response three weeks after administration (Wang et al., 2005). [Also see, PCT/CA2003/000622. filed May 2, 2003, published Nov. 13, 2003, herein incorporated by reference.]
[0005]Currently, it is thought that following initial trauma, neurons die by necrosis, apoptosis or a combination of the two (Thompson, 1995; Columbano., 1995; Rosser and Gores, 1995; Watson, 1995). Necrosis has been defined as unprogrammed cell death induced by physiological trauma, such as hypoxia, injury, infection and cancer. The role of pH in the brain during these times of stress depends upon the trauma inflicted as both phenomenon can occur simultaneously depending upon pathological conditions, physiological activators, physical trauma, environmental toxins and carcinogenic chemicals (Wyllie et al., 1980; Arends and Willie, 1991; Buja et al., 1993; Majno and Jorris, 1995). Various neurodegenerative diseases, such as brain ischemia and Huntington's Disease, exist contingent upon various forms of cell death that in turn are mediated by their environments' surrounding pH. Although extracellular pH changes under normal metabolic circumstances, a number of pathological conditions affect pH and lead to cell death.
[0006]One of the logistical problems in understanding cell death and its corroborating factors is the ambiguity surrounding cell death. The current research indicates that many characteristics that were once thought to pertain only to apoptosis, now apply to necrosis as well. The current consensus is that following the initial insult such as during brain ischemia, brain cells die by necrosis, apoptosis or a combination of the two and pH plays a pivotal role during these times, specifically alkaline pH (Levine et al., 1992; Robertson, 2002).
[0007]Although, the literature on brain acidosis is extensive, brain alkalosis, is not well understood (Robertson, 2002). Intracellular alkalinization has been observed in cells undergoing cytokine deprivation (Khaled, 1999) as well as hypoxia-ischemia (HI) (Robertson, 2002). For example, during brain ischemia, brain pH levels indicated a progression from early acidosis to subacute alkalosis (Levine et al., 1992).
[0008]There is a need to counteract the effects of stress, such as pH induced cellular stress on the brain and to develop methods and compounds to protect cells against said effects, accordingly. Further there is a need to regulate neurite growth which may be beneficial in the diagnosis and treatment of various neuronal conditions.
SUMMARY OF THE INVENTION
[0009]In one aspect the invention provides a method for inhibiting neuronal cells against cell death. The inventors have surprisingly found that TCAP treated cells survive better in stress conditions, for instance in pH induced stress conditions, and in one aspect in alkaline pH conditions compared to vehicle treated cells.
[0010]As such, in one aspect the invention provides a method for inhibiting neuronal cells against cell death by administering an effective amount of TCAP, pharmaceutically acceptable salt or ester thereof or obvious chemical equivalent thereof to the cells. In another embodiment, administration of TCAP to the cells is administration of TCAP to a patient in need thereof comprising said cells. In one aspect the patient in need thereof is a patient who sustained or is suspected to have sustained a physiological trauma. In one aspect, a pharmaceutical composition comprising TCAP, pharmaceutically acceptable salt or ester or obvious chemical equivalent thereof and a pharmaceutically acceptable carrier is administered.
[0011]In one aspect, the invention provides a method of inhibiting and/or preventing neuronal cell death comprising administering to the cell an effective amount of TCAP, a pharmaceutical acceptable salt or ester thereof or obvious chemical equivalent thereof.
[0012]In one embodiment, inhibiting neuronal cell death comprises inhibiting and/or protecting and/or preventing neuronal cells from cell death under conditions where cell death may occur, such as a result of physiological trauma.
[0013]In one embodiment, conditions wherein cell death may occur are conditions conducive to necrosis. As such, in one aspect the invention provides a method of inhibiting, preventing or protecting neuronal cells from cell death by necrosis by administering an effective amount of TCAP, pharmaceutically acceptable salt or ester thereof or obvious chemical equivalent thereof.
[0014]In one embodiment, conditions where cell death may occur is stress-induced neuronal cell death, such as pH-induced neuronal cell death. In one aspect, pH-induced neuronal cell death is alkalosis-stress induced neuronal cell death or cell death as a result of high pH conditions. In one aspect, high pH conditions are conditions wherein pH is greater than 7.4. In another aspect, the pH is 8.0 or greater. In another aspect, the pH is from 8.0 to 9.0, 8.0 to 8.5, or 8.0 to 8.4. In another aspect, one condition of pH induced stress is from 6.0 to 7.4 or at pH 6.8.
[0015]In another aspect, the physiological trauma is selected from the group consisting of: hypoxia, injury, infection, cytokine deprivation, carcinogenic agents and cancer and/or is related to or the result of a neurodegenerative disease.
[0016]In one aspect, the neurodegenerative disease is selected from the group consisting of: Alzheimer's, Parkinson's, Huntington's, Multiple Sclerosis and brain ischemia.
[0017]In yet another embodiment, the physiological trauma is selected from the group consisting of: hypothermia, hypoxia, acute ischemia, hypoxia-ischemia, respiratory alkalosis, metabolic alkalosis and brain alkalosis. In another embodiment, it is traumatic injury to the brain or spinal cord or a result of secondary energy failure post the physiological trauma.
[0018]In one embodiment, the invention provides a method for using an effective amount of TCAP, pharmaceutical acceptable salt or ester thereof or obvious chemical equivalent thereof in the treatment of a neuronal condition associated with alkaline neuronal cell pH, by administering said TCAP to the patient in need thereof. In one aspect said condition is related to pH conditions greater than 7.4, 8.0 or greater, from 8.0 to 9.0, or from 8.0 to 8.4.
[0019]In one embodiment of the aforementioned methods of the invention, the neuronal cell is a immortalized mouse hypothalamic cell.
[0020]In one embodiment, the invention provides a method of screening of modulators of the neuronal cell death inhibitory effects of TCAP, comprising administering TCAP to neuronal cells under conditions that would normally induce neuronal cell death if TCAP were not present (e.g. pH induced cell death, alkalosis induced cell death); administering a suspected modulator of said TCAP function and determining the effects of said suspected modulator on TCAP inhibition of neuronal cell death. If said suspected modulator enhances TCAP inhibition of neuronal cell death or decreases TCAP inhibition of neuronal cell death, then it is a modulator of TCAP inhibition of neuronal cell death. In one embodiment, said suspected modulator is administered to the cells prior to, simultaneously with and/or after administration of TCAP. In another embodiment, determining the effects of said modulator comprises comparing the levels of neuronal cell death and/or survival with a control, such as cell death absent the presence of TCAP or modulator; in the presence of TCAP alone or modulator alone, or compared to established baseline effects of neuronal cell death under various conditions.
[0021]In another aspect of the invention, the invention provides a method for increasing neuronal cell proliferation under conditions of neutral pH or acidosis pH conditions. In one embodiment, the pH conditions are pH of 7.4 or less. In another embodiment, the pH conditions are 6.8 or less. In yet another embodiment the pH conditions are between 6.8 and 7.4.
[0022]In another embodiment, the invention provides a method to regulate neurite growth by administering TCAP to neuronal cells. In another embodiment, the invention provides of a method of inducing neuronal growth by administering an effective amount of TCAP to neuronal cells. In another aspect, the invention provides a use of TCAP to increase β-tubulin and/or β-actin levels in neuronal cells. In one aspect, the invention provides a method for treating conditions related to β-tubulin and/or β-actin levels, such as memory loss, learning disorders, neurodegenerative diseases and necrosis or inflammation resulting from trauma to the central nervous system.
[0023]In another aspect, the invention provides a method or use of TCAP to induce fasciculation of neuronal cells, cultures or tissue, such as primary embryonic hippocampal cultures. In yet another embodiment, the invention provides a method for treating a condition that can be treated by increasing fasciculation among neuronal cells, such as in the treatment of physiological or physical trauma to neuronal cells, such brain d injuries.
[0024]In another embodiment, the invention provides a method or use of TCAP as a guidance molecule. Axonal guidance and pathfinding is anormal and necessary aspect of neuroregeneration and restoration of function following a trauma As such, in one aspect, the invention includes a method for axonal guidance or neurogeneration comprising administering an effective amount of TCAP to a neuron or patient in need thereof.
[0025]Additional aspects and advantages of the present invention will be apparent in view of the description which follows. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0026]The invention will now be described in relation to the drawings, in which:
[0027]FIG. 1a. Cell Morphology of N38 cells at 48 hrs as a function of pH treatment.
[0028]FIG. 1b. Cell Morphology of N38 cells at 72 hrs as a function of pH treatment.
[0029]FIG. 1c. Example of necrotic cell.
[0030]FIG. 1d. Quantification of small crenated (necrotic) cells. The levels of significance were determined by two-way ANOVA using Bonferroni's Post Test.
[0031]FIG. 2a. Proliferation of N38 cells a function of pH. TCAP-1(10-7M) increased the number of cells post 48 hrs after treatment at pH extremes 6.8, 8.0, 8.4. The level of significance was determined using a two-way analysis of variance (ANOVA).
[0032]FIG. 2b. Changes in cell viability, over 48 hours as determined by trypan blue. TCAP increased the number of viable cells at pH 6.8 (p<0.10) pH 8.0 (p<0.001) and pH 8.4 (p<0.05). The level of significance was determined using a two-way analysis of variance (ANOVA).
[0033]FIG. 3. Changes in mitochondrial metabolism of N38 cells as determined by the MTT assay. TCAP-1(10-7M) increased the number of viable cells post 48 hrs after treatment at pH extremes 8.0 and 8.4. The level of significance was determined using a two-way analysis of variance (ANOVA).
[0034]FIG. 4a. Apoptotic, necrotic and healthy cells fluorescent microscopy quantification analyses post 48 hrs. Cell types are characterized by colour: apoptosis (green) necrosis (red) healthy (blue).
[0035]FIG. 4b. Example of apoptotic cell.
[0036]FIG. 4c. Apoptotic, necrotic and healthy cells fluorescent microscopy quantification analyses. TCAP significantly decreased the amount of necrotic cells post 48 hrs at pH extremes 6.8(P<0.0001), 8.0 (P<0.0001), 8.4 (P<0.0001). A two way ANOVA was used to determine levels of significance.
[0037]FIG. 5a Caspase 8 colorimetric assay at pH extremes.
[0038]FIG. 5b. Caspase 3 colorimetric assay at pH extremes.
[0039]FIG. 5c. Caspase 3 western blot.
[0040]FIG. 6a. PARP quantification using transformed data.
[0041]FIG. 6b. PARP western blot detection at pH extremes. Post 48 hrs TCAP-1 (10-7M).
[0042]FIG. 6c. PARP optical density quantification.
[0043]FIG. 7a. Akt quantification using transformed data
[0044]FIG. 7b. Akt western blot detection at pH extremes. Post 48 hrs TCAP-1 (10-7M).
[0045]FIG. 7c. Akt optical density quantification
[0046]FIG. 7d. Phospho-Akt western blot detection at pH extremes. There was no indication of AKT phosphorylation in any sample except for the control, thus TCAP is not rescuing cells through the AKT cell survival pathway.
[0047]FIG. 8. BrdU colorimetric assay at pH extremes.
[0048]FIG. 9 illustrates immortalized mouse hypothalamic N38 cells treated with 1 nM and 100 nM mouse TCAP-1 and measurements of neurite lengths. FIG. 9A illustrates untreated cells at 8 hours. FIG. 9B illustrates cells treated with 100 nM of TCAP-1 at 8 hours. FIG. 9C illustrates percent change in neurite length in control (untreated), 1 nM TCAP-1 and 100 nM TCAP-1 at 0, 4 and 8 hours post TCAP administration. FIG. 9D illustrates percent change of number of neurites in control (untreated), 1 nM TCAP-1 and 100 nM TCAP-1 at 0, 4, and 8 hours post TCAP administration. FIGS. 9E and 9F illustrate the frequency distribution in neurite length of the cell population in untreated (9E) and 100 nM TCAP-1 treated (9F) samples.
[0049]FIG. 10: Analysis of gene expression following TCAP stimulation. N-38 immortalized neurons were treated with 1 or 100 nM TCAP or vehicle over a 8 h timecourse. Total RNA was isolated at the indicated timepoints. Real-time RT-PCR was performed for β- actin; α-actinin 4; and β-tubulin . All genes were normalized to 18S rRNA levels as a loading control. Statistical significance was determined using a two way analysis of variance (n=5-8).
[0050]FIG. 11: β-tubulin protein expression is increased after 1 hour of 100 nM TCAP-1 treatment. A. Protein levels in N38 cells were assayed using western blotting. 100 nM TCAP-1 induced a significant increase at 1 hour (two-way ANOVA with Bonferroni's post-hoc test p<0.05) B. Representative blots for the different time-points C. Mean and SE of the optical density of the blots at 1 hour.
[0051]FIG. 12; Cytoskeletal β-actin protein expression is upregulated in N38 cells after 1 hour of TCAP-1 treatment. A. 1 and 100 nM TCAP-1 induces a significant increase in β-actin levels in cells treated for one hour (two way ANOVA with Bonferroni's post-hoc test p<0.001) B. Representative blots for the different time points C. Mean and SE of the optical density of the blots at 1 hour.
[0052]FIG. 13 Effect of TCAP-1 treatment on α-actinin-4 protein levels. A. TCAP-1 treatment did not cause any real change over 8 hours. B. Representative western blots.
[0053]FIG. 14: Confocal immunofluorescence of beta tubulin in N38 cells Confocal analysis of 100 nM TCAP-1 effects on localization of β-tubulin in N38 cells. Immunofluorescence analysis of cells treated with 1 hour TCAP-1 show an increase in β-tubulin expression both in the perinuclear and the whole cell region. A. Ten central cells from each image was analyzed for the number of pixels at maximal intensity (149) and expressed as a ratio of total pixels in B. the perinuclear region and C. the whole cell ( Student's t-test with Welch's correction for unequal variances p=0.05, minimum 30 cells per group). Perinculear region and cell size not different in control and treated cells. Bar=20 μm.
[0054]FIG. 15 illustrates that 100 nM TCAP administered to a developing axon of an N38 cell in the direction of the arrow (13A) causes expansion of the growth cone area followed by repulsion away from the source of TCAP (13B). Bar=1 μm.
[0055]FIG. 16. Effect of TCAP-1 on primary cultures of hippocampal cells. A.Reverse image of hippocampal cultures. Top panel, vehicle treated cells, bottom panel TCAP treated cells. B. There were significantly (p<0.01) greater number of cells and cell clusters in the TCAP-1 treated cultures (n=4, two way students t test). C. Histograms of mean pixel intensity (n=4). Standard error of the mean is indicated.
[0056]FIG. 17: TCAP-1 in culture medium caused an increase in dendritic density and fasciculation in primary E18 hippocampal cultures. Anti-β-tubulin III immunocytochemistry cultured in the presence of vehicle or 100 nM TCAP-1 for seven days. Boxes indicate regions shown in the subsequent image. A. ×40 magnification, bar=0.25mm B. ×100 magnification, bar=100 μm C. ×4000 magnification, arrows point to areas of fasciculation, bar=25 μm.
[0057]FIG. 18 illustrates the results as described in Example 8, wherein FIG. 18A illustrates the presence of the superoxide radical measured indirectly by the conversion of a soluble tetrazolium salt in cells after 48 hours. Absorbance of the substrate is proportional to superoxide radical activity. FIGS. 18B and 18C illustrate the presence of superoxide dismutase directly by western blot (FIG. 18C) and change relative to vehicle treated cells (percent) versus pH (FIG. 18B). FIG. 18D illustrates superoxide dismustase gene expression as measured by real-time PCR, while FIG. 18E illustrate superoxide copper chaperone expression as measured by real-time PCR.
[0058]FIG. 19 illustrates the results as described in Example 8. FIG. 19A illustrates that TCAP-1 showed a significant increase in MTT activity relative to the vehicle-treated at 6-48 hours in cells treated with 50 uM H2O2. FIG. 19B illustrates the results of a catalase assay on pH treated cells. FIG. 19C illustrates catalase gene expression as determined by real-tie PCR.
DETAILED DESCRIPTION OF THE INVENTION
[0059]As described herein, teneurin C-terminus associated peptide (TCAP) inhibits neuronal cell death, such as during timed of pH induced cellular stress in the brain. In another aspect, TCAP has a neuroprotective effect, protecting neuronal cells from cell death, such as, during times of pH induced cellular stress in the brain. In one aspect of the invention, such pH induced cellular stress in the brain is related to hypoxia-ischemia and/or brain alkalosis. In the examples described herein, an immortal hypothalamic mouse cell line (N38) was treated with medium buffered at pHs 6.8, 7.4, 8.0 and 8.4 treated with 100 nM TCAP and examined at 24 and 48 hours. TCAP significantly increased cell proliferation at pH 6.8 and inhibited declines in cell proliferation at pHs 8.0 and 8.4 as determined by direct cell viability assays. TCAP did not significantly alter caspase 8 and 3 activity, nor induce PARP cleavage. TCAPs effects on the S phase of cell cycling were investigated through a bromodeoxyuridine (BrdU) uptake assay, the results showing that TCAP does not have a major effect during the S phase of cell proliferation. The incidence of necrosis was tested via cell viability (Trypan Blue) assay and fluorescence microscopy utilizing fluorophores to Annexin V andethidium Homodimer III as well as morphological analyses. The results indicate that TCAP can protect cells from necrosis. In one aspect, TCAP has a neuroprotective role during times of cellular stress, such as induced pH stress. As such, TCAP can be used in the treatment of physiological effects of pH in the brain during trauma, such as hypoxia-ischemia.
[0060]In another aspect of the invention, TCAP was shown to enhance neurite length, β-tubulin and β-actin levels in neuronal cells and to enhance fasciculation of neuronal cells in cell culture or tissue. All these can contribute to TCAP's neuronal protective effects against death and to inhibit neuronal cell death. In another aspect, it illustrates the use of TCAP in the treatment neuronal conditions resulting from traumatic or epigenetically associated necrosis. In one aspect, TCAP can regulate neurite and axonal growth. In another aspect, it was shown that TCAP can alter interneuron communication via changes in neurite and axon outgrowth.
Definitions
[0061]Administering to the cell(s)" as used herein means both in vitro and in vivo administration to the cells and can be direct or indirect administration, as long as the cells are at some point exposed to the substance being administered. In the case of a peptide, it can also include methods to increase expression of the peptide or peptides to enhance exposure of the desired target to said peptide.
[0062]Apoptosis" as used herein means "programmed cell death" and is a necessary event of normal development. It is a normal process for eliminating unwanted cells.
[0063]Effective Amount" and "Therapeutically Effective Amount" as used herein means an amount effective, at dosages and for periods of time necessary to achieve the desired results. For example, an effective amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance to elicit a desired response in the individual. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
[0064]Homeostasis" as used herein means the inherent tendency in an organism or cell toward maintenance of physiological stability and making automatic adjustments in relation to its environment. Other wise known as normal stability of the internal environment (Sapolsky, 1992).
[0065]Inhibiting Neuronal Cell Death" as used herein include inhibiting, preventing, and protecting neuronal cells (including rescuing neuronal cells) from, cell death.
[0066]Necrosis" as used herein means unprogrammed cell death induced by physiological trauma, such as hypoxia, injury, infection and cancer/carcinogenic agents.
[0067]Neuronal Cells" as used herein includes immortalized mouse hypothalamic neurons.
[0068]Obvious Chemical Equivalents" as used herein means , in the case of TCAP, any variant that does not have a material effect upon the way the invention works and would be known to a person skilled in the art. For instance, this could include but not necessarily be limited to any salts, esters, conjugated molecules comprising TCAP, truncations or additions to TCAP.
[0069]Pharmaceutically Acceptable Carrier" as used herein means any medium which does not interfere with the effectiveness or activity of an active ingredient and which is not toxic to the hosts to which it is administered. It includes any carrier, excipient, or vehicle, which further includes diluents, binders, adhesives, lubricants, disintegrates, bulking agents, wetting or emulsifying agents, pH buffering agents, and miscellaneous materials such as absorbants that may be needed in order to prepare a particular composition. Examples of carriers, excipient or vehicles include but are not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The use of such media and agents for an active substance is well known in the art (e.g., "Remington: The Sciences and Practice of Pharmacy, 21st Edition", (University of the Sciences in Philadelphia, 2005)
[0070]Neuronal condition associated with alkaline neuronal cell pH" as used herein means any neuronal condition that is caused by or causes or results in or is associated with alkaline neuronal cell pH. Such conditions include, but are not limited to brain ischemia, neurodegenerative diseases such as, Alzheimer's, Parkinson's, Huntington's, brain ischemia and multiple sclerosis, and brain injury associated with physiological trauma.
[0071]Stressor" is defined as anything that disrupts physiological balance, be it physical or psychological (Sapolsky, 1992)
[0072]Stress-related brain or neuronal condition" as used herein means any brain neuronal condition associated with neuronal cells not being in a state of homeostasis.
[0073]TCAP" as used herein means a 38- 41 amino acid sequence, preferably a 40-41 amino acid sequence from the C-terminal end of a teneurin peptide and all analogs, homologs, fragments, derivatives, salts, esters of the TCAP peptide which have the desired activity, and obvious chemical equivalents thereto, as described in PCT/CA2003/000622. filed May 2, 2003, published Nov. 13, 2003, and which is herein incorporated by reference. For instance, in one embodiment, TCAP includes human or mouse TCAP, such as TCAP 1, such as SEQ. ID. NOs. 37-44 (mouse) or 69-76 (human) of PCT/CA2003/000622 and analogs, homologs, fragments, derivatives, salts, esters and obvious chemical equivalents thereof. In one embodiment the TCAP is mouse TCAP-1 having the amino acid sequence:
[0074]QQLLGTGRVQGYDGYFVLSVEQYLELSDSANNIHFMRQSEI-NH2 (a number nm 011855) (SEQ. ID. NO. 38).
In one embodiment TCAP is prepared by solid phase synthesis and stored as a lyophilized powder at -80° C. reconstituted by alkalinizing with ammonium hydroxide and dissolved into physiological saline at 10-4 M stock solution.
[0075]A nucleotide encoding TCAP" as used herein means a nucleotide sequence that encodes TCAP, including DNA and RNA. Such suitable sequences are described in PCT/CA2003/000622, which is herein incorporated by reference.
Applications: The Use of TCAP to Inhibit Neuronal Cell Death
[0076]The invention broadly contemplates the use of TCAP, including an isolated TCAP, or a nucleotide encoding TCAP to inhibit neuronal cell death. In another aspect, the invention broadly contemplates the use of TCAP to increase fasciculation of neuronal cells in culture or in tissue, and in another aspect to increase β-tubulin and/or β-actin levels.
(a) Necrosis in Neurodegenerative Diseases
[0077]Necrotic cell death in the central nervous system follows acute ischemia or traumatic injury to the brain or spinal cord (Linnik, 1993; Emery, 1998). It occurs in areas that are most severely affected by abrupt biochemical collapse, which leads to the generation of free radicals and excitotoxins (e.g., glutamate, cytotoxic cytokines, and calcium). The histologic features of necrotic cell death are mitochondrial and nuclear swelling, dissolution of organelles, and condensation of chromatin around the nucleus. These events are followed by the rupture of nuclear and cytoplasmic membranes and the degradation of DNA by random enzymatic cuts in the molecule (Martin, 2001). Given these mechanisms and the rapidity with which the process occurs, necrotic cell death is extremely difficult to treat or prevent. The present inventors herein describe a method of treating and/or preventing necrotic cell death using TCAP.
(b) pH in Necrosis
[0078]According to Potapenko et al., brain alkalinization induces an increase of Ca2+ in neurons due to Ca2+ sequestering structures, such as the mitochondria and endoplasmic reticulum, and elevated cytoplasmic Ca2+ is implicated in neuronal cell death, more specifically, necrosis during brain ischemia (Yuan et al., 2003). As mentioned previously such excessive rises in Ca2+ may be induced by excitoxicity caused by brain ischemia, subsequently over stimulating postsynaptic glutamate receptors; of these glutamate-gated channels, NMDA receptor channels play a key role in excitotoxicity as they conduct both Na.sup.+ and Ca2+ (Bonfoco et al. 1995).
(c) Brain Injuries Related to Alkalosis
[0079]Insults to the brain can quite often lead to shifts in pH and based on the data presented it appears that TCAP is rescuing neurons from necrosis consistently at high pH extremes, specifically pH 8.0 and 8.4. Dying neurons are a clear indication of many neurodegenerative diseases including Alzheimer's, Parkinson's, Huntington's, brain ischemia and multiple sclerosis (Siao, 2002). These neurodegenerative conditions are characterized by their deleterious effects on brain function resulting from deterioration of neurons. The destruction of neurons in these conditions may be regulated by various forms of cell death and can be caused by damaged mitochondrion, increased levels of excitotoxins such as glutamate, which increases calcium influx into the neurons and activates calcium dependent enzymes such as calpain and caspases (Randall & Thayer, 1992; Brorson et al., 1995) and pH. Brain pH during times of neurodegenerative stress is not well understood, however, calcium and pH are not mutually exclusive, during both respiratory and metabolic alkalosis, increases in calcium occur in rat neurons due to intracellular calcium accumulating structures such as the mitochondrion (Potapenko, 2004), this is also substantiated by the fact that glutamate induced neuron death requires mitochondrial calcium uptake (Stout et al., 1998).
[0080]Recent studies on brain energy metabolism using phosphorous and proton magnetic resonance (MR) spectroscopy have allowed an understanding of energy changes within the brain following (HI) (Thornton, 1998; Moon, 1973). A phenomenon named the "secondary energy failure" that occurs some 8-24 hours after the initial insult has been recently discovered, and have correlated the magnitude of this disruption with the eventual neurodevelopmental outcome (Thornton, 1998). A similar relationship between intracellular alkalosis and the severity of brain injury in infants has also found that babies with the most alkaline brain cells had more severe changes on MR imaging within the first 2 weeks of life and the worst neurodevelopmental outcome at one year (Roberstson, 2002). Thus, a means of identifying neuropeptides with pH protective properties would be a pivotal finding as it would provide novel therapeutic treatments. The inventors have shown herein that TCAP is a neuroprotective peptide and can inhibit neuronal cell death. As such, it can be used to treat a number of neuronal conditions, such as a neuronal condition associated with alkaline neuronal cell pH.
(d) Neuronal Cell Death Inhibition/Neuroprotective Role of TCAP During Times of Stress
[0081]The potential for neuropeptides to regulate brain processes during times of stress (e.g. as a result of a stress-related brain or neuronal condition) is an important paradigm in the search for novel ways of coping with neurodegenerative diseases and physiological stress and examples of neuropeptides being connected with therapeutic uses are plentiful. (Gozes et al., 1994; Glazer et al. 1994; Zhang et al., 2001) The teneurin C-terminus associated peptides (TCAP) have a neuroprotective effect from cell death, during times of pH induced cellular stress in the brain such as during hypoxia-ischemia. The present inventors herein describe a method of treatment or use of TCAP in the treatment of such stress-related brain or neuronal conditions and the use of TCAP in the preparation of a medicament for the treatment of such conditions.
(e) Screening for Potential Modulators of TCAP Inhibition of Neuronal Cell Death.
[0082]In on embodiment, the invention provides a method for screening compounds that modulate TCAP inhibition of neuronal cell death, comprising, administering TCAP to neuronal cells under conditions that promote inhibition of neuronal cell death in the presence of a potential TCAP modulator and monitoring the affects of said potential modulator on the viability of the neuronal cells. In one embodiment, this can be done in comparison to a control, such as the potential modulator with or without TCAP and/or with TCAP but no potential modulator. In one aspect of the invention the administration of TCAP can occur in a number of ways including, but not necessarily limited to: administering the TCAP in a suitable form of peptide to the cells, administering a substance that will enhance TCAP expression and availability of TCAP to the cell; administration of a nucleic acid encoding TCAP that will result in enhanced TCAP expression to the cell.
(f) The Use of TCAP to Regulate Neurite Growth--TCAP As A Neuroplastic Agent
[0083]In one embodiment of the invention, TCAP alters interneuron communication via changes in neurite and axon outgrowth. Synthetic mouse/rat TCAP-1 was used to treat cultured immortalized mouse hypothalamic cells to determine if TCAP-1 could directly regulate neurite and axon growth. TCAP-1 treated cells showed a significant increase in the length of neurites, accompanied by a marked increase in β-tubulin transcription and translation as determined by real-time PCR and western blot analysis, respectively, although changes in α-actinin 4 transcription and β-actin translation were also noted. Immunofluorescence confocal microscopy using β-tubulin antisera showed enhanced resolution of β-tubulin cytoskeletal elements throughout the cell. In order to determine if the effects of TCAP-1 could be reproduced in primary neuronal cultures, primary cultures of day E18 rat hippocampal cells were treated with 100 nM TCAP-1. The TCAP-1 treated hippocampal cultures showed a significant increase in both the number of cells and the presence of large and fasciculated β-tubulin immunoreactive axons. The data indicates the TCAP acts as a functional region of the teneurins to regulate neurite and axonal growth of neurons.
[0084]It is also herein shown that TCAP-1 increases neurite length and alters the levels and distribution of key cytoskeletal proteins and genes associated with axon outgrowth in immortalized neuronal cell lines. Moreover, because both TCAP-1 expression (Wang et al., 2005) and teneurin-1 (Zhou et al., 2003) expression is high in hippocampal cells the effects of TCAP-1 was studied on primary cultures of hippocampal cells. In these cultures TCAP-1 dramatically increased the incidence of axon formation, e.g. in primary cultures of hippocampal cells. The TCAP/teneurin system represents a new mechanism in neuroplasticity.
[0085]This has implications in the treatment of certain conditions and inducing changes in the brain, such as changes in acoustic startle response, learning, memory, anxiety or other brain or neuronal conditions. TCAP can be used to treat such conditions.
[0086]One can screen for modulators of TCAP, neurite growth or neuroplasticity, by administering the suspected modulator to a neuron or neurons or tissue comprising neurons in the presence of TCAP under conditions that promote neurite growth or neuroplasticity and monitoring the effects of the suspected modulator on said activities. The effect can be compared to a control, such as known baseline levels of activity, or a control such as in the presence or absence of TCAP and/or the suspected modulator. In one embodiment, a modulator can enhance the effects of TCAP. In another embodiment, the modulator can diminish the effects of TCAP.
Pharmaceutical Compositions and Modes of Administration
[0087]TCAP, pharmaceutically acceptable salts or esters thereof or obvious chemical equivalents thereof can be administered by any means that produce contact of said active agent with the agent's sites of action in the body of a subject or patient to produce a therapeutic effect, in particular a beneficial effect, in particular a sustained beneficial effect. The active ingredients can be administered simultaneously or sequentially and in any order at different points in time to provide the desired beneficial effects. A compound and composition of the invention can be formulated for sustained release, for delivery locally or systemically. It lies with the capability of a skilled physician or veterinarian to select a form and route of administration that optimizes the effects of the compositions and treatments of the present invention to provide therapeutic effects, in particular beneficial effects, more particularly sustained beneficial effects.
[0088]In one embodiment, administration of TCAP includes any mode that produce contact of said active agent with the agent's sites of action in vitro or in the body of a subject or patient to produce the desired or therapeutic effect, as the case may be. As such it includes administration of the peptide to the site of action--directly or through a mode of delivery (e.g. sustained release formulations, delivery vehicles that result in site directed delivery of the peptide to a particular cell or site in the body. It also includes administration of a substance that enhances TCAP expression and leads to delivery of TCAP to a desired cell or site in the body. This would include but is not limited to the use of an oligonucleotide encoding TCAP, e.g. via gene therapy or through a TCAP expression system in vitro or in vivo, as the case may be that results in enhanced expression of TCAP. It can also include administration of a substance to the cell or body that enhances TCAP levels at the desired site.
[0089]The above described substances including TCAP and nucleic acids encoding TCAP or other substances that enhance TCAP expression may be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. By "biologically compatible form suitable for administration in vivo" is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects. The substances may be administered to living organisms including humans, and animals.
[0090]Thus in one embodiment, the invention provides the use of TCAP or modulator thereof in the preparation of a medicament for the inhibition of neuronal cell death and/or the treatment of related conditions. In one embodiment, a therapeutically effective amount of TCAP or a pharmaceutical composition as described herein is administered to a patient in need thereof. A patient in need thereof is any animal, in one embodiment a human, that may benefit from TCAP and its effect on inhibition of neuronal cell death or increase neuronal growth, β-tubulin or β-actin levels, increase in fasciculation or as a guidance molecule.
[0091]An active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions that may inactivate the compound. In one embodiment, TCAP is administered directly to or proximate to the desired site of action, by injection or by intravenous. If the active substance is a nucleic acid encoding, for example, a TCAP peptide it may be delivered using techniques known in the art.
[0092]The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutical acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutical acceptable vehicle or carrier. Suitable vehicles or carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985 or Remington's The Sciences and Practice of Pharmacy, 21st Edition", (University of the Sciences in Philadelphia, 2005) or Handbook of Pharmaceutical Additives (compiled by Michael and Irene Ash, Gower Publishing Limited, Aldershot, England (1995)). On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutical acceptable vehicles, carriers or diluents, and may be contained in buffered solutions with a suitable pH and/or be iso-osmotic with physiological fluids. In this regard, reference can be made to U.S. Pat. No. 5,843,456.
[0093]As will also be appreciated by those skilled, administration of substances described herein may be by an inactive viral carrier. In one embodiment TCAP can be administered in a vehicle comprising saline and acetic acid.
[0094]Further, in one embodiment, TCAP may be administered in a form that is conjugated to another peptide to facilitate delivery to a desired site, or in a vehicle, eg. a liposome or other vehicle or carrier for delivery. For instance, in one embodiment TCAP can be conjugated to a brain targeting vector, which is a peptide or peptidomimetic monoclonal antibody (MAb), that is transported into brain from blood via an endogenous blood brain barrier (bBB) transport system, which has shown to significantly reduce stroke volume (e.g. see Zhang et al. (2001)). Thus, in one embodiment, brain ischemia can be treated by neuropeptides, such as TCAP, with noninvasive intravenous administration. In one embodiment, the peptide is conjugated to a BBB drug targeting system such as transferrin, for example as described in Vuisser et al. (2004) or Kang et al. (1994). In another embodiment, TCAP does not require a transport mechanism to cross the blood brain barrier.
[0095]The present invention is described in the following Examples, which are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.
EXAMPLES
Example 1
Peptide Synthesis
[0096]Mouse TCAP-1 (i.e., SEQ. ID. NO. 38) was prepared by solid phase synthesis as previously described (Qian et al., 2004). The peptide was solubilized in phosphate buffered saline (PBS) at a concentration of 2×10-7 M before being diluted in the appropriate medium.
[0097]More particularly, a mouse paralogue of the putative peptide sequence from teneurin-1 was synthesized on an automated peptide synthesizer, Model Novayn Crystal (NovaBiochem, UK Ltd., Nottingham, UK) on PEG-PS resin using continuous flow Fmoc chemistry (Calbiochem-Novabiochem Group, San Diego, Calif.). Eight times excess diisopropyl ethylamine (Sigma-Aldrich Canada Ltd.) and four times excess Fmoc-amino acid activated with HATU (O-(7-azabenzotriazol)-1-3,3-tetramethyluronium hexfluorophosphate; Applied Biosystems, Foster City, Calif.) at a 1:1 (mol/mol) ratio were used during the coupling reaction. The reaction time was 1 h. A solution of 20% piperidine (Sigma-Aldrich Canada Ltd.) in N,N-dimethylformide (DMF; Caledon Laboratories Ltd., Canada) was used for the deprotection step in the synthesis cycle. The DMF was purifiedin-house and used fresh each time as a solvent for the synthesis. The cleavage/deprotection of the final peptide was carried out with trifluoroacetic acid (TFA), thioanisole, 1,2 ethandithiol, m-cresole, triisopropylsilane, and bromotrimethyl silane (Sigma-Aldrich Canada Ltd.) at a ratio of 40:10:5:1:1:5. Finally, it was desalted on a Sephadex G-10 column using aqueous 0.1% TFA solution and lyophilized. The peptide was solubilized by exposure to ammonium hydroxide vapors for 2 minutes before dilution in phosphate-buffered saline (PBS) pH 7.4 with 10 nM sodium phosphate.
Example 2
Cell Morphology Analysis
[0098]The effect of TCAP-1 on cell morphology was conducted using the N38 cells immortalized mouse hypothalamic cell line (Belsham et al, 2004). Cells were grown in six-well culture plate with 2 ml of Dulbeco's Modified Eagle Medium (DMEM) with high glucose, L-glutamate, 25 mM HEPES buffer, pyridoxine hydrochloride in the absence of sodium pyruvate, 5 ml penicillin with 10% fetal bovine serum (FBS) at pH 7.4 (all from Gibco-Invitrogen, Burlington, Canada).
[0099]At 24 and 48 hrs, the medium was replaced with medium buffered at pH 6.8, 7.4, 8.0 or 8.4. Half of the cell groups received (10-7M) TCAP-1, whereas the other half received phosphate buffered saline (PBS) pH 7.4 containing 8g NaCl, 0.2 g KCl, 1.4 g Na2HPO4, 0.2 g KH2PO4 in 800 mL ddH2O. For all groups, 4 replicates were run. Digital pictures were taken at 24, 48 and 72 hrs using an Olympus IX&1 inverted microscope at magnification and analyzed using Lab Works 4.0 Image Acquisition and Analysis Software (Ultraviolet Products Ltd., Calif.)
Results
[0100]TCAP did not induce any observable morphological changes in the cells cultured at pH 7.4. However, there was significant increase in the number of small round cell types (necrotic cells) in the vehicle-treated cultures at pH 6.8 (p<0.05), 8.0 (p<0.001) and 8.4 (p<0.001) as compared to the TCAP-treated samples at 48 hrs (F=96.16). At 72 hrs, TCAP significantly decreased the number of rounded cells in pH 8.0 (p<0.001) and pH 8.4 (p<0.001) (F=51.13) relative to the vehicle-treated cells. (FIG. 1).
Example 3
Effect of TCAP on Cell Proliferation and Viability
[0101]The effect of TCAP-1 on cell proliferation at each pH was examined by direct counts using a hemocytometer and indirectly by assessing mitochondrial activity using a colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay on cultured N38 cells. For hemocytometer counts, the cultures were incubated for 24 and 48 hrs. The cells were suspended using 1 ml of 0.25% Trypsin with EDTA (Gibco-Invitrogen, Burlington, Canada), centrifuged at 1600 RPM for 4 min, and resuspended with PBS. The cells in 50111 aliquots were vortexed and counted on a hemocytometer.
[0102]The proportion of viable cells in the samples was determined by measuring Trypan Blue uptake. At 48 hrs, the cells from the four pH treatments were suspended using 1 ml of Trypsin EDTA, centrifuged at 1600 RPM for 4 min and resuspended in 1 ml of BSS (Hank's Balanced Salt Solution) (Sigma, St. Louis). An aliquot of 0.5 ml of 0.04% Trypan Blue solution was transferred to a 1.5 ml tube, 0.03 ml of BSS was added to 0.2 ml of the cell suspension; the samples were mixed thoroughly and the cell suspension-Trypan Blue mixture was allowed to stand for 10 minutes and then counted on a hemocytometer. Separate counts were kept for both viable and non viable cells.
[0103]A (3-4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) MTT assay was conducted using the In Vitro Toxicology Assay Kit: MTT based (Sigma, St. Louis). The cells were cultured for the MTT assay at 24 and 48 hours and were incubated at 37° C. in 5% CO2 for 3 hrs in the presence of MTT 200 μl/2 ml medium. The samples were mixed by shaking the plate horizontally for 30 min. The background absorbance of the multi-well plates was determined at 690nm and subtracted from the 570 nm measurement.
Results
[0104]There were no significant differences in the total number of cells, as determined by direct hemocytometer counts, between the vehicle- and TCAP-treated cells at 24 hrs under any pH condition (FIG. 2a). There was a marked reduction in the number of total cells at pH 8.0 and 8.4 in the vehicle-treated cells at both 24 and 48 hrs. However, TCAP inhibited the decrease in total cell numbers relative to the vehicle-treated cells at pH 6.8 (P<0.001) 8.0 (P<0.001) and 8.4 (P<0.01) (F=38.10) after 48 hrs of incubation.
[0105]A Trypan Blue stain was conducted in order to estimate the proportion of viable cells in a population (FIG. 2b). TCAP-1 treatment caused a significant decrease in the number of cells that took up the trypan blue stain at 48hrs in cells cultured at pH 6.8 (p<0.05), pH 8.0 (p<0.0001) and at pH 8.4 (p<0.001)(F=58.27) but not pH 7.4. Although TCAP did not induce a significant effect on MTT activity at pH 7.4 or pH 6.8 there was a significant increase in optical density at 48hrs in TCAP-1-treated samples cultured in pH 8.0 (p<0.01) and pH 8.4 (p<0.001) (F=21.19) (FIG. 3).
Example 4
Fluorescent Microscopy of Necrosis and Apoptosis Markers
[0106]N38 cells were cultured on poly-D-Lysine treated coverslips (VWR, Mississauga) in each of the four pH condition, and cells were washed twice with PBS, each fluorochrome was added to each well: 5 μl Fluorescein (FITC)-Annexin V in Tris EDTA buffer containing 0.1% BSA (Bovine serum albumin) and 0.1% NaN3, pH 7.5, 5 μl rhodamine EtD-III 200 μM in PBS and 5 μl 4',6-Diamidino-2-phenylindole (DAPI) Hoechst 33342 5 μg/mL in PBS (Biotium, Inc. Hayward). The samples were incubated in the dark for 15 min, then washed before being placed on slides. The cells were viewed under a LEICA DM 4500 inverted fluorescent microscope and digitally analyzed using OpenLab software.
Results
[0107]Annexin V labelled with fluorescein (FITC) was used to identify apoptotic cells in green. Ethidium homodimer III (EtD-III) is a positively charged nucleic acid probe, which is impermeable to live or apoptotic cells but stains necrotic cells with red fluorescence (rhodamine) and Hoechst 3342 (4',6-Diamidino-2-phenylindole (DAPI) emits bright blue fluorescence upon binding to DNA in living cells.
[0108]TCAP-1 decreased the number of rhodanine-fluorescing cells at pH 6.8 (p<0.001), 8.0 (p<0.001) and 8.4 (p<0.001) (F=348.2) but not in the pH 7.4 samples (FIG. 4). There were nominal amounts of FITC-labelled cells located intermittently throughout samples where only a total of 3 green cells were counted (see inset, FIG. 4).
Summary of Examples 3 and 4
Necrosis
[0109]Necrosis occurs when cells are exposed to extreme variance from physiological conditions such as hypothermia and hypoxia, which may result in damage to the plasma membrane (Majno and Jorris, 1995). Necrosis begins with an impairment of the cell's ability to maintain homeostasis, leading to an influx of water and extracellular ions. Intracellular organelles, most notably the mitochondria, and the entire cell swell and rupture (cell lysis)(Linnik et al, 1993). Due to the ultimate degeneration of the plasma membrane, the cytoplasmic contents including lysosomal enzymes are released into the extracellular fluid. Therefore, in vivo, necrotic cell death is often associated with extensive tissue damage resulting in an intense inflammatory response (Emery et al, 1993). Necrosis was determined as the form of cell death occurring based on expected morphological alterations affecting the plasma membrane including massive production of small surface evaginations (bubbles) caused by the cells inability to control water influx through the plasma membrane (Rello et al., 2005). The Trypan Blue Stain (Example 3) is based on an acid dye that contains two azo chromophores. The reactivity of this dye is dependent on the negatively charged chromophore binding to cytoplasmic material when the membrane is damaged. Staining facilitates the visualization of cell morphology since it is only the dead cells that take up the dye, thus identifying cells that are necrotic or are in the very late stages of apoptosis. The fluorescent microscopy study (Example 4) also solidifies this assumption as TCAP decreases the number of necrotic cells and not apoptotic cells. These findings are significant as necrosis plays an integral role in neurodegenerative diseases.
Example 5
Apoptosis (Caspase and PARP) Markers
[0110]Apoptosis, otherwise known as "programmed cell death" is a necessary event of normal development. The apoptotic pathway is mediated by a family of death proteins, caspases, These signaling proteins are proteolytic enzymes that when inactive, lay dormant as zymogens until they are activated by various triggers (Hengartner, 2000). Upon activation of caspase 3 certain nuclear proteins are cleaved such as Poly ADP-ribose polymerase (PARP). PARP, a 116 kDa nuclear polymerase, is involved in DNA repair usually in response to environmental stress (Hengartner, 2000; Willie, 1980; Kerr, 1972). The protein can be cleaved by many interleukin-converting enzyme-like (ICE-like) proteases (Willie, 1980; Liu, 1997). (PARP) was one of the first proteins reported to be cleaved during apoptosis, and is a target of the Yama/CPP32 protease, caspase-3 (Kaufmann, 1989; Kaufman et al, 1993). Cleavage products occurring due to apoptosis result in western blot bands at 89 KDa The following experiments were conducted to determine whether TCAP works through the apoptotic pathway.
(a) Colorimetric Caspase Assays
[0111]Caspase 8 and 3 colorimetric assays were performed on the N38 cells at all pH conditions. The assay was based on the detection of the chromophore pNA after cleavage from the labeled substrate IETD-pNA and DEVD-pNA for caspase 8 and 3, respectively. Comparison of the pNA absorbance from the suspected apoptotic sample was compared to the uninduced neutral pH sample. Caspase 8 and 3 were analysed using the Caspase-3 Colorimetric Activity Assay (Chemicon, Temecula USA) and Caspase-8 Colorimetric Activity Assay (Chemicon, Temecula USA). The cells from each pH treatment described previously at 24 and 48 hrs were removed using a cell scraper and centrifuged at 1500 rpm for 10 minutes. The cells were resuspended in 350 μl of chilled cell lysis buffer containing 500 μl PBS, 5 μl 1% Triton×100 (Sigma, St. Louis), 25 μl proteinase inhibitor cocktail set III (VWR, Mississauga), 0.5 μl M dithiothreitol (DTT) (Sigma, St. Louis) and 2.5 μl phenylmethylsulphonylfluoride (PMSF) diluted in 1 mL of methanol (EM Science, Gibbstown), then incubated on ice for 10 min and centrifuged for 5 minutes at 10,000 rpm. The supernatant, consisting of cytosolic extracts, was transferred to a new tube and a bicinchoninic acid (BCA) protein assay (Pierce, Rockford) was conducted to determine total protein concentration. The absorbance of each sample was measured on a SPECTRAmax Microplate spectrophotometer at 405 nm after an incubation period of 2 hours at 37° C. Changes in caspase 3 activity were determined by comparing the absorbance reading from the induced sample with the level of the uninduced control. Background readings from the buffer were subtracted from the reading of both the induced (pH 6.8, 8.0, 8.4) and uninduced (pH 7.4) samples before calculating changes in caspase 3 activity. The same was done for the detection of caspase 8. As a control, N38 cells were cultured with pH 7.4 DMEM and incubated for 4 days, apoptosis was then induced using 10 μM/ml etoposide and lysed according to the above protocol and used a control for all subsequent caspase 3 detection. All assays were performed with 4 replications.
(b) Caspase 3 and Poly(ADP-ribose)Polymerase (PARP) Cleavage B Immunoblot
[0112]Detection of caspase 3 cleavage was determined at 48 hrs. The samples at each pH and control (see above) were lysed using total protein isolation lysis buffer (described above). An aliquot of 25 μl of each sample was combined with 25 μl of 2×20% sodium dodecyl sulphate (SDS) sample buffer and loaded onto a 4-10% HCL-Tris pre cast polyacrylamide gel (BioRad, Mississauga). The gel was run at 200 v for 35 min and proteins were electrotransfered to a Hybond-C nitrocellulose membrane (Amershain, Baie d'Urfe) for 75 min at 100 v. After transfer, the membrane was washed with 10 ml of PBS with 0.05% Tween 20 (PBST) for 5 min at room temperature (RT) and the membrane was incubated in 10 ml of PBST-milk for one hour at RT followed by 3 times for 5 min washes with 10 ml of PBST. The membrane was then incubated with cleaved caspase 3 primary antiserum (Cell Signaling Technology, Beverly) at a titre of 1:500 in 6ml of PBST-milk with gentle agitation overnight at 4° C. The membranes were washed 3 times for 5 min with 10ml of PBST followed by membrane incubation with anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Amersham, Baie d'Urfe) at 1:3000 in 6 ml of PBST-milk with gentle agitation for 1 hr at RT. The membranes were then washed 3 times for 5 min with 10 ml of PBST then exposed to Kodak X-OMAT Blue scientific imaging film (Perkin Elmer Canada Inc, Vaudreuil-Dorion) for 30 min.
[0113]Using the same protocol, changes in PARP expression were determined at 48 hrs. The membrane was incubated with PARP primary antibody (Cell Signaling Technology, Beverly) at a titre of 1:100. The membranes were washed 3 times for 5 min with 10 ml of PBST followed by membrane incubation with anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Amersham, Baie d'Urfe) at 1:3000 in 6 ml of PBST-milk with gentle agitation for 1 hr at RT. The membranes were then washed 3 times for 5 min with 10 ml of PBST then exposed to Kodak X-OMAT Blue scientific imaging film (Perkin Elmer Canada Inc, Vaudreuil-Dorion) for 30 min. Total optical density of the blots, were quantified using LabWorks 4.0 Image Acquisition and Analysis Software from Ultra-Violet Products Ltd. (UVP).
Results
[0114]Etoposide was used to determine the amount of caspase 8 (FIG. 5a) and 3 (FIG. 5b) activation under apoptotic conditions. Etoposide induced a greater than 3-fold increase in caspase 8 and 3.5-fold increase in caspase-3 relative to the vehicle-treated cells at pH 7.4. Although TCAP-1 increased caspase 8 activity in pH 7.4 samples (P<0.001)(F=20.80) and increased caspase 3 activity in pH 6.8 samples (P<0.05) (F=2.117), the relative level of caspase activity was about 70% and 40% of the etoposide-induced increase for caspase 8 and 3 respectively. There were no significant differences in caspase 8 and 3 activity between the TCAP-1- and vehicle-treated cells at pH 8.0 and 8.4. As a further determination of caspase 3 activity, four replicates of western blots were conducted on pH treated N38 cells at the 48 hr mark in order to detect the cleaved and activated caspase 3 (17/19 kDa) (FIG. 5c). The caspase 3 cleavage product was clearly visible in the protein extracts of the etoposide-treated cell but could not be observed in any of the TCAP-1 or vehicle-treated cells at any of the pH conditions.
[0115]Four replicates of western blots were conducted on pH treated N38 cells at the 48 hour mark in order to detect endogenous levels of full length PARP, as well as the large fragment (89 kDa) and small fragment (24 kDa) of PARP resulting from caspase cleavage. The western blot revealed endogenous PARP at all pH treatments as well as vehicles samples and based on a two way ANOVA using Bonferroni's Post Test, there were no significant differences between vehicle and TCAP treated samples (FIG. 6).
[0116]Based on the studies conducted and described in Example 5, TCAP is not protecting neuronal cells by inhibiting the apoptotic pathway.
Example 6
Kinase B/Akt Cell Survival Pathway
[0117]Protein kinase B or Akt (PKB/Akt) is a serine/threonine kinase, which functions to promote cell survival by inhibiting apoptosis by means of its ability to phosphorylate and inactivate several targets including BAD and forkhead transcription factors (Crowder, 1998). AKT, also referred to as PKB or Rac, plays a critical role in controlling the balance between cell survival and cell death in neurons (Dudek, 1997). The present example was conducted to determine whether TCP acts through this particular survival pathway.
[0118]Western blots using Akt and phosphorylated Akt (P-Akt) primary antibodies were conducted on all conditions of the cultured N38 cells to determine whether TCAP was preventing cell death by phosphorylation. The same western blot procedure outlined above was repeated with an Akt primary antibody (Cell Signalling, Beverly) at a titer of 1:500, followed by membrane incubation with anti-rabbit HRP-conjugated secondary antibody (Amersham, Baie d'Urfe) at 1:3000, followed by exposure on Kodak X-OMAT Blue film (Perkin Elmer Canada Inc, Vaudreuil-Dorion) for 30 min. Phospho Akt expression at 48 hrs was determined using the method described above with a PAkt primary antibody (Cell Signalling 9271) at 1:1000 followed by membrane incubation with anti-rabbit HRP-conjugated secondary antibody (Amersham, Baie d'Urfe) at 1:2000 followed by exposure on Kodak X-OMAT Blue film (Perkin Elmer Canada Inc, Vaudreuil-Dorion) overnight. Cultured N38 cells were serum-starved for 48 hours in order to induce phosphorylation and following the same protocol above were loaded as a control. Total optical density of the blots, were quantified using LabWorks 4.0 Image Acquisition and Analysis Software from Ultra-Violet Products Ltd. (UVP).
Results
[0119]Western blots were conducted using an Akt antibody, which detected total levels of endogenous Akt. The blot revealed endogenous Akt in all treatments as well as the vehicle, however according to a two way ANOVA using Bonferroni's Post Test, there appears to be no difference in endogenous Akt between vehicle and TCAP treated samples. Total optical density of the blots were quantified using LabWorks 4.0 Image Acquisition and Analysis Software from Ultra-Violet Products Ltd. (UVP) (FIG. 7b).
[0120]Western blots were conducted using a Phospho-Akt antibody, which detected total levels of endogenous Akt1 only when phosphorylated at serine 473. The blot revealed no bands in any samples, thus phosphylation of Akt is not occurring. Phsophorylation of cells was induced by serum starvation and loaded as a control, the blot revealed a band, however no other bands were detected (FIG. 7d)
Example 7
The Effect of TCAP On Cell Cycling: Bromodeoxyuridine (BrdU) Incorporation Assay
[0121]The evaluation of cell cycle progression is important when assessing the viability of a cell population. The cell cycle is a sequence of stages that a cell passes through between one division and the next. The cell cycle oscillates between mitosis and the interphase, which is divided into G, S, and G 2. In the G phase there is a high rate of biosynthesis and growth; in the S phase there is the doubling of the DNA content as a consequence of chromosome replication; in the G 2 phase the final preparations for cell division (cytokinesis) are made (Raza, 1985). In order to determine whether TCAP was increasing cell cycle efficiency, a bromodeoxyuridine (BrdU) non-isotopic enzyme immunoassay was conducted (Calbiochem, Canada). BrdU incorporation into newly synthesized DNA of actively proliferating cells enables one to quantify cell cycle progression and the population of cells entering the S phase (Gratzner, 1982; Raza, 1985).
[0122]N38 cells were grown in a 96-well culture plate using 100 μl at an initial density of 2×105 cells/ml. Controls consisted of a blank, one well containing only DMEM with no cells and background, and one well with cells but with no BrdU label added. A working stock of BrdU was prepared by diluting the BrdU label 1:2000 into fresh DMEM, 20 μl of the working stock was added to each well to be labelled, the BrdU was allowed to incubate with the cells for 2 hrs at 37° C. The contents of the wells were then removed and 200 μl of the enclosed Fixative/Dentauring solution was added to each well and incubated for 30 min at Room Temperature (RT). The contents of the wells were removed and Anti-BrdU Antibody (1:100) was added to each well and incubated for 1 hr at RT. Wells were washed 3 times with wash buffer, the plate was then gently blotted on paper towel. The conjugate was prepared by diluting the reconstituted in (1×PBS) peroxidase goat anti-Mouse IgG HRP conjugate in the enclosed conjugate diluent and loaded onto a syringe filter through 0.2 μm filter and a 100 μl aliquot of this solution was transferred to each well and incubated for 30 min at RT. The wells were washed with wash buffer, the entire plate was then flooded with double deonized water and the contents of the wells were removed. An aliquot of 100 μl of BrdU substrate solution was added to each well, the plate was then incubated in the dark at RT for 15 min. 100 μl of stop solution containing 2.5N sulphuric acid was added to each well in the same order as the previously added substrate solution. Absorbance was measured on a SPECTRAmax Microplate spectrophotometer at dual wavelengths at 450-540nm.
Results
[0123]Based on a two way ANOVA using Bonferroni's Post Test there were no significant results at 24 or 48 hrs (FIG. 8).
[0124]This investigation indicates that synthetic TCAP-1 has a neuroprotective effect on immortalized hypothalamic mouse cells. The data described in this study suggest a significant neuroprotective role for TCAP during times of pH induced cellular stress. Several lines of evidence point to this. Based on haemocytometer counts and an MTT assay conducted on pH stressed N38 cell samples, TCAP has a positive affect on cell viability during pH induced cellular stress, suggesting that TCAP could be inhibiting cells from undergoing apoptosis, acting through a cell survival pathway or rescuing cells from necrosis. The Examples herein indicate that this neuroprotective effect occurs by the inhibition of mechanisms regulating necrosis and to a lesser extent by regulating apoptotic, survival, or cell cycle pathways.
Example 8
TCAP Modulates Neurite Length in Immortalized Hypothalamic N38 Cells
[0125]Immortalized mouse hypothalamic N38 cells were treated with 1 nM and 100 nM mouse TCAP-1 and measurements of neurite lengths were taken over 8 hours post TCAP administration. FIG. 9A illustrates untreated cells at 8 hours. FIG. 9B illustrates cells treated with 100 nM of TCAP-1 at 8 hours. FIG. 9C illustrates percent change in neurite length in control (untreated), 1 nM TCAP-1 and 100 nM TCAP-1 at 0, 4 and 8 hours post TCAP administration. FIG. 9D illustrates the percent change of number of neuritis in control (untreated), 1 nM TCAP-1 and 100 nM TCAP-1 at 0, 4, and 8 hours post TCAP administration. FIGS. 9E and 9F illustrate the frequency distribution in neurite length of the cell population in untreated (9E) and 100 nM TCAP-1 treated (9F) samples.
Results
[0126]The results of these experiments illustrate that TCAP is useful in enhancing neurite length. TCAP-1 induced 25% and 31% increase (p<0.001, one way ANOVA with Bonferroni's post-test, n=4) in neurite length at 100 nM, at 4 hr and 8 hr, respectively, relative to the length at the beginning of treatment. After 8 hrs, 100 nM TCAP-1 induced about a 45% (p<0.001) reduction in the number of neurites per cell. A frequency distribution of the neurite length indicated that 100 nM TCAP-1 promoted longer but fewer neurites per cell
Example 9
TCAP Upregulates β-Tubulin and β-Actin Levels In Immortalized N38 Cells
[0127]-tubulin and β-actin expression levels in immortalized mouse N38 cells were studied.
Materials and Methods
Primary Antisera
[0128]All antisera used in this study are rabbit polyclonal antisera. β-Actin and GAPDH were purchased from Abcam (Cambridge, Mass.). α-actinin 4 antisera were purchased from Alexis Biochemicals (Lausen, Switzerland). The β-tubulin antisera was purchased from Neomarkers, Lab Vision (Fremont, Calif.) and β-tubulin III was purchased from Sigma-Aldrich Canada (Oakville, ON).
Morphological Analyses of Immortalized Neurons
[0129]N-38 immortalized mouse hypothalamic cells were cultured in quadruplet in 6 well tissue culture plates until 70% confluent at which time fresh DMEM with 10% fetal bovine serum (Invitrogen Canada, Burlington, ON) containing 1 nM TCAP-1, 100 nM TCAP-1 or vehicle (PBS) was added. Each well was digitally imaged twice at 0, 4 and 8 hours using an inverted Zeiss Axiovert 200M. A minimum of 90 cells were analyzed per condition using Labworks V4.0.0.8 (UVP, Upland, Calif.) and scored for number of neurites per cell, neurite length and cell size.
Quantitative Real Time-PCR
[0130]Total RNA from N-38 cells was isolated by the guanidinium thiocyanate phenol chloroform extraction method (Chomczynski and Sacchi, 1987). First strand cDNA was synthesized from 1 μg deoxyribonuclease I-treated RNA, using SuperScript reverse transcriptase (RT) and random primers (Invitrogen, Carlsbad, Calif.), as described in the Superscript cDNA Synthesis Kit (Invitrogen, Carlsbad, Calif.). The specificity of each amplification reaction was monitored in control reactions, where amplification was carried out on samples in which the RT was omitted (RT-). Quantitative "real time" RTPCR was performed as described in the SYBR Green PCR Master Mix and PCR Protocol (Applied Biosystems, Foster City, Calif.). Briefly, cDNA was synthesized from 1 μg total RNA in a total volume of 20 ul. 50-100 ng cDNA as template was amplified with SYBR Green Master Mix (Applied Biosystems) and 300 nM primers in a 10 μl reaction for 40 cycles (15 sec at 95 C., 1 min 60 C.). The primers used for RT-PCR are: 18s rRNA; gtaacccgttgaaccccatt, ccatccaatcggtagtagcg: α-actinin 4; gagaagcagcagcgcaaga, ccgaagatgagagttgcacca: β-actin; ggccaaccgtgaaaagatga, cacagcctggatggctacgt: β-catenin; agcagtttgtggagggcgt, cgagcaaggatgtggagagc: α-tubulin 1; acaggattcgcaagctggc, ccaagaagccctggagacc: and β-tubulin 4; tgaggccacaggtggaaactatgt, aagttgtctggccgaaagatctgg. All primers were designed using Primer Express software (Applied Biosystems) and synthesized by ACGT Corp. (Toronto, ON) or Integrated DNA Technologies, Inc. (Coralville, Iowa). Data was represented as mean quantity, defined as the average of the replicate group (n>3), analyzed using ABI Prism 7000 SDS software package (Applied Biosystems). Copy number of amplified gene was standardized to 18S rRNA levels. The final fold differences in expression were relative to the vehicle treatment at each individual timepoint.
Western Blot Analysis of Cytoskeletal Proteins
[0131]N38 immortalized hypothalamic cells were cultured as described previously (Belsham et al, 2004) in Dulbecco's Modified Eagle Medium (DMEM) with 5% fetal bovine serum (Invitrogen Canada, Burlington, ON). At 70% confluency, cells were treated with medium containing 1 nM TCAP-1, 100 nM TCAP-1 or vehicle (phosphate buffered saline (PBS) pH 7.4) for 0.5, 1, 4 or 8 hours after which total cell proteins were extracted. Briefly, cells were removed in the presence of cold PBS and centrifuged. The cells were resuspended in PBS containing 1% Triton X-100 (Sigma), 1 mM dithiothreitol (DTT) and protease inhibitors ((5% Protease inhibitor cocktail set III (Calbiochem, EMD Biosciences, San Diego, Calif.) and 1 mM phenylmethyl sulfonyl fluoride (PMSF, EM Science)). Following vortex mixing, the cells were spun for 15 minutes at 15,300 g at 4° C. The supernatant containing total proteins was stored at -20° C. until further analysis. The protein concentration was determined using a BCA protein assay kit (Pierce Chemical Co, Rockford, Ill.). For SDS PAGE, the appropriate μg loading volumes were determined for each antiserum. Samples were mixed with sample buffer containing SDS and boiled for 5 minutes at 90° C. and were run in duplicate to test for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. The proteins were resolved on 4-20% Tris-HCl Ready gels (Bio-Rad, Hercules, Calif.) using a Mini-PROTEAN 3 Cell (Bio-Rad) electrophoresis unit for 35 minutes at 200V. Transfers were performed using the Mini Trans-Blot Electrophoretic Transfer cell (Bio-Rad) with Hybond C Nitrocellulose membranes (GE Healthcare, Piscataway, N.J.) at 100V for 2 hours. Membranes were blocked in 0.2% PBS-Tween 20(v/v) containing 5% nonfat milk (w/v) and probed with primary antiserum overnight at 4° C. at the appropriate dilution. The dilutions are as follows: β-actin, 1:4000; β-tubulin, 1:500; α-actinin-4,1:5000. The secondary antibody conjugated to horse radish peroxidase was used at a concentration of 1:5000. For all analyses the GAPDH antiseraum was used at a dilution of 1:2000. A ECL Western Blotting Analysis System (GE Healthcare, Piscataway, N.J.) was used to detect the proteins using X OMAT Blue XB1 film. Blots were scanned and optical density was determined using an Epi Chemi II Darkroom and Lab works V4.0.0.8 (UVP, Upland, Calif.).
Gene and Protein Expression and Confocal Studies
Gene Expression
[0132]Significant changes in mRNA expression, as determined by real-time PCR were not observed in any of the 1 nM TCAP treatments (FIG. 10). However there were indications of expression increase in α-actinin-4 and β-tubulin MRNA after 4 hours, although these changes were not statistically significant. In contrast, at a concentration of 100 nM TCAP, there was a significant increase in synthesis as determined by a two-way analysis of variance (ANOVA) for β-catenin (p=0.0158; F=6.192), α-actinin-4 (p=0.0265; F=5.329) and α-tubulin (p=0.0042; F=9.320). Expression of MRNA for β-catenin and α-actinin-4 showed increases between 30 and 40% within 1 hr of treatment and remained high for 8 h. β-tubulin expression was more modest with a maximal increase of 25 to 30%, although inter-experimental variability as assessed by standard errors were low.
Protein Expression
[0133]Treatment of cells with 1 nM TCAP-1 did not result in any significant changes in β-tubulin protein levels over 8 hours (FIG. 11). In contrast, cells treated with 100 nM showed a significant increase of 60% at 1 hour in β-tubulin relative to the vehicle treated cells at the same time point (two-way ANOVA with Bonferroni post-test, p<0.05, F=1.48). TCAP-1 treated cells also experienced a significant change with regards to β-actin expression (FIG. 12). A concentration of 1 nM TCAP-1 showed an expression level of 184±4.1% of vehicle at 0.5 hrs. Similarly 100 nM TCAP-1 induced an expression level of 192±10.5%. No significant effects were noted at any other time points or on α-actinin-4 (FIG. 13). Due to the consistent high levels of both β-tubulin mRNA and protein levels, βtubulin immunoreactivity was used as a marker to examine subsequent TCAP induced effects on cellular morphology.
Immunofluorescence Confocal Microscopy
[0134]In one study, a confocal analysis of 100 nM TCAP-1 effects on localization of B-tubulin in N38 cells was conducted.
[0135]FIG. 14A show B-tubulin immunoreactivity with Alexa fluor 488 in vehicle and TCAP-1 treated cells after 1 hour post-vehicle or mouse TCAP-1 treatment, respectively. Ten central cells from each image were then analyzed for number of pixels at maximal intensity (149) and expressed as a ratio of total pixels in the perinuclear region (FIG. 14B) and the whole cell (FIG. 14C) (Student's t-test with Welch's correction for unequal variances P=0.05; bar=20 μm). Perinuclear region and cell size were not different in 30 cells per group.
Results
[0136]Overall, the TCAP-treated cells were characterized by greater clarity and number of observable β-tubulin strands in the cells and the neuritis (FIG. 14A). The 100 nM TCAP-1 treatment resulted in a significant increase in whole cell immunofluorescence (FIG. 14B).
[0137]The results indicate that cells treated with TCAP show an increased expression of B-tubulin in the cell and perinuclear region of neuronal cells and increase in B-tubulin protein levels. The results further illustrate that cytoskeletal B-actin is upregulated in TCAP-treated cells. Actin synthesis and expression is a normal and required componet of neuron function, migration and axon elongfation. Regulation of actin synthesis and expression is required for restoration of function following necrotic or inflammatory degenerative conditions.
Example 10
TCAP Induces Repulsion In Growing Axons
[0138]100 μM TCAP-1 was puffed on the neurite of an N38 cell in the direction of the arrow in FIG. 15. The neurite was imaged over one hour. TCAP caused expansion of the growth cone area followed by repulsion away from the source of TCAP. Bar-1 um.
Results
[0139]The images clearly show that TCAP induce repulsion in growing axons and can be used as a guidance molecule for neuronal growth and potentially fasciculation.
Example 11
Increases Growth and Fasciculation of Primary Embryonic Hippocampal Cultures
[0140]This example illustrates the immunohistochemistry of B-tubulin III in primary hippocampal E18 cultures treated with vehicle or 100 nM TCAP-1 for seven days.
[0141]Timed-pregnant Sprague-Dawley rats (Charles River, Boston, Mass.) on day 18 (E18) of gestation were euthanized in a CO2 chamber. The uteri were surgically removed and embryos were collected in Hank's balanced salts solution (HBSS) with 15 mM HEPES and 10 mM sodium bicarbonate (Sigma-Aldrich Canada, Oakville, ON). The embryos were decapitated the hippocampi dissected. The hippocampi were trypsinized for 15 minutes at 37° C., centrifuged for 5 minutes at 1600 rpm and the pellets washed two times in HBSS. The cell pellets were suspended in Neurobasal medium supplemented with B27, 0.5 mM Glutamax, and penicillin/streptomycin and this medium was subsequently used for culturing. Following trituration with a fire polished glass pipette, 300,000 cells were plated into 6-well plates containing 12 mm glass coverslips coated with poly-D-lysine (VWR, Mississauga, ON). After 24 hours, fresh medium containing 100 nM TCAP or vehicle was used. The medium was replaced twice a week. On the eighth day of culture, coverslips were processed.
[0142]The coverslips with cells were rinsed with PBS (pH 7.4) twice before fixing with 1 ml 4% paraformaldehyde for 15 minutes. Following two washes for 5 minutes, cells were permiabilized by addition with 0.2% Triton X100 solution in PBS for 90 seconds. After washing twice for 2 minutes, the cells were incubated with 0.5% normal goat serum (NGS) in PBS. A 1:100 dilution of β-tubulin III antiserum in 0.5% NGS was applied to the coverslips and incubated at room temperature for 1 hour. The detection and staining was done according to instructions provided by the Vectastain ABC kit (Vector Laboratories, Burlington, ON, Canada). The biotinylated goat anti-rabbit serum was applied at 1:200 dilution in serum for 1 hour as well. The Vectastain reagents, avidin DH and biotinlyated horse radish peroxidase H were mixed and incubated with the cells for 30 minutes before washing for 5 minutes with PBS. The DAB substrate (Vector Laboratories) was then added for 8 minutes and cells washed with distilled water for 5 minutes. The cells were dehydrated with ethanol, cleared with Xylene and the coverslip mounted on a slide using Vectamount mounting medium (Vector Laboratories). The stained cells were visualized using an Olympus (BX60) microscope and imaged with a CCD CoolSNAP camera (Photometrics, Tuscon, Ariz.).
Results
[0143]The results shown in FIGS. 16 and 17 clearly show that TCAP caused an increase in dendritic density and fasciculation compared to control.
[0144]The β-tubulin-III immunoreactivity in primary hippocampal cultures treated with 100 nM TCAP-1 was enhanced (FIG. 16). A frequency distribution of pixel intensity indicated a significant (p<0.05) effect of TCAP-1. using a Chi square test for trends. The increase in immunoreactivity was due to both an increase in total number of cells and cell processes as indicated by the increase in the number of pixels in the dark gray to black regions (FIG. 16A). TCAP-1 treated cultures show a significantly greater (p=0.0142) mean number of cell clusters (270±22) over the vehicle treated cells (175±17) as determined by a two-tailed Students t-test. A much denser mesh of cell processes were observed in TCAP treated cells.
[0145]TCAP treated hippocampal cells showed a much greater incidence in the number of large axons and axon bundles relative to the vehicle treated cells. Higher magnification of both groups of cultures revealed that the TCAP treated cells showed a much greater tendency for fasciculation along with a greater incidence of neural processes outgrowth (FIG. 17).
[0146]The results further illustrate that TCAP can be used to increase fasciculation among neurons and in addition to supporting the effects of TCAP on inhibiting neuronal cell death, it illustrates that TCAP may be used in the treatment of a number of conditions, such as brain injury, especially if administered with 8 or 24 hours of said injury to minimize any secondary injury effects.
[0147]TCAP 1 is a novel putative neuropeptide that bears the structural hallmarks of a bioactive peptide. TCAP-1 can modulate cell growth and anxiety-related behaviors. The present study shows that TCAP-1 has the ability to stimulate neurite outgrowth in part by increasing the synthesis of components of the cytoskeleton. The TCAP-1 mediated neurite outgrowth is coupled with an increase in the synthesis and translation of β-tubulin and possibly the enhanced translation of β-actin. In primary hippocampal cultures, the increase in β-tubulin expression is associated with an increase in the number of immunoreactive β-tubulin cells and large axonal processes. Because many long term behavioural effects are associated with changes in neuronal circuitry, the effects observed with TCAP can be explained by changes in the morphological properties of neurons.
[0148]The morphological characteristics of cells treated with TCAP were examined. An immortalized hypothalamic cell line (N38) previously known to be responsive to TCAP-1 (Belsham et al, 2004; Wang et al, 2005) was used in the Examples. Cell cultures were held at 70-80% confluency as a maximal as beyond that, the cells went into a stress response. TCAP-1 treated cells showed a dose-dependent increase in the number of longer neurites and a decrease in the number of shorter neurites.
[0149]Together, the present studies with the N38 cell line indicate that TCAP 1 stimulates neurite outgrowth and increases the synthesis and translation of β-tubulin while enhancing β-actin translation only. TCAP induced an increase in the incidence of axon formation and fasciculation. In one embodiment, TCAP and the teneurins can be used to regulate neuronal process outgrowth in the hippocampus and in the potentiation of learning and memory.
[0150]In one embodiment, the Examples indicate that TCAP may exert its effects at least in part by inducing changes in axonal and dendritic outgrowth. Changes in dendritic morphology are important since they are the mechanism behind many diseases and disorders. Specifically, the hippocampus is a neuroplastic part of the brain whose cells when exposed to effectors can undergo morphological changes associated with disorders such as stress and depression (McEwen, 1999). The present Examples also indicate that the TCAP and teneurin system is associated with neuroplasticity, learning and anxiety.
Example 12
Superoxide Dismutase-Catalase Data
Superoxide Dismutase Detection and Measurement
[0151]Examination of the superoxide dismutase-associated system was investigated as a possible mechanism for necrosis after the apoptotic, survival and cell cycle experiments did not show a robust effect. The presence of the superoxide radical was measured indirectly by the conversion of a soluble tetrazolium salt in cells after 48 hours (FIG. 18A). The TCAP-1 treated cells showed a 40% (p<0.05) and 60% (p<0.01) decrease in the absorbance of the substrate, which is proportional to superoxide radical activity, at pHs 8.0 and 8.4, respectively. However, because this method shows only the indirect presence of the superoxide radical, and by inference, the presence of superoxide dismutase, we also examined the presence of this enzyme protein directly by western blot (FIG. 18 B,C). Relative to the vehicle-treated cells at pH 7.4, superoxide dismutase levels in the vehicle-treated cells showed a significant (p<0.05) decrease as a function of pH, as determined by a one-way ANOVA. There were no significant differences in the expression of the superoxide dismutase protein at pHs 6.8 and 7.4. In contrast, at pH 8.0 and 8.4, TCAP-1 significantly (p<0.05 and p<0.01, respectively) reduced the pH-induced decline in superoxide dismutase levels. The superoxide dismutase expression levels at pH 8.0 and 8.4 were not significantly different than that of the vehicle-treated cells at pH 7.4.
[0152]Superoxide dismustase gene expression as measured by real-time PCR indicated a significant (p<0.01) increase over the vehicle treated cells at pH 7.4 and 8.4 (FIG. 18D). A greater effect on gene expression was noted in superoxide copper chaperone (CCSD) expression where CCSD expression levels in the TCAP-1 treated cells at pH 8.4 was increased almost 4.5 fold over the vehicle treated cells (FIG. 18E).
[0153]H2O2 toxicity and catalase activity
[0154]TCAP-1 showed a significant increase in MTT activity relative to the vehicle-treated at 6 to 48 hours in cells treated with 50 μM H2O 2 (FIG. 19A). The results indicate that TCAP-1 significantly increased mitochondrial activity at 6, 12 and 48 hours (p<0.001) (F=168.2) as compared with the vehicle-treated cells. There was also a less significant effect at 24 hours (p<0.05) and no effect at 0 hours.
[0155]A catalase assay was performed on the pH treated cells in order to determine whether TCAP-1 was conferring survivability to the cells via upregulation of catalase and thus increasing H2O 2 breakdown into H2O and O2 (FIG. 19B). The results indicate that TCAP-1 significantly increased catalase levels at pH 8.4 (p<0.001)(F=24.42) as compared to the vehicle treated cells according to a two-way ANOVA with a Bonferroni's post hoc test. There was also a significant TCAP-1 effect at pH 8.0 (p<0.01) but no significant effects at either pH 6.8 or pH 7.4 compared to the vehicle treated cells. Bovine liver was also assayed as a positive control. Catalase gene expression, as determined by real-time PCR indicated that TCAP induced mRNA levels by 3 fold (p<0.001) and 5 fold (p<0.001) at pHs 8.0 and 8.4, respectively (FIG. 19C).
[0156]Superoxide dismutase is an enzyme that is responsible for catalyzing the highly reactive oxygen radical, superoxide (O2-) into hydrogen peroxide (H2O2). Hydrogen peroxide is in turn, catalysed to water by the enzyme catalase. Superoxide dismutase is bound to copper atoms for full activity. The protein superoxide dismutase copper chaperone acts to effect the transfer of copper to superoxide dismutase. Together, these three proteins act to protect the cells from the toxic effects of reactive oxygen species (ROS). High concentrations of ROS have been implicated in the destruction of cellular membranes and proteins and play a significant role in the onset of neurodegenerative disorders. The findings that TCAP enhances the activity and expression of the superoxide dismutase-catalase system is indicative that TCAP inhibits cellular necrosis.
[0157]While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art, from a reading of the disclosure, that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.
[0158]All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
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Pawloski, J., Kraft, A. Bax induced apoptotic cell death. PNAS. 97(2000):529-531. [0192]34. Potapenko E, Kostyuk E, Voitenko N, Kostyuk P. Alkalinization-induced changes in intracellular calcium in rat spinal cord neurons. Neurochem Res. 2004 Sep;29(9):1659-65. [0193]35. Priest, J. M, Fischbeck, K. H., Nouri, N & B. J., Keats, A locus for axonal motorsensory neuropathy with deafness and mental retardation maps to Xq24-q26. Genomics 29 (1995) 409-412. [0194]36. Qian X, Barsyte-Lovejoy D, Wang L, Chewpoy B, Gautam N, Al Chawaf A, D. A Lovejoy, Cloning and characterization of teneurin C-terminus associated peptide TCAP)-3 from the hypothalamus of an adult rainbow trout (Oncorhynchus mykiss). Gen Comp Endocrinol. 137(2004):205-16. [0195]37. Raza, A., Ukar, K., Preisler, H D. Double labeling and in vitro versus in vivo incorporation of bromodeoxyuridine in patients with acute nonlymphocytic leukemia Cytometry 6(1985):633-40. [0196]38. Rello, S., Stockert, J., Moreno, V., Gamez, A., Pacheco, M., Juarranz, A., Canete, M., Villanueva, A. Morphological criteria to distinguish cell death induced by apoptotic and necrotic treatments. Apoptosis. 10(2005):201-208. [0197]39. Robertson N J, Cowan F M, Cox U, Edwards A D Brain alkaline intracellular pH after neonatal encephalopathy. Ann. Neurol. 52(2002):732-742. [0198]40. Rosser B G and Gores G J. Liver cell necrosis: cellular mechanisms and clinical implications. Gastroenterology. 108(1995):252-275. [0199]41. Rubin, B., Tucker, P., Martin, D., Chiquet-Ehrismann, R. Teneurins: a novel family of neuronal cell surface proteins in vertebrates, homologous to the Drosophila pair-rule gene product, Ten-m. Dev Biol. 216 (1999): 195-209. [0200]42. Sapolsky, R. M. (1992) Stress, the aging brain and the mechanisms of neuronal death. MIT Press. Cambridge Mass. 428 pages. [0201]43. Stout, A., Raphael, H., Kanterewicz, B., Klann, E., Reynolds, I. Glutamate-induced neuron death requires mitochondrial calcium uptake. Nature Neuroscience. 1 (1998): 366-373. [0202]44. Thornton J S, Ordidge R J, Penrice J, Cady E B, Amess P N, Punwani S, Clemence M, Wyatt J S, Temporal and anatomical variations of brain water apparent diffusion coefficient in perinatal cerebral hypoxic-ischemic injury: relationships to cerebral energy metabolism. Magn Reson. Med. 39 (1998):920-927. [0203]45. Thompson, C B. Apoptosis in the pathogenesis and treatment of disease. Science 267(1995):1465-1462. [0204]46. Traynelis S, Cull-Candy S. Proton inhibition of NMDA receptors in cerebellar neurons. Nature. 356(1990): 347-349. [0205]47. Vomov J J, Thomas A G, Jo D. Protective effects of extracellular acidosis and blockade of sodium/hydrogen ion exchange during recovery from metabolic inhibition in neuronal tissue culture. J Neurochem. 67(1996): 2379-2389. [0206]48. Wang, L. S. Rotzinger, A. AlChawaf, R. B. Chewpoy D. Barsyte-Lovejoy, X. Qian, C. F. Elias, N. C. Wang, J. C. Bittencourt, A. De Cristefaro, D. Belsham, F. Vaccarino, D. A. Lovejoy, Teneurin proteins possess a carboxy terminal (CRF)-like sequence that modulates emotionality and neuronal growth, Submitted to Molecular Brain Research (2004). [0207]49. Watson, A J M. Necrosis and apoptosis in the gastrointestinal tract. Gut 37(1995):165-167. [0208]50. Willie, A H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284(1980):555-6. [0209]51. Willie, A H., Kerr, J F R and Currie A C. Cell death: the significance of apoptosis. Int.Rev.Cytol. 68 (1980):251-305. [0210]52. Xu L, Glassford A J M, Giaccia A J, Giffard R G. Acidosis reduces neuronal apoptosis. Neuroreport. 9(1998): 875-879. [0211]53. Zhang Y, Pardridge W M. Conjugation of brain-derived neurotrophic factor to a blood-brain barrier drug targeting system enables neuroprotection in regional brain ischemia following intravenous injection of the neurotrophin. Brain Res 889(2001): 49-56. [0212]54. Zhou X H, Brandau O, Feng K, Oohashi T, Ninomiya Y, Rauch U, Fassler R (2003) The murine Ten-m/Odz genes show distinct by overlapping expression patterns during development and in adult brain. Gene Expression Patterns 3:397-405. 21
Sequence CWU
1
13611490DNAArtificial SequenceRainbow Trout Ten M3 carboxy termini'
1tccatctcgg gggtgcaaca ggaagtgacc cggcaagcca aggctttcct gtccttcgag
60aggatgccgg agatccagct gagccgccgg cgctccaacc gggagaaacc ctggctgtgg
120ttcgccaccg ccaagtctct gatcggtaag ggtgtcatgt tggcggtgac gcagggccgt
180gtggtcacca acgctctgaa catcgccaac gaggactgca tcaaggtcgc cgccgtcctc
240aacaatgcgt tctacctgga ggacctgcac ttcacggtgg agggacgcga cacgcactac
300ttcatcaaga ccagcctccc ggagagcgac ctgggagcgc tgaggctgac aagcgggagg
360aagtcgctgg agaacggaag tcaacgtgac tgtgtcccag tccaccaccg tggtgaacgg
420cagaaccggc gcttcgccga cgtggagctg cagtacggcg ctctagcgct ccacgtgcgc
480tatggcatga ctctggacga ggagaaggcg cgtgtgctgg agcaggccag gcagaaggcg
540ttgtcgagtg cctggtccag ggagcaacaa cgggtgaggg agggggagga gggggtgagg
600ctgtggacgg agggggagaa gaggcagctg ctgagcggga ggaaggttct gggctacgac
660gggtactacg tcctctccat agagcagtac cccgagctag cagactccgc taacaacatc
720cagttcctca ggcagagcga aatagggaag aggtaacaga cagaatcctc ggcactggcc
780gccaaagaga ctaccccctc caaatcctgc cccccaacct ccctcgcctc cccccttttc
840tctaaaaagg gggagggtcc aggctagtgc tgtgtttagc gccgactagc tgaaacaaac
900agtaaaatgt agaatatctt aaactgaact atacctaata ctaccactgt ggggcctgaa
960aatcaaacaa aacggctcca actgacgcaa atgtttgtcc catgtgctat acagcgttga
1020atggactgtg gactctcttg aaaagagaga aaaaaaagtc aaaactctcg gtttgtgaaa
1080ggagaaaaaa acgttttttt tttttttaaa tagacttcct gaatttgctt tcggaaaaaa
1140tattttaaaa agaaagaaga aatgtgttta catacgcata acactacaac acgtctggac
1200taatagaaga aaagccttct ggtttcttac acaggacaac gtctataatc tgattctaca
1260tcctgacgac tgacctttga ttgacctttg cgtactgaaa aaggtagtgt tgttgttcgc
1320agtaggacca tgggtctcca atggtggtaa ctagacagtt aaaaccactt gttgaaacca
1380cttgcttgtt cttctgcttt tctttccaaa agggacaaaa cagctcccac caagtgactt
1440ctttaccaat actagatcaa agtgggacgt tttgggctcg tgccgaattc
14902756DNAArtificial SequenceRainbow Trout Ten M3 coding sequence of
carboxy termini of Ten M3 2tccatctcgg gggtgcaaca ggaagtgacc
cggcaagcca aggctttcct gtccttcgag 60aggatgccgg agatccagct gagccgccgg
cgctccaacc gggagaaacc ctggctgtgg 120ttcgccaccg ccaagtctct gatcggtaag
ggtgtcatgt tggcggtgac gcagggccgt 180gtggtcacca acgctctgaa catcgccaac
gaggactgca tcaaggtcgc cgccgtcctc 240aacaatgcgt tctacctgga ggacctgcac
ttcacggtgg agggacgcga cacgcactac 300ttcatcaaga ccagcctccc ggagagcgac
ctgggagcgc tgaggctgac aagcgggagg 360aagtcgctgg agaacggaag tcaacgtgac
tgtgtcccag tccaccaccg tggtgaacgg 420cagaaccggc gcttcgccga cgtggagctg
cagtacggcg ctctagcgct ccacgtgcgc 480tatggcatga ctctggacga ggagaaggcg
cgtgtgctgg agcaggccag gcagaaggcg 540ttgtcgagtg cctggtccag ggagcaacaa
cgggtgaggg agggggagga gggggtgagg 600ctgtggacgg agggggagaa gaggcagctg
ctgagcggga ggaaggttct gggctacgac 660gggtactacg tcctctccat agagcagtac
cccgagctag cagactccgc taacaacatc 720cagttcctca ggcagagcga aatagggaag
aggtaa 7563251PRTArtificial SequenceRainbow
Trout Ten M3 carboxy termini of Ten M3 3Ser Ile Ser Gly Val Gln Gln Glu
Val Thr Arg Gln Ala Lys Ala Phe1 5 10
15Leu Ser Phe Glu Arg Met Pro Glu Ile Gln Leu Ser Arg Arg
Arg Ser20 25 30Asn Arg Glu Lys Pro Trp
Leu Trp Phe Ala Thr Ala Lys Ser Leu Ile35 40
45Gly Lys Gly Val Met Leu Ala Val Thr Gln Gly Arg Val Val Thr Asn50
55 60Ala Leu Asn Ile Ala Asn Glu Asp Cys
Ile Lys Val Ala Ala Val Leu65 70 75
80Asn Asn Ala Phe Tyr Leu Glu Asp Leu His Phe Thr Val Glu
Gly Arg85 90 95Asp Thr His Tyr Phe Ile
Lys Thr Ser Leu Pro Glu Ser Asp Leu Gly100 105
110Ala Leu Arg Leu Thr Ser Gly Arg Lys Ser Leu Glu Asn Gly Val
Asn115 120 125Val Thr Val Ser Gln Ser Thr
Thr Val Val Asn Gly Arg Thr Arg Arg130 135
140Phe Ala Asp Val Glu Leu Gln Tyr Gly Ala Leu Ala Leu His Val Arg145
150 155 160Tyr Gly Met Thr
Leu Asp Glu Glu Lys Ala Arg Val Leu Glu Gln Ala165 170
175Arg Gln Lys Ala Leu Ser Ser Ala Trp Ser Arg Glu Gln Gln
Arg Val180 185 190Arg Glu Gly Glu Glu Gly
Val Arg Leu Trp Thr Glu Gly Glu Lys Arg195 200
205Gln Leu Leu Ser Gly Arg Lys Val Leu Gly Tyr Asp Gly Tyr Tyr
Val210 215 220Leu Ser Ile Glu Gln Tyr Pro
Glu Leu Ala Asp Ser Ala Asn Asn Ile225 230
235 240Gln Phe Leu Arg Gln Ser Glu Ile Gly Lys Arg245
2504252PRTArtificial SequenceMouse Ten M1 4Met Ile Leu Gly
Ile Gln Cys Glu Leu Gln Lys Gln Leu Arg Asn Phe1 5
10 15Ile Ser Leu Asp Gln Leu Pro Met Thr Pro
Gln Tyr Asn Glu Gly Arg20 25 30Cys Leu
Glu Gly Gly Lys Gln Pro Arg Phe Ala Ala Val Pro Ser Val35
40 45Phe Gly Lys Gly Ile Lys Phe Ala Ile Lys Glu Gly
Ile Val Thr Ala50 55 60Asp Ile Ile Gly
Val Ala Asn Glu Asp Ser Arg Arg Leu Ala Ala Ile65 70
75 80Leu Asn Asn Ala His Tyr Leu Glu Asn
Leu His Phe Thr Ile Glu Gly85 90 95Arg
Asp Thr His Tyr Phe Ile Lys Leu Gly Ser Leu Glu Glu Asp Leu100
105 110Val Leu Ile Gly Asn Thr Gly Gly Arg Arg Ile
Leu Glu Asn Gly Val115 120 125Asn Val Thr
Val Ser Gln Met Thr Ser Val Leu Asn Gly Arg Thr Arg130
135 140Arg Phe Ala Asp Ile Gln Leu Gln His Gly Ala Leu
Cys Phe Asn Ile145 150 155
160Arg Tyr Gly Thr Thr Val Glu Glu Glu Lys Asn His Val Leu Glu Met165
170 175Ala Arg Gln Arg Ala Val Ala Gln Ala
Trp Thr Gln Glu Gln Arg Arg180 185 190Leu
Gln Glu Gly Glu Glu Gly Thr Arg Val Trp Thr Glu Gly Glu Lys195
200 205Gln Gln Leu Leu Gly Thr Gly Arg Val Gln Gly
Tyr Asp Gly Tyr Phe210 215 220Val Leu Ser
Val Glu Gln Tyr Leu Glu Leu Ser Asp Ser Ala Asn Asn225
230 235 240Ile His Phe Met Arg Gln Ser
Glu Ile Gly Arg Arg245 2505253PRTArtificial SequenceMouse
Ten M2 5Leu Ile Thr Gly Val Gln Gln Thr Thr Glu Arg His Asn Gln Ala Phe1
5 10 15Leu Ala Leu Glu
Gly Gln Val Ile Thr Lys Lys Leu His Ala Ser Ile20 25
30Arg Glu Lys Ala Gly His Trp Phe Ala Thr Thr Thr Pro Ile
Ile Gly35 40 45Lys Gly Ile Met Phe Ala
Ile Lys Glu Gly Arg Val Thr Thr Gly Val50 55
60Ser Ser Ile Ala Ser Glu Asp Ser Arg Lys Val Ala Ser Val Leu Asn65
70 75 80Asn Ala Tyr Tyr
Leu Asp Lys Met His Tyr Ser Ile Glu Gly Lys Asp85 90
95Thr His Tyr Phe Val Lys Ile Gly Ala Ala Asp Gly Asp Leu
Val Thr100 105 110Leu Gly Thr Thr Ile Gly
Arg Lys Val Leu Glu Ser Gly Val Asn Val115 120
125Thr Val Ser Gln Pro Thr Leu Leu Val Asn Gly Arg Thr Arg Arg
Phe130 135 140Thr Asn Ile Glu Phe Gln Tyr
Ser Thr Leu Leu Leu Ser Ile Arg Tyr145 150
155 160Gly Leu Thr Pro Asp Thr Leu Asp Glu Glu Lys Ala
Arg Val Leu Asp165 170 175Gln Ala Gly Gln
Arg Ala Leu Gly Thr Ala Trp Ala Lys Glu Gln Gln180 185
190Lys Ala Arg Asp Gly Arg Glu Gly Ser Arg Leu Trp Thr Glu
Gly Glu195 200 205Lys Gln Gln Leu Leu Ser
Thr Gly Arg Val Gln Gly Tyr Glu Gly Tyr210 215
220Tyr Val Leu Pro Val Glu Gln Tyr Pro Glu Leu Ala Asp Ser Ser
Ser225 230 235 240Asn Ile
Gln Phe Leu Arg Gln Asn Glu Met Gly Lys Arg245
2506251PRTArtificial SequenceMouse Ten M3 6Pro Ile Phe Gly Val Gln Gln
Gln Val Ala Arg Gln Ala Lys Ala Phe1 5 10
15Leu Ser Leu Gly Lys Met Ala Glu Val Gln Val Ser Arg
Arg Lys Ala20 25 30Gly Ala Glu Gln Ser
Trp Leu Trp Phe Ala Thr Val Lys Ser Leu Ile35 40
45Gly Lys Gly Val Met Leu Ala Val Ser Gln Gly Arg Val Gln Thr
Asn50 55 60Val Leu Asn Ile Ala Asn Glu
Asp Cys Ile Lys Val Ala Ala Val Leu65 70
75 80Asn Asn Ala Phe Tyr Leu Glu Asn Leu His Phe Thr
Ile Glu Gly Lys85 90 95Asp Thr His Tyr
Phe Ile Lys Thr Thr Thr Pro Glu Ser Asp Leu Gly100 105
110Thr Leu Arg Leu Thr Ser Gly Arg Lys Ala Leu Glu Asn Gly
Ile Asn115 120 125Val Thr Val Ser Gln Ser
Thr Thr Val Val Asn Gly Arg Thr Arg Arg130 135
140Phe Ala Asp Val Glu Met Gln Phe Gly Ala Leu Ala Leu His Val
Arg145 150 155 160Tyr Gly
Met Thr Leu Asp Glu Glu Lys Ala Arg Ile Leu Glu Gln Ala165
170 175Arg Gln Arg Ala Leu Ala Arg Ala Trp Ala Arg Glu
Gln Gln Arg Val180 185 190Arg Asp Gly Glu
Glu Gly Ala Arg Leu Trp Thr Glu Gly Glu Lys Arg195 200
205Gln Leu Leu Ser Ala Gly Lys Val Gln Gly Tyr Asp Gly Tyr
Tyr Val210 215 220Leu Ser Val Glu Gln Tyr
Pro Glu Leu Ala Asp Ser Ala Asn Asn Ile225 230
235 240Gln Phe Leu Arg Gln Ser Glu Ile Gly Lys
Arg245 2507243PRTArtificial SequenceMouse Ten M4 7Ser Ile
Leu Gly Val Gln Cys Glu Val Gln Lys Gln Leu Lys Ala Phe1 5
10 15Val Thr Leu Glu Arg Phe Asp Gln
Leu Tyr Gly Ser Thr Ile Thr Ser20 25
30Cys Gln Gln Ala Pro Glu Thr Lys Lys Phe Ala Ser Ser Gly Ser Ile35
40 45Phe Gly Lys Gly Val Lys Phe Ala Leu Lys
Asp Gly Arg Val Thr Thr50 55 60Asp Ile
Ile Ser Val Ala Asn Glu Asp Gly Arg Arg Ile Ala Ala Ile65
70 75 80Leu Asn Asn Ala His Tyr Leu
Glu Asn Leu His Phe Thr Ile Asp Gly85 90
95Val Asp Thr His Tyr Phe Val Lys Pro Gly Pro Ser Glu Gly Asp Leu100
105 110Ala Ile Leu Gly Leu Ser Gly Gly Arg
Arg Thr Leu Glu Asn Gly Val115 120 125Asn
Val Thr Val Ser Gln Ile Asn Thr Met Leu Ile Gln Leu Gln Tyr130
135 140Arg Ala Leu Cys Leu Asn Thr Arg Tyr Gly Thr
Thr Val Asp Glu Glu145 150 155
160Lys Val Arg Val Leu Glu Leu Ala Arg Gln Arg Ala Val Arg Gln
Ala165 170 175Trp Ala Arg Glu Gln Gln Arg
Leu Arg Glu Gly Glu Glu Gly Leu Arg180 185
190Ala Trp Thr Asp Gly Glu Lys Gln Gln Val Leu Asn Thr Gly Arg Val195
200 205Gln Gly Tyr Asp Gly Phe Phe Val Thr
Ser Val Glu Gln Tyr Pro Glu210 215 220Leu
Ser Asp Ser Ala Asn Asn Ile His Phe Met Arg Gln Ser Glu Met225
230 235 240Gly Arg
Arg8252PRTArtificial SequenceHuman Ten M1 8Thr Ile Leu Gly Ile Gln Cys
Glu Leu Gln Lys Gln Leu Arg Asn Phe1 5 10
15Ile Ser Leu Asp Gln Leu Pro Met Thr Pro Arg Tyr Asn
Asp Gly Arg20 25 30Cys Leu Glu Gly Gly
Lys Gln Pro Arg Phe Ala Ala Val Pro Ser Val35 40
45Phe Gly Lys Gly Ile Lys Phe Ala Ile Lys Asp Gly Ile Val Thr
Ala50 55 60Asp Ile Ile Gly Val Ala Asn
Glu Asp Ser Arg Arg Leu Ala Ala Ile65 70
75 80Leu Asn Asn Ala His Tyr Leu Glu Asn Leu His Phe
Thr Ile Glu Gly85 90 95Arg Asp Thr His
Tyr Phe Ile Lys Leu Gly Ser Leu Glu Glu Asp Leu100 105
110Val Leu Ile Gly Asn Thr Gly Gly Arg Arg Ile Leu Glu Asn
Gly Val115 120 125Asn Val Thr Val Ser Gln
Met Thr Ser Val Leu Asn Gly Arg Thr Arg130 135
140Arg Phe Ala Asp Ile Gln Leu Gln His Gly Ala Leu Cys Phe Asn
Ile145 150 155 160Arg Tyr
Gly Thr Thr Val Glu Glu Glu Lys Asn His Val Leu Glu Ile165
170 175Ala Arg Gln Arg Ala Val Ala Gln Ala Trp Thr Lys
Glu Gln Arg Arg180 185 190Leu Gln Glu Gly
Glu Glu Gly Ile Arg Ala Trp Thr Glu Gly Glu Lys195 200
205Gln Gln Leu Leu Ser Thr Gly Arg Val Gln Gly Tyr Asp Gly
Tyr Phe210 215 220Val Leu Ser Val Glu Gln
Tyr Leu Glu Leu Ser Asp Ser Ala Asn Asn225 230
235 240Ile His Phe Met Arg Gln Ser Glu Ile Gly Arg
Arg245 2509253PRTArtificial SequenceHuman Ten M2 9Leu Ile
Thr Gly Val Gln Gln Thr Thr Glu Arg His Asn Gln Ala Phe1 5
10 15Met Ala Leu Glu Gly Gln Val Ile
Thr Lys Lys Leu His Ala Ser Ile20 25
30Arg Glu Lys Ala Gly His Trp Phe Ala Thr Thr Thr Pro Ile Ile Gly35
40 45Lys Gly Ile Met Phe Ala Ile Lys Glu Gly
Arg Val Thr Thr Gly Val50 55 60Ser Ser
Ile Ala Ser Glu Asp Ser Arg Lys Val Ala Ser Val Leu Asn65
70 75 80Asn Ala Tyr Tyr Leu Asp Lys
Met His Tyr Ser Ile Glu Gly Lys Asp85 90
95Thr His Tyr Phe Val Lys Ile Gly Ser Ala Asp Gly Asp Leu Val Thr100
105 110Leu Gly Thr Thr Ile Gly Arg Lys Val
Leu Glu Ser Gly Val Asn Val115 120 125Thr
Val Ser Gln Pro Thr Leu Leu Val Asn Gly Arg Thr Arg Arg Phe130
135 140Thr Asn Ile Glu Phe Gln Tyr Ser Thr Leu Leu
Leu Ser Ile Arg Tyr145 150 155
160Gly Leu Thr Pro Asp Thr Leu Asp Glu Glu Lys Ala Arg Val Leu
Asp165 170 175Gln Ala Arg Gln Arg Ala Leu
Gly Thr Ala Trp Ala Lys Glu Gln Gln180 185
190Lys Ala Arg Asp Gly Arg Glu Gly Ser Arg Leu Trp Thr Glu Gly Glu195
200 205Lys Gln Gln Leu Leu Ser Thr Gly Arg
Val Gln Gly Tyr Glu Gly Tyr210 215 220Tyr
Val Leu Pro Val Glu Gln Tyr Pro Glu Leu Ala Asp Ser Ser Ser225
230 235 240Asn Ile Gln Phe Leu Arg
Gln Asn Glu Met Gly Lys Arg245 25010251PRTArtificial
SequenceHuman Ten M3 10Pro Ile Phe Gly Val Gln Gln Gln Val Ala Arg Gln
Ala Lys Ala Phe1 5 10
15Leu Ser Leu Gly Lys Met Ala Glu Val Gln Val Ser Arg Arg Arg Ala20
25 30Gly Gly Ala Gln Ser Trp Leu Trp Phe Ala
Thr Val Lys Ser Leu Ile35 40 45Gly Lys
Gly Val Met Leu Ala Val Ser Gln Gly Arg Val Gln Thr Asn50
55 60Val Leu Asn Ile Ala Asn Glu Asp Cys Ile Lys Val
Ala Ala Val Leu65 70 75
80Asn Asn Ala Phe Tyr Leu Glu Asn Leu His Phe Thr Ile Glu Gly Lys85
90 95Asp Thr His Tyr Phe Ile Lys Thr Thr Thr
Pro Glu Ser Asp Leu Gly100 105 110Thr Leu
Arg Leu Thr Ser Gly Arg Lys Ala Leu Glu Asn Gly Ile Asn115
120 125Val Thr Val Ser Gln Ser Thr Thr Val Val Asn Gly
Arg Thr Arg Arg130 135 140Phe Ala Asp Val
Glu Met Gln Phe Gly Ala Leu Ala Leu His Val Arg145 150
155 160Tyr Gly Met Thr Leu Asp Glu Glu Lys
Ala Arg Ile Leu Glu Gln Ala165 170 175Arg
Gln Arg Ala Leu Ala Arg Ala Trp Ala Arg Glu Gln Gln Arg Val180
185 190Arg Asp Gly Glu Glu Gly Ala Arg Leu Trp Thr
Glu Gly Glu Lys Arg195 200 205Gln Leu Leu
Ser Ala Gly Lys Val Gln Gly Tyr Asp Gly Tyr Tyr Val210
215 220Leu Ser Val Glu Gln Tyr Pro Glu Leu Ala Asp Ser
Ala Asn Asn Ile225 230 235
240Gln Phe Leu Arg Gln Ser Glu Ile Gly Arg Arg245
25011252PRTArtificial SequenceHuman Ten M4 11Ser Ile Leu Gly Val Gln Cys
Glu Val Gln Lys Gln Leu Lys Ala Phe1 5 10
15Val Thr Leu Glu Arg Phe Asp Gln Leu Tyr Gly Ser Thr
Ile Thr Ser20 25 30Cys Leu Gln Ala Pro
Lys Thr Lys Lys Phe Ala Ser Ser Gly Ser Val35 40
45Phe Gly Lys Gly Val Lys Phe Ala Leu Lys Asp Gly Arg Val Thr
Thr50 55 60Asp Ile Ile Ser Val Ala Asn
Glu Asp Gly Arg Arg Val Ala Ala Ile65 70
75 80Leu Asn His Ala His Tyr Leu Glu Asn Leu His Phe
Thr Ile Asp Gly85 90 95Val Asp Thr His
Tyr Phe Val Lys Pro Gly Pro Ser Glu Gly Asp Leu100 105
110Ala Ile Leu Gly Leu Ser Gly Gly Arg Arg Thr Leu Glu Asn
Gly Val115 120 125Asn Val Thr Val Ser Gln
Ile Asn Thr Val Leu Ser Gly Arg Thr Arg130 135
140Arg Tyr Thr Asp Ile Gln Leu Gln Tyr Gly Ala Leu Cys Leu Asn
Thr145 150 155 160Arg Tyr
Gly Thr Thr Leu Asp Glu Glu Lys Ala Arg Val Leu Glu Leu165
170 175Ala Arg Gln Arg Ala Val Arg Gln Ala Trp Ala Arg
Glu Gln Gln Arg180 185 190Leu Arg Glu Gly
Glu Glu Gly Leu Arg Ala Trp Thr Glu Gly Glu Lys195 200
205Gln Gln Val Leu Ser Thr Gly Arg Val Gln Gly Tyr Asp Gly
Phe Phe210 215 220Val Ile Ser Val Glu Gln
Tyr Pro Glu Leu Ser Asp Ser Ala Asn Asn225 230
235 240Ile His Phe Met Arg Gln Ser Glu Met Gly Arg
Arg245 25012252PRTArtificial SequenceZebrafish Ten M3
12Ser Ile Ser Gly Val Gln Gln Glu Val Met Arg Gln Ala Lys Ala Phe1
5 10 15Leu Ser Phe Glu Arg Met
Pro Glu Ile Gln Leu Ser Arg Arg Arg Ser20 25
30Ser Arg Glu Lys Pro Trp Leu Trp Phe Ala Thr Val Lys Ser Leu Ile35
40 45Gly Lys Gly Val Met Leu Ala Ile Thr
Ser Lys Gly Gln Val Ala Thr50 55 60Asn
Ala Leu Asn Ile Ala Asn Glu Asp Cys Ile Lys Val Val Thr Val65
70 75 80Leu Asn Asn Ala Phe Tyr
Leu Glu Asp Leu His Phe Thr Val Glu Gly85 90
95Arg Asp Thr His Tyr Phe Ile Lys Thr Ser Leu Pro Glu Ser Asp Leu100
105 110Gly Ala Leu Arg Leu Thr Ser Gly
Arg Lys Ser Leu Glu Asn Gly Val115 120
125Asn Val Thr Val Ser Gln Ser Thr Thr Val Val Asn Gly Arg Thr Arg130
135 140Arg Phe Ala Asp Val Glu Leu Gln Tyr
Gly Ala Leu Ala Leu His Val145 150 155
160Arg Tyr Gly Met Thr Leu Asp Glu Glu Lys Ala Arg Val Leu
Glu Gln165 170 175Ala Arg Gln Arg Ala Leu
Ser Ser Ala Trp Ala Arg Glu Gln Gln Arg180 185
190Val Arg Asp Gly Glu Glu Gly Val Arg Leu Trp Thr Glu Gly Glu
Lys195 200 205Arg Gln Leu Leu Ser Ser Gly
Lys Val Leu Gly Tyr Asp Gly Tyr Tyr210 215
220Val Leu Ser Val Glu Gln Tyr Pro Glu Leu Ala Asp Ser Ala Asn Asn225
230 235 240Val Gln Phe Leu
Arg Gln Ser Glu Ile Gly Lys Arg245 2501340PRTArtificial
SequenceRainbow Trout TCAP3 (40a.a.) 13Gln Leu Leu Ser Gly Arg Lys Val
Leu Gly Tyr Asp Gly Tyr Tyr Val1 5 10
15Leu Ser Ile Glu Gln Tyr Pro Glu Leu Ala Asp Ser Ala Asn
Asn Ile20 25 30Gln Phe Leu Arg Gln Ser
Glu Ile35 401441PRTArtificial SequenceRainbow Trout TCAP
3 (41a.a.) 14Arg Gln Leu Leu Ser Gly Arg Lys Val Leu Gly Tyr Asp Gly Tyr
Tyr1 5 10 15Val Leu Ser
Ile Glu Gln Tyr Pro Glu Leu Ala Asp Ser Ala Asn Asn20 25
30Ile Gln Phe Leu Arg Gln Ser Glu Ile35
401543PRTArtificial SequenceRainbow Trout preTCAP3 (43 a.a.) 15Gln Leu
Leu Ser Gly Arg Lys Val Leu Gly Tyr Asp Gly Tyr Tyr Val1 5
10 15Leu Ser Ile Glu Gln Tyr Pro Glu
Leu Ala Asp Ser Ala Asn Asn Ile20 25
30Gln Phe Leu Arg Gln Ser Glu Ile Gly Lys Arg35
401644PRTArtificial SequenceRainbow Trout preTCAP3 (44 a.a.) 16Arg Gln
Leu Leu Ser Gly Arg Lys Val Leu Gly Tyr Asp Gly Tyr Tyr1 5
10 15Val Leu Ser Ile Glu Gln Tyr Pro
Glu Leu Ala Asp Ser Ala Asn Asn20 25
30Ile Gln Phe Leu Arg Gln Ser Glu Ile Gly Lys Arg35
4017120DNAArtificial SequenceRainbow Trout TCAP3 (120 n.a.) 17cagctgctga
gcgggaggaa ggttctgggc tacgacgggt actacgtcct ctccatagag 60cagtaccccg
agctagcaga ctccgctaac aacatccagt tcctcaggca gagcgaaata
12018123DNAArtificial SequenceRainbow Trout TCAP3 (123 n.a.) 18aggcagctgc
tgagcgggag gaaggttctg ggctacgacg ggtactacgt cctctccata 60gagcagtacc
ccgagctagc agactccgct aacaacatcc agttcctcag gcagagcgaa 120ata
12319129DNAArtificial SequenceRainbow Trout preTCAP3 (129 n.a.)
19cagctgctga gcgggaggaa ggttctgggc tacgacgggt actacgtcct ctccatagag
60cagtaccccg agctagcaga ctccgctaac aacatccagt tcctcaggca gagcgaaata
120gggaagagg
12920132DNAArtificial SequenceRainbow Trout preTCAP3 (132 n.a.)
20aggcagctgc tgagcgggag gaaggttctg ggctacgacg ggtactacgt cctctccata
60gagcagtacc ccgagctagc agactccgct aacaacatcc agttcctcag gcagagcgaa
120atagggaaga gg
1322140PRTArtificial SequenceZebrafish TCAP3 (40 a.a.) 21Gln Leu Leu Ser
Ser Gly Lys Val Leu Gly Tyr Asp Gly Tyr Tyr Val1 5
10 15Leu Ser Val Glu Gln Tyr Pro Glu Leu Ala
Asp Ser Ala Asn Asn Val20 25 30Gln Phe
Leu Arg Gln Ser Glu Ile35 402241PRTArtificial
SequenceZebrafish TCAP3 (41 a.a.) 22Arg Gln Leu Leu Ser Ser Gly Lys Val
Leu Gly Tyr Asp Gly Tyr Tyr1 5 10
15Val Leu Ser Val Glu Gln Tyr Pro Glu Leu Ala Asp Ser Ala Asn
Asn20 25 30Val Gln Phe Leu Arg Gln Ser
Glu Ile35 402343PRTArtificial SequenceZebrafish preTCAP3
(43 a.a.) 23Gln Leu Leu Ser Ser Gly Lys Val Leu Gly Tyr Asp Gly Tyr Tyr
Val1 5 10 15Leu Ser Val
Glu Gln Tyr Pro Glu Leu Ala Asp Ser Ala Asn Asn Val20 25
30Gln Phe Leu Arg Gln Ser Glu Ile Gly Lys Arg35
402444PRTArtificial SequenceZebrafish preTCAP3 (44 a.a.) 24Arg
Gln Leu Leu Ser Ser Gly Lys Val Leu Gly Tyr Asp Gly Tyr Tyr1
5 10 15Val Leu Ser Val Glu Gln Tyr
Pro Glu Leu Ala Asp Ser Ala Asn Asn20 25
30Val Gln Phe Leu Arg Gln Ser Glu Ile Gly Lys Arg35
4025120DNAArtificial SequenceZebrafish TCAP3 (120 n.a.) 25cagttgctca
gctctgggaa ggtgctgggt tacgatggtt actatgtact atcagtggag 60caataccctg
aactggccga cagtgccaac aatgtccagt tcttgaggca gagtgagata
12026123DNAArtificial SequenceZebrafish TCAP3 (123 n.a.) 26aggcagttgc
tcagctctgg gaaggtgctg ggttacgatg gttactatgt actatcagtg 60gagcaatacc
ctgaactggc cgacagtgcc aacaatgtcc agttcttgag gcagagtgag 120ata
12327129DNAArtificial SequenceZebrafish TCAP3 (129 n.a.) 27cagttgctca
gctctgggaa ggtgctgggt tacgatggtt actatgtact atcagtggag 60caataccctg
aactggccga cagtgccaac aatgtccagt tcttgaggca gagtgagata 120gggaagagg
12928132DNAArtificial SequenceZebrafish preTCAP3 (132 n.a.) 28aggcagttgc
tcagctctgg gaaggtgctg ggttacgatg gttactatgt actatcagtg 60gagcaatacc
ctgaactggc cgacagtgcc aacaatgtcc agttcttgag gcagagtgag 120atagggaaga
gg
1322940PRTArtificial SequenceZebrafish TCAP4 (40 a.a.) 29Gln Leu Leu Ser
Ser Gly Arg Val Gln Gly Tyr Glu Gly Phe Tyr Ile1 5
10 15Val Ser Val Asp Gln Phe Pro Glu Leu Thr
Asp Asn Ile Asn Asn Val20 25 30His Phe
Trp Arg Gln Thr Glu Met35 403041PRTArtificial
SequenceZebrafish TCAP4 (41 a.a.) 30Gln Gln Leu Leu Ser Ser Gly Arg Val
Gln Gly Tyr Glu Gly Phe Tyr1 5 10
15Ile Val Ser Val Asp Gln Phe Pro Glu Leu Thr Asp Asn Ile Asn
Asn20 25 30Val His Phe Trp Arg Gln Thr
Glu Met35 403143PRTArtificial SequenceZebrafish preTCAP4
(43 a.a.) 31Gln Leu Leu Ser Ser Gly Arg Val Gln Gly Tyr Glu Gly Phe Tyr
Ile1 5 10 15Val Ser Val
Asp Gln Phe Pro Glu Leu Thr Asp Asn Ile Asn Asn Val20 25
30His Phe Trp Arg Gln Thr Glu Met Gly Arg Arg35
403244PRTArtificial SequenceZebrafish preTCAP4 (44 a.a.) 32Gln
Gln Leu Leu Ser Ser Gly Arg Val Gln Gly Tyr Glu Gly Phe Tyr1
5 10 15Ile Val Ser Val Asp Gln Phe
Pro Glu Leu Thr Asp Asn Ile Asn Asn20 25
30Val His Phe Trp Arg Gln Thr Glu Met Gly Arg Arg35
4033120DNAArtificial SequenceZebrafish TCAP4 (120 n.a.) 33cagctcctaa
gctctggacg tgtacagggc tacgaaggct tctacatagt atcagtcgac 60cagttcccag
agttgactga caacataaat aacgtccatt tctggcgaca gactgagatg
12034123DNAArtificial SequenceZebrafish TCAP4 (123 n.a.) 34cagcagctcc
taagctctgg acgtgtacag ggctacgaag gcttctacat agtatcagtc 60gaccagttcc
cagagttgac tgacaacata aataacgtcc atttctggcg acagactgag 120atg
12335129DNAArtificial SequenceZebrafish preTCAP4 (129 n.a.) 35cagctcctaa
gctctggacg tgtacagggc tacgaaggct tctacatagt atcagtcgac 60cagttcccag
agttgactga caacataaat aacgtccatt tctggcgaca gactgagatg 120ggacgcagg
12936132DNAArtificial SequenceZebrafish preTCAP4 (132 n.a.) 36cagcagctcc
taagctctgg acgtgtacag ggctacgaag gcttctacat agtatcagtc 60gaccagttcc
cagagttgac tgacaacata aataacgtcc atttctggcg acagactgag 120atgggacgca
gg
1323740PRTArtificial SequenceMouse TCAP1 (40 a.a.) 37Gln Leu Leu Gly Thr
Gly Arg Val Gln Gly Tyr Asp Gly Tyr Phe Val1 5
10 15Leu Ser Val Glu Gln Tyr Leu Glu Leu Ser Asp
Ser Ala Asn Asn Ile20 25 30His Phe Met
Arg Gln Ser Glu Ile35 403841PRTArtificial SequenceMouse
TCAP1 (41 a.a.) 38Gln Gln Leu Leu Gly Thr Gly Arg Val Gln Gly Tyr Asp Gly
Tyr Phe1 5 10 15Val Leu
Ser Val Glu Gln Tyr Leu Glu Leu Ser Asp Ser Ala Asn Asn20
25 30Ile His Phe Met Arg Gln Ser Glu Ile35
403943PRTArtificial SequenceMouse preTCAP1 (43 a.a.) 39Gln Leu Leu
Gly Thr Gly Arg Val Gln Gly Tyr Asp Gly Tyr Phe Val1 5
10 15Leu Ser Val Glu Gln Tyr Leu Glu Leu
Ser Asp Ser Ala Asn Asn Ile20 25 30His
Phe Met Arg Gln Ser Glu Ile Gly Arg Arg35
404044PRTArtificial SequenceMouse preTCAP1 (44 a.a.) 40Gln Gln Leu Leu
Gly Thr Gly Arg Val Gln Gly Tyr Asp Gly Tyr Phe1 5
10 15Val Leu Ser Val Glu Gln Tyr Leu Glu Leu
Ser Asp Ser Ala Asn Asn20 25 30Ile His
Phe Met Arg Gln Ser Glu Ile Gly Arg Arg35
4041120DNAArtificial SequenceMouse TCAP1 (120 n.a.) 41cagcttttgg
gcaccgggag ggtgcagggg tatgatgggt attttgtctt gtctgttgag 60cagtatttag
aactttcaga cagtgccaac aatattcact tcatgagaca gagtgaaata
12042123DNAArtificial SequenceMouse TCAP1 (123 n.a.) 42cagcagcttt
tgggcaccgg gagggtgcag gggtatgatg ggtattttgt cttgtctgtt 60gagcagtatt
tagaactttc agacagtgcc aacaatattc acttcatgag acagagtgaa 120ata
12343129DNAArtificial SequenceMouse preTCAP1 (129 n.a.) 43cagcttttgg
gcaccgggag ggtgcagggg tatgatgggt attttgtctt gtctgttgag 60cagtatttag
aactttcaga cagtgccaac aatattcact tcatgagaca gagtgaaata 120ggcaggagg
12944132DNAArtificial SequenceMouse preTCAP1 (132 n.a.) 44cagcagcttt
tgggcaccgg gagggtgcag gggtatgatg ggtattttgt cttgtctgtt 60gagcagtatt
tagaactttc agacagtgcc aacaatattc acttcatgag acagagtgaa 120ataggcagga
gg
1324540PRTArtificial SequenceMouse TCAP2 (40 a.a.) 45Gln Leu Leu Ser Thr
Gly Arg Val Gln Gly Tyr Glu Gly Tyr Tyr Val1 5
10 15Leu Pro Val Glu Gln Tyr Pro Glu Leu Ala Asp
Ser Ser Ser Asn Ile20 25 30Gln Phe Leu
Arg Gln Asn Glu Ile35 404641PRTArtificial SequenceMouse
TCAP2 (41 a.a.) 46Gln Gln Leu Leu Ser Thr Gly Arg Val Gln Gly Tyr Glu Gly
Tyr Tyr1 5 10 15Val Leu
Pro Val Glu Gln Tyr Pro Glu Leu Ala Asp Ser Ser Ser Asn20
25 30Ile Gln Phe Leu Arg Gln Asn Glu Met35
404743PRTArtificial SequenceMouse preTCAP2 (43 a.a) 47Gln Leu Leu Ser
Thr Gly Arg Val Gln Gly Tyr Glu Gly Tyr Tyr Val1 5
10 15Leu Pro Val Glu Gln Tyr Pro Glu Leu Ala
Asp Ser Ser Ser Asn Ile20 25 30Gln Phe
Leu Arg Gln Asn Glu Met Gly Lys Arg35
404844PRTArtificial SequenceMouse preTCAP2 (44 a.a.) 48Gln Gln Leu Leu
Ser Thr Gly Arg Val Gln Gly Tyr Glu Gly Tyr Tyr1 5
10 15Val Leu Pro Val Glu Gln Tyr Pro Glu Leu
Ala Asp Ser Ser Ser Asn20 25 30Ile Gln
Phe Leu Arg Gln Asn Glu Met Gly Lys Arg35
4049120DNAArtificial SequenceMouse TCAP2 (120 n.a.) 49caactcctga
gcacgggacg ggtacaaggt tatgagggct attacgtact tccggtggaa 60cagtacccgg
agctggcaga cagtagcagc aacatccagt tcttaagaca gaatgagagg
12050123DNAArtificial SequenceMouse TCAP 2 (123 n.a.) 50cagcaactcc
tgagcacggg acgggtacaa ggttatgagg gctattacgt acttccggtg 60gaacagtacc
cggagctggc agacagtagc agcaacatcc agttcttaag acagaatgag 120atg
12351129DNAArtificial SequenceMouse preTCAP2 (129 n.a.) 51caactcctga
gcacgggacg ggtacaaggt tatgagggct attacgtact tccggtggaa 60cagtacccgg
agctggcaga cagtagcagc aacatccagt tcttaagaca gaatgagatg 120ggaaagagg
12952132DNAArtificial SequenceMouse preTCAP2 (132 n.a.) 52cagcaactcc
tgagcacggg acgggtacaa ggttatgagg gctattacgt acttccggtg 60gaacagtacc
cggagctggc agacagtagc agcaacatcc agttcttaag acagaatgag 120atgggaaaga
gg
1325340PRTArtificial SequenceMouse TCAP3 (40 a.a.) 53Gln Leu Leu Ser Ala
Gly Lys Val Gln Gly Tyr Asp Gly Tyr Tyr Val1 5
10 15Leu Ser Val Glu Gln Tyr Pro Glu Leu Ala Asp
Ser Ala Asn Asn Ile20 25 30Gln Phe Leu
Arg Gln Ser Glu Ile35 405441PRTArtificial SequenceMouse
TCAP3 (41 a..a) 54Arg Gln Leu Leu Ser Ala Gly Lys Val Gln Gly Tyr Asp Gly
Tyr Tyr1 5 10 15Val Leu
Ser Val Glu Gln Tyr Pro Glu Leu Ala Asp Ser Ala Asn Asn20
25 30Ile Gln Phe Leu Arg Gln Ser Glu Ile35
405543PRTArtificial SequenceMouse preTCAP3 (43 a.a.) 55Gln Leu Leu
Ser Ala Gly Lys Val Gln Gly Tyr Asp Gly Tyr Tyr Val1 5
10 15Leu Ser Val Glu Gln Tyr Pro Glu Leu
Ala Asp Ser Ala Asn Asn Ile20 25 30Gln
Phe Leu Arg Gln Ser Glu Ile Gly Lys Arg35
405644PRTArtificial SequenceMouse preTCAP3 (44 a.a.) 56Arg Gln Leu Leu
Ser Ala Gly Lys Val Gln Gly Tyr Asp Gly Tyr Tyr1 5
10 15Val Leu Ser Val Glu Gln Tyr Pro Glu Leu
Ala Asp Ser Ala Asn Asn20 25 30Ile Gln
Phe Leu Arg Gln Ser Glu Ile Gly Lys Arg35
4057120DNAArtificial SequenceMouse TCAP3 (120 n.a.) 57cagctgctga
gcgctggcaa ggtgcagggc tacgatgggt actacgtact gtcggtggag 60cagtaccccg
agctggctga cagtgccaac aacatccagt tcttgcgaca aagtgagatc
12058123DNAArtificial SequenceMouse TCAP3 (123 n.a.) 58cggcagctgc
tgagcgctgg caaggtgcag ggctacgatg ggtactacgt actgtcggtg 60gagcagtacc
ccgagctggc tgacagtgcc aacaacatcc agttcttgcg acaaagtgag 120atc
12359129DNAArtificial SequenceMouse preTCAP3 (129 n.a.) 59cagctgctga
gcgctggcaa ggtgcagggc tacgatgggt actacgtact gtcggtggag 60cagtaccccg
agctggctga cagtgccaac aacatccagt tcttgcgaca aagtgagatc 120ggcaagagg
12960132DNAArtificial SequenceMouse preTCAP3 (132 n.a.) 60cggcagctgc
tgagcgctgg caaggtgcag ggctacgatg ggtactacgt actgtcggtg 60gagcagtacc
ccgagctggc tgacagtgcc aacaacatcc agttcttgcg acaaagtgag 120atcggcaaga
gg
1326140PRTArtificial SequenceMouse TCAP4 (40 a.a.) 61Gln Val Leu Asn Thr
Gly Arg Val Gln Gly Tyr Asp Gly Phe Phe Val1 5
10 15Thr Ser Val Glu Gln Tyr Pro Glu Leu Ser Asp
Ser Ala Asn Asn Ile20 25 30His Phe Met
Arg Gln Ser Glu Met35 406241PRTArtificial SequenceMouse
TCAP4 (41 a.a.) 62Gln Gln Val Leu Asn Thr Gly Arg Val Gln Gly Tyr Asp Gly
Phe Phe1 5 10 15Val Thr
Ser Val Glu Gln Tyr Pro Glu Leu Ser Asp Ser Ala Asn Asn20
25 30Ile His Phe Met Arg Gln Ser Glu Met35
406343PRTArtificial SequenceMouse preTCAP4 (43 a.a.) 63Gln Val Leu
Asn Thr Gly Arg Val Gln Gly Tyr Asp Gly Phe Phe Val1 5
10 15Thr Ser Val Glu Gln Tyr Pro Glu Leu
Ser Asp Ser Ala Asn Asn Ile20 25 30His
Phe Met Arg Gln Ser Glu Met Gly Arg Arg35
406444PRTArtificial SequenceMouse preTCAP4 (44 a.a.) 64Gln Gln Val Leu
Asn Thr Gly Arg Val Gln Gly Tyr Asp Gly Phe Phe1 5
10 15Val Thr Ser Val Glu Gln Tyr Pro Glu Leu
Ser Asp Ser Ala Asn Asn20 25 30Ile His
Phe Met Arg Gln Ser Glu Met Gly Arg Arg35
4065120DNAArtificial SequenceMouse TCAP4 (120 n.a.) 65caggtgctga
acacggggcg ggtgcaaggc tacgacggct tctttgtgac ctcggtcgag 60cagtacccag
aactgtcaga cagcgccaac aatatccact tcatgagaca gagcgagatg
12066123DNAArtificial SequenceMouse TCAP4 (123 n.a.) 66cagcaggtgc
tgaacacggg gcgggtgcaa ggctacgacg gcttctttgt gacctcggtc 60gagcagtacc
cagaactgtc agacagcgcc aacaatatcc acttcatgag acagagcgag 120atg
12367129DNAArtificial SequenceMouse preTCAP4 (129 n.a.) 67caggtgctga
acacggggcg ggtgcaaggc tacgacggct tctttgtgac ctcggtcgag 60cagtacccag
aactgtcaga cagcgccaac aatatccact tcatgagaca gagcgagatg 120ggccgaagg
12968132DNAArtificial SequenceMouse preTCAP4 (132 n.a.) 68cagcaggtgc
tgaacacggg gcgggtgcaa ggctacgacg gcttctttgt gacctcggtc 60gagcagtacc
cagaactgtc agacagcgcc aacaatatcc acttcatgag acagagcgag 120atgggccgaa
gg
1326940PRTArtificial SequenceHuman TCAP1 (40 a.a.) 69Gln Leu Leu Ser Thr
Gly Arg Val Gln Gly Tyr Asp Gly Tyr Phe Val1 5
10 15Leu Ser Val Glu Gln Tyr Leu Glu Leu Ser Asp
Ser Ala Asn Asn Ile20 25 30His Phe Met
Arg Gln Ser Glu Ile35 407041PRTArtificial SequenceHuman
TCAP1 (41 a.a.) 70Gln Gln Leu Leu Ser Thr Gly Arg Val Gln Gly Tyr Asp Gly
Tyr Phe1 5 10 15Val Leu
Ser Val Glu Gln Tyr Leu Glu Leu Ser Asp Ser Ala Asn Asn20
25 30Ile His Phe Met Arg Gln Ser Glu Ile35
407143PRTArtificial SequenceHuman preTCAP1 (43 a.a.) 71Gln Leu Leu
Ser Thr Gly Arg Val Gln Gly Tyr Asp Gly Tyr Phe Val1 5
10 15Leu Ser Val Glu Gln Tyr Leu Glu Leu
Ser Asp Ser Ala Asn Asn Ile20 25 30His
Phe Met Arg Gln Ser Glu Ile Gly Arg Arg35
407244PRTArtificial SequenceHuman preTCAP1 (44 a.a.) 72Gln Gln Leu Leu
Ser Thr Gly Arg Val Gln Gly Tyr Asp Gly Tyr Phe1 5
10 15Val Leu Ser Val Glu Gln Tyr Leu Glu Leu
Ser Asp Ser Ala Asn Asn20 25 30Ile His
Phe Met Arg Gln Ser Glu Ile Gly Arg Arg35
4073120DNAArtificial SequenceHuman TCAP1 (120 n.a.) 73cagcttttga
gcactgggcg ggtacaaggt tacgatgggt attttgtttt gtctgttgag 60cagtatttag
aactttctga cagtgccaat aatattcact ttatgagaca gagcgaaata
12074123DNAArtificial SequenceHuman TCAP1 (123 n.a.) 74cagcagcttt
tgagcactgg gcgggtacaa ggttacgatg ggtattttgt tttgtctgtt 60gagcagtatt
tagaactttc tgacagtgcc aataatattc actttatgag acagagcgaa 120ata
12375129DNAArtificial SequenceHuman preTCAP1 (129 n.a.) 75cagcttttga
gcactgggcg ggtacaaggt tacgatgggt attttgtttt gtctgttgag 60cagtatttag
aactttctga cagtgccaat aatattcact ttatgagaca gagcgaaata 120ggcaggagg
12976132DNAArtificial SequenceHuman preTCAP1 (132 n.a.) 76cagcagcttt
tgagcactgg gcgggtacaa ggttacgatg ggtattttgt tttgtctgtt 60gagcagtatt
tagaactttc tgacagtgcc aataatattc actttatgag acagagcgaa 120ataggcagga
gg
1327740PRTArtificial SequenceHuman TCAP2 (40 a.a.) 77Gln Leu Leu Ser Thr
Gly Arg Val Gln Gly Tyr Glu Gly Tyr Tyr Val1 5
10 15Leu Pro Val Glu Gln Tyr Pro Glu Leu Ala Asp
Ser Ser Ser Asn Ile20 25 30Gln Phe Leu
Arg Gln Asn Glu Met35 407841PRTArtificial SequenceHuman
preTCAP2 (41 a.a.) 78Gln Gln Leu Leu Ser Thr Gly Arg Val Gln Gly Tyr Glu
Gly Tyr Tyr1 5 10 15Val
Leu Pro Val Glu Gln Tyr Pro Glu Leu Ala Asp Ser Ser Ser Asn20
25 30Ile Gln Phe Leu Arg Gln Asn Glu Met35
407943PRTArtificial SequenceHuman preTCAP2 (43 a.a.) 79Gln Leu
Leu Ser Thr Gly Arg Val Gln Gly Tyr Glu Gly Tyr Tyr Val1 5
10 15Leu Pro Val Glu Gln Tyr Pro Glu
Leu Ala Asp Ser Ser Ser Asn Ile20 25
30Gln Phe Leu Arg Gln Asn Glu Met Gly Lys Arg35
408044PRTArtificial SequenceHuman preTCAP2 (44 a.a.) 80Gln Gln Leu Leu
Ser Thr Gly Arg Val Gln Gly Tyr Glu Gly Tyr Tyr1 5
10 15Val Leu Pro Val Glu Gln Tyr Pro Glu Leu
Ala Asp Ser Ser Ser Asn20 25 30Ile Gln
Phe Leu Arg Gln Asn Glu Met Gly Lys Arg35
4081120DNAArtificial SequenceHuman TCAP2 (120 n.a.) 81cagcttctga
gcaccgggcg cgtgcaaggg tacgagggat attacgtgct tcccgtggag 60caatacccag
agcttgcaga cagtagcagc aacatccagt ttttaagaca gaatgagatg
12082123DNAArtificial SequenceHuman TCAP2 (123 n.a.) 82cagcagcttc
tgagcaccgg gcgcgtgcaa gggtacgagg gatattacgt gcttcccgtg 60gagcaatacc
cagagcttgc agacagtagc agcaacatcc agtttttaag acagaatgag 120atg
12383129DNAArtificial SequenceHuman preTCAP2 (129 n.a.) 83cagcttctga
gcaccgggcg cgtgcaaggg tacgagggat attacgtgct tcccgtggag 60caatacccag
agcttgcaga cagtagcagc aacatccagt ttttaagaca gaatgagatg 120ggaaagagg
12984132DNAArtificial SequenceHuman preTCAP2 (132 n.a.) 84cagcagcttc
tgagcaccgg gcgcgtgcaa gggtacgagg gatattacgt gcttcccgtg 60gagcaatacc
cagagcttgc agacagtagc agcaacatcc agtttttaag acagaatgag 120atgggaaaga
gg
1328540PRTArtificial SequenceHuman TCAP3 (40 a.a.) 85Gln Leu Leu Ser Ala
Gly Lys Val Gln Gly Tyr Asp Gly Tyr Tyr Val1 5
10 15Leu Ser Val Glu Gln Tyr Pro Glu Leu Ala Asp
Ser Ala Asn Asn Ile20 25 30Gln Phe Leu
Arg Gln Ser Glu Ile35 408641PRTArtificial SequenceHuman
TCAP3 (41 a.a.) 86Arg Gln Leu Leu Ser Ala Gly Lys Val Gln Gly Tyr Asp Gly
Tyr Tyr1 5 10 15Val Leu
Ser Val Glu Gln Tyr Pro Glu Leu Ala Asp Ser Ala Asn Asn20
25 30Ile Gln Phe Leu Arg Gln Ser Glu Ile35
408743PRTArtificial SequenceHuman preTCAP3 (43 a.a.) 87Gln Leu Leu
Ser Ala Gly Lys Val Gln Gly Tyr Asp Gly Tyr Tyr Val1 5
10 15Leu Ser Val Glu Gln Tyr Pro Glu Leu
Ala Asp Ser Ala Asn Asn Ile20 25 30Gln
Phe Leu Arg Gln Ser Glu Ile Gly Arg Arg35
408844PRTArtificial SequenceHuman preTCAP3 (44 a.a.) 88Arg Gln Leu Leu
Ser Ala Gly Lys Val Gln Gly Tyr Asp Gly Tyr Tyr1 5
10 15Val Leu Ser Val Glu Gln Tyr Pro Glu Leu
Ala Asp Ser Ala Asn Asn20 25 30Ile Gln
Phe Leu Arg Gln Ser Glu Ile Gly Arg Arg35
4089120DNAArtificial SequenceHuman TCAP3 (120 n.a.) 89cagctgctga
gcgccggcaa ggtgcagggc tacgacgggt actacgtact ctcggtggag 60cagtaccccg
agctggccga cagcgccaac aacatccagt tcctgcggca gagcgagatc
12090123DNAArtificial SequenceHuman TCAP3 (123 n.a.) 90cggcagctgc
tgagcgccgg caaggtgcag ggctacgacg ggtactacgt actctcggtg 60gagcagtacc
ccgagctggc cgacagcgcc aacaacatcc agttcctgcg gcagagcgag 120atc
12391129DNAArtificial SequenceHuman preTCAP (129 n.a.) 91cagctgctga
gcgccggcaa ggtgcagggc tacgacgggt actacgtact ctcggtggag 60cagtaccccg
agctggccga cagcgccaac aacatccagt tcctgcggca gagcgagatc 120ggcaggagg
12992132DNAArtificial SequenceHuman preTCAP3 (132 n.a.) 92cggcagctgc
tgagcgccgg caaggtgcag ggctacgacg ggtactacgt actctcggtg 60gagcagtacc
ccgagctggc cgacagcgcc aacaacatcc agttcctgcg gcagagcgag 120atcggcagga
gg
1329340PRTArtificial SequenceHuman TCAP4 (40 a.a.) 93Gln Val Leu Ser Thr
Gly Arg Val Gln Gly Tyr Asp Gly Phe Phe Val1 5
10 15Ile Ser Val Glu Gln Tyr Pro Glu Leu Ser Asp
Ser Ala Asn Asn Ile20 25 30His Phe Met
Arg Gln Ser Glu Met35 409441PRTArtificial SequenceHuman
TCAP4 (41 a.a.) 94Gln Gln Val Leu Ser Thr Gly Arg Val Gln Gly Tyr Asp Gly
Phe Phe1 5 10 15Val Ile
Ser Val Glu Gln Tyr Pro Glu Leu Ser Asp Ser Ala Asn Asn20
25 30Ile His Phe Met Arg Gln Ser Glu Met35
409543PRTArtificial SequenceHuman preTCAP4 (43 a..a) 95Gln Val Leu
Ser Thr Gly Arg Val Gln Gly Tyr Asp Gly Phe Phe Val1 5
10 15Ile Ser Val Glu Gln Tyr Pro Glu Leu
Ser Asp Ser Ala Asn Asn Ile20 25 30His
Phe Met Arg Gln Ser Glu Met Gly Arg Arg35
409644PRTArtificial SequenceHuman preTCAP4 (44 a.a.) 96Gln Gln Val Leu
Ser Thr Gly Arg Val Gln Gly Tyr Asp Gly Phe Phe1 5
10 15Val Ile Ser Val Glu Gln Tyr Pro Glu Leu
Ser Asp Ser Ala Asn Asn20 25 30Ile His
Phe Met Arg Gln Ser Glu Met Gly Arg Arg35
4097120DNAArtificial SequenceHuman TCAP4 (120 n.a.) 97caggtgctga
gcacagggcg ggtgcaaggc tacgacggct ttttcgtgat ctctgtcgag 60cagtacccag
aactgtcaga cagcgccaac aacatccact tcatgagaca gagcgagatg
12098123DNAArtificial SequenceHuman TCAP4 (123 n.a.) 98cagcaggtgc
tgagcacagg gcgggtgcaa ggctacgacg gctttttcgt gatctctgtc 60gagcagtacc
cagaactgtc agacagcgcc aacaacatcc acttcatgag acagagcgag 120atg
12399129DNAArtificial SequenceHuman preTCAP4 (129 n.a.) 99caggtgctga
gcacagggcg ggtgcaaggc tacgacggct ttttcgtgat ctctgtcgag 60cagtacccag
aactgtcaga cagcgccaac aacatccact tcatgagaca gagcgagatg 120ggccggagg
129100132DNAArtificial SequenceHuman preTCAP4 (132 n.a.) 100cagcaggtgc
tgagcacagg gcgggtgcaa ggctacgacg gctttttcgt gatctctgtc 60gagcagtacc
cagaactgtc agacagcgcc aacaacatcc acttcatgag acagagcgag 120atgggccgga
gg
13210141PRTArtificial SequenceG. gallus TCAP-1 101Gln Gln Leu Leu Asn Thr
Gly Arg Val Gln Gly Tyr Asp Gly Tyr Phe1 5
10 15Val Leu Ser Val Glu Gln Tyr Leu Glu Leu Ser Asp
Ser Ala Asn Asn20 25 30Ile His Phe Met
Arg Gln Ser Glu Ile35 4010241PRTArtificial
SequenceZebrafish TCAP-4 102Gln Gln Leu Leu Ser Ser Gly Arg Val Gln Gly
Tyr Glu Gly Phe Tyr1 5 10
15Ile Val Ser Val Asp Gln Phe Pro Glu Leu Thr Asp Asn Ile Asn Asn20
25 30Val His Phe Trp Arg Gln Thr Glu Met35
4010337PRTArtificial SequenceD. melanogaster Ten-m gene
product 103Glu Leu Val Gln His Gly Asp Val Asp Gly Trp Asn Gly Asp Ile
His1 5 10 15Ser Ile His
Lys Tyr Pro Gln Leu Ala Asp Pro Gly Asn Val Ala Phe20 25
30Gln Arg Asp Ala Lys3510441PRTArtificial SequenceHuman
CRF TCAP like region 104Ser Glu Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe
His Leu Leu Arg1 5 10
15Glu Val Leu Glu Met Ala Arg Ala Glu Gln Leu Ala Gln Gln Ala His20
25 30Ser Asn Arg Lys Leu Met Glu Ile Ile35
4010540PRTArtificial SequenceHuman urocortin TCAP-like
region 105Asp Asn Pro Ser Leu Ser Ile Asp Leu Thr Phe His Leu Leu Arg
Thr1 5 10 15Leu Leu Glu
Leu Ala Arg Thr Gln Ser Gln Arg Glu Arg Ala Glu Gln20 25
30Asn Arg Ile Ile Phe Asp Ser Val35
4010638PRTArtificial SequenceHuman urocortin 2 TCAP-like region 106Ile
Val Leu Ser Leu Asp Val Pro Ile Gly Leu Leu Gln Ile Leu Leu1
5 10 15Glu Gln Ala Arg Ala Arg Ala
Ala Arg Glu Gln Ala Thr Thr Asn Ala20 25
30Arg Ile Leu Ala Arg Val3510738PRTArtificial SequenceHuman urocortin 3
TCAP=like region 107Phe Thr Leu Ser Leu Asp Val Pro Thr Asn Ile Met Asn
Leu Leu Phe1 5 10 15Asn
Ile Ala Lys Ala Lys Asn Leu Arg Ala Gln Ala Ala Ala Asn Ala20
25 30His Leu Met Ala Gln Ile3510846PRTArtificial
SequenceL. migratoria DP 108Met Gly Met Gly Pro Ser Leu Ser Ile Val Asn
Pro Met Asp Val Leu1 5 10
15Arg Gln Arg Leu Leu Leu Glu Ile Ala Arg Arg Arg Leu Arg Asp Ala20
25 30Glu Glu Gln Ile Lys Ala Asn Lys Asp Phe
Leu Gln Gln Ile35 40
4510946PRTArtificial SequenceA. domesticus DP 109Thr Gly Ala Gln Ser Leu
Ser Ile Val Ala Pro Leu Asp Val Leu Arg1 5
10 15Gln Arg Leu Met Asn Glu Leu Asn Arg Arg Arg Met
Arg Glu Leu Gln20 25 30Gly Ser Arg Ile
Gln Gln Asn Arg Gln Leu Leu Thr Ser Ile35 40
4511039PRTArtificial SequenceT. molitor DP 110Ser Pro Thr Ile Ser
Ile Thr Ala Pro Ile Asp Val Leu Arg Lys Thr1 5
10 15Trp Glu Gln Glu Arg Ala Arg Lys Gln Met Val
Ala Gln Asn Asn Arg20 25 30Glu Phe Leu
Asn Ser Leu Asn3511141PRTArtificial SequenceM. sexta DP-1 111Arg Met Pro
Ser Leu Ser Ile Asp Leu Pro Met Ser Val Leu Arg Gln1 5
10 15Lys Leu Ser Leu Glu Lys Glu Arg Lys
Val His Ala Leu Arg Ala Ala20 25 30Ala
Asn Arg Asn Phe Leu Asn Asp Ile35 4011230PRTArtificial
SequenceM. sexta DP-II 112Ser Leu Ser Val Asn Pro Ala Val Asp Ile Leu Gln
His Arg Tyr Met1 5 10
15Glu Lys Val Ala Gln Asn Asn Arg Asn Phe Leu Asn Arg Val20
25 3011345PRTArtificial SequenceP. Americana 113Thr
Gly Ser Gly Pro Ser Leu Ser Ile Val Asn Pro Leu Asp Val Leu1
5 10 15Arg Gln Arg Leu Leu Leu Glu
Ile Ala Arg Arg Arg Met Arg Gln Ser20 25
30Gln Asp Gln Ile Gln Asn Arg Glu Ile Leu Gln Thr Ile35
40 4511441PRTArtificial SequenceO. keta CRP 114Ser Asp
Asp Pro Pro Ile Ser Leu Asp Leu Thr Phe His Met Leu Arg1 5
10 15Gln Met Asn Glu Met Ser Arg Ala
Glu Gln Leu Gln Gln Gln Ala His20 25
30Ser Asn Arg Lys Met Met Glu Ile Phe35
4011540PRTArtificial SequenceR. norvegicus 115Asp Asp Pro Pro Leu Ser Ile
Asp Leu Thr Phe His Leu Leu Arg Thr1 5 10
15Leu Leu Glu Leu Ala Arg Thr Gln Ser Gln Arg Glu Arg
Ala Glu Gln20 25 30Asn Arg Ile Ile Phe
Asp Ser Val35 4011637PRTArtificial SequenceP. sauvageii
116Gln Gly Pro Pro Ile Ser Ile Asp Leu Ser Leu Glu Leu Leu Arg Lys1
5 10 15Met Ile Glu Ile Glu Lys
Gln Glu Lys Glu Lys Gln Gln Ala Ala Asn20 25
30Asn Arg Leu Leu Leu3511741PRTArtificial SequenceC. carpio US
117Asn Asp Asp Pro Pro Ile Ser Ile Asp Leu Thr Phe His Leu Leu Arg1
5 10 15Asn Met Ile Glu Met Ala
Arg Asn Glu Asn Gln Arg Glu Gln Ala Gly20 25
30Leu Asn Arg Lys Tyr Leu Asp Glu Val35
4011838PRTArtificial SequenceM. Musculus UCN2 118Val Ile Leu Ser Leu Asp
Val Pro Ile Gly Leu Leu Arg Ile Leu Leu1 5
10 15Glu Gln Ala Arg Tyr Lys Ala Ala Arg Asn Gln Ala
Ala Thr Asn Ala20 25 30Gln Ile Leu Ala
His Val3511938PRTArtificial SequenceR. dano UCN2 119Leu Thr Leu Ser Leu
Asp Val Pro Thr Asn Ile Met Asn Val Leu Phe1 5
10 15Asp Val Ala Lys Ala Lys Asn Leu Arg Ala Lys
Ala Ala Glu Asn Ala20 25 30Arg Leu Leu
Ala His Ile35120305DNAArtificial SequenceHamster 305bp urocortin cDNA
probe examples "cloning mRNA" 120attcaccgcc gctcgggatc tgagcctgca
ggcgagcggc agcgacggga agaccttccg 60ctgtccatcg acctcacatt ccacctgcta
cggaccctgc tggagatggc ccggacacag 120agccaacgcg agcgagcaga gcagaaccga
atcatactca acgcggtggg caagtgatcg 180gcccggtgtg ggaccccaaa aggctcgacc
ctttccccta cctaccccgg ggctgaagtc 240acgcgaccga agtcggctta gtcccgcggt
gcagcgcctc ccagagttac cctgaacaat 300cccgc
30512124DNAArtificial SequenceTCAP1 fwd
primer 121acgtcagtgt tgatgggagg acta
2412227DNAArtificial SequenceTCAP1 rvs primer 122cctcctgcct
atttcactct gtctcat
2712325DNAArtificial SequenceTCAP2 Fwd primer 123tcgagggcaa ggacacacac
tactt 2512426DNAArtificial
SequenceTCAP2 rvs primer 124aagaactgga tgttgctgct actgtc
2612525DNAArtificial SequenceTCAP3 fwd primer
125caacaacgcc ttctacctgg agaac
2512621DNAArtificial SequenceTCAP3 rvs primer 126tgttgttggc actgtcagcc a
2112723DNAArtificial
SequenceTCAP4 fwd primer 127tttgcctcca gtggttccat ctt
2312824DNAArtificial SequenceTCAP4 rvs primer
128tggatattgt tggcgctgtc tgac
241296PRTArtificial SequenceConserved motif between CRF and TCAP I/L S X
X (X)-L/V at amino terminus 129Xaa Ser Xaa Xaa Xaa Xaa1
51304PRTArtificial SequenceConserved motif between CRF and TCAP - In
middle L/V-L/I-X-V/ali phatic residue 130Xaa Xaa Xaa
Xaa11314PRTArtificial SequenceConserved motif between CRF and TCAP
N/I/A-H/basic residue -I/L/F/-aliphatic at carboxy terminus 131Asn Xaa
Xaa Xaa11328964DNAMus musculusexon(50)..(8197) 132aagttctaag aagccggacc
gatgtgcaca gagaaggaat gaaggaagt atg gat gtg 58Met Asp Val1aag gaa cgc
agg cct tac tgc tcc ttg acc aag agc aga cgg gaa aag 106Lys Glu Arg
Arg Pro Tyr Cys Ser Leu Thr Lys Ser Arg Arg Glu Lys5 10
15gaa agg cgc tat aca aat tcg tcc gcg gac aat gag gag
tgt agg gtc 154Glu Arg Arg Tyr Thr Asn Ser Ser Ala Asp Asn Glu Glu
Cys Arg Val20 25 30
35ccc acg cag aag tcc tat agt tcc agt gaa acc ttg aaa gct ttc gat
202Pro Thr Gln Lys Ser Tyr Ser Ser Ser Glu Thr Leu Lys Ala Phe Asp40
45 50cat gat tat tca cgg ctg ctt tat gga aac
aga gta aag gat ttg gtc 250His Asp Tyr Ser Arg Leu Leu Tyr Gly Asn
Arg Val Lys Asp Leu Val55 60 65cac aga
gaa gcc gac gag tat act aga caa gga cag aat ttt acc cta 298His Arg
Glu Ala Asp Glu Tyr Thr Arg Gln Gly Gln Asn Phe Thr Leu70
75 80agg cag tta gga gtg tgt gaa tcc gca act cga aga
gga gtg gca ttc 346Arg Gln Leu Gly Val Cys Glu Ser Ala Thr Arg Arg
Gly Val Ala Phe85 90 95tgt gcg gaa atg
ggg ctc cct cac aga ggt tac tcc atc agt gca ggg 394Cys Ala Glu Met
Gly Leu Pro His Arg Gly Tyr Ser Ile Ser Ala Gly100 105
110 115tca gat gcg gat acg gaa aac gaa gca
gtg atg tcc cct gag cat gcc 442Ser Asp Ala Asp Thr Glu Asn Glu Ala
Val Met Ser Pro Glu His Ala120 125 130atg
aga ctt tgg ggc agg ggg gtc aaa tcg ggc cgc agt tcc tgc ctg 490Met
Arg Leu Trp Gly Arg Gly Val Lys Ser Gly Arg Ser Ser Cys Leu135
140 145tca agc cgg tcc aac tcc gcc ctc acc ctg aca
gac acg gag cac gag 538Ser Ser Arg Ser Asn Ser Ala Leu Thr Leu Thr
Asp Thr Glu His Glu150 155 160aac agg tcg
gac agt gag agc gag caa cct tca aac aac cca ggg caa 586Asn Arg Ser
Asp Ser Glu Ser Glu Gln Pro Ser Asn Asn Pro Gly Gln165
170 175ccc acc ctg cag cct ttg ccg cca tcc cac aag cag
cac ccg gcg cag 634Pro Thr Leu Gln Pro Leu Pro Pro Ser His Lys Gln
His Pro Ala Gln180 185 190
195cat cac ccg tcc atc act tcc ctc aat aga aac tcc ctg acc aat aga
682His His Pro Ser Ile Thr Ser Leu Asn Arg Asn Ser Leu Thr Asn Arg200
205 210agg aac cag agt ccg gcc ccg ccg gct
gct ttg ccc gcc gag ctg caa 730Arg Asn Gln Ser Pro Ala Pro Pro Ala
Ala Leu Pro Ala Glu Leu Gln215 220 225acc
aca ccc gag tcc gtc cag ctg cag gac agc tgg gtc ctt ggc agt 778Thr
Thr Pro Glu Ser Val Gln Leu Gln Asp Ser Trp Val Leu Gly Ser230
235 240aat gta cca ctg gaa agc agg cat ttc cta ttc
aaa aca ggg aca ggg 826Asn Val Pro Leu Glu Ser Arg His Phe Leu Phe
Lys Thr Gly Thr Gly245 250 255acg acg cca
ctg ttc agt acg gca acc ccg gga tac aca atg gca tct 874Thr Thr Pro
Leu Phe Ser Thr Ala Thr Pro Gly Tyr Thr Met Ala Ser260
265 270 275ggc tct gtt tat tct ccg cct
acc cgg cca ctt cct aga aac acc cta 922Gly Ser Val Tyr Ser Pro Pro
Thr Arg Pro Leu Pro Arg Asn Thr Leu280 285
290tca aga agt gct ttt aaa ttc aag aag tct tca aag tac tgc agc tgg
970Ser Arg Ser Ala Phe Lys Phe Lys Lys Ser Ser Lys Tyr Cys Ser Trp295
300 305agg tgc acc gca ctg tgt gct gta ggg
gtc tca gtg ctc ctg gcc att 1018Arg Cys Thr Ala Leu Cys Ala Val Gly
Val Ser Val Leu Leu Ala Ile310 315 320ctc
ctc tcc tat ttt ata gca atg cat cta ttt ggc ctc aac tgg cac 1066Leu
Leu Ser Tyr Phe Ile Ala Met His Leu Phe Gly Leu Asn Trp His325
330 335tta cag cag acg gaa aat gac aca ttc gag aat
gga aaa gtg aat tct 1114Leu Gln Gln Thr Glu Asn Asp Thr Phe Glu Asn
Gly Lys Val Asn Ser340 345 350
355gac acc gtg cca aca aac act gta tcg tta cct tct ggc gac aat gga
1162Asp Thr Val Pro Thr Asn Thr Val Ser Leu Pro Ser Gly Asp Asn Gly360
365 370aaa tta ggt gga ttt aca cat gaa aat
aac acc ata gat tcc gga gaa 1210Lys Leu Gly Gly Phe Thr His Glu Asn
Asn Thr Ile Asp Ser Gly Glu375 380 385ctt
gat att ggc cgg aga gca att caa gag gtt ccc ccc ggg atc ttc 1258Leu
Asp Ile Gly Arg Arg Ala Ile Gln Glu Val Pro Pro Gly Ile Phe390
395 400tgg aga tcg cag ctc ttt att gat cag cca cag
ttt ctt aag ttc aac 1306Trp Arg Ser Gln Leu Phe Ile Asp Gln Pro Gln
Phe Leu Lys Phe Asn405 410 415atc tct ctt
cag aag gat gca ttg atc gga gtg tac ggc cgg aag ggc 1354Ile Ser Leu
Gln Lys Asp Ala Leu Ile Gly Val Tyr Gly Arg Lys Gly420
425 430 435tta ccg cct tcc cat act cag
tac gac ttt gtg gaa cta ctg gat ggt 1402Leu Pro Pro Ser His Thr Gln
Tyr Asp Phe Val Glu Leu Leu Asp Gly440 445
450agc agg tta att gcg aga gag cag cgg aac ctg gtg gag tcc gaa aga
1450Ser Arg Leu Ile Ala Arg Glu Gln Arg Asn Leu Val Glu Ser Glu Arg455
460 465gcc ggg cgg cag gcg aga tct gtc agc
ctg cac gaa gct ggc ttc atc 1498Ala Gly Arg Gln Ala Arg Ser Val Ser
Leu His Glu Ala Gly Phe Ile470 475 480cag
tac ttg gat tct gga atc tgg cat ctg gct ttt tat aac gac ggg 1546Gln
Tyr Leu Asp Ser Gly Ile Trp His Leu Ala Phe Tyr Asn Asp Gly485
490 495aaa aac cca gag cag gtc tcc ttt aac acg atc
gtt ata gag tct gtg 1594Lys Asn Pro Glu Gln Val Ser Phe Asn Thr Ile
Val Ile Glu Ser Val500 505 510
515gtg gaa tgc ccc cga aat tgc cat gga aat gga gag tgt gtt tct gga
1642Val Glu Cys Pro Arg Asn Cys His Gly Asn Gly Glu Cys Val Ser Gly520
525 530act tgc cat tgt ttc ccc ggg ttt cta
ggt ccg gat tgt tca aga gca 1690Thr Cys His Cys Phe Pro Gly Phe Leu
Gly Pro Asp Cys Ser Arg Ala535 540 545gcc
tgt ccg gtg ctc tgt agt ggc aac ggg caa tac tcc aag ggc cgc 1738Ala
Cys Pro Val Leu Cys Ser Gly Asn Gly Gln Tyr Ser Lys Gly Arg550
555 560tgc ctg tgc ttc agt ggc tgg aag ggc acc gag
tgt gac gtg ccg acg 1786Cys Leu Cys Phe Ser Gly Trp Lys Gly Thr Glu
Cys Asp Val Pro Thr565 570 575acc cag tgc
att gac ccg cag tgc ggg ggt cgt ggg att tgc atc atg 1834Thr Gln Cys
Ile Asp Pro Gln Cys Gly Gly Arg Gly Ile Cys Ile Met580
585 590 595ggc tct tgc gct tgt aac tcg
gga tac aaa gga gaa aac tgt gag gaa 1882Gly Ser Cys Ala Cys Asn Ser
Gly Tyr Lys Gly Glu Asn Cys Glu Glu600 605
610gcg gac tgt cta gac cct gga tgt tct aat cac ggg gtg tgt atc cat
1930Ala Asp Cys Leu Asp Pro Gly Cys Ser Asn His Gly Val Cys Ile His615
620 625ggg gaa tgt cac tgc aat cca ggc tgg
ggt ggc agc aac tgt gaa ata 1978Gly Glu Cys His Cys Asn Pro Gly Trp
Gly Gly Ser Asn Cys Glu Ile630 635 640ctg
aag act atg tgt gca gac cag tgc tca ggc cac ggg act tac ctt 2026Leu
Lys Thr Met Cys Ala Asp Gln Cys Ser Gly His Gly Thr Tyr Leu645
650 655caa gaa agc ggc tcc tgc act tgc gac cca aat
tgg act ggc ccc gac 2074Gln Glu Ser Gly Ser Cys Thr Cys Asp Pro Asn
Trp Thr Gly Pro Asp660 665 670
675tgc tca aat gaa ata tgt tca gtg gac tgc ggc tca cac ggc gtc tgc
2122Cys Ser Asn Glu Ile Cys Ser Val Asp Cys Gly Ser His Gly Val Cys680
685 690atg ggg ggc tcc tgt cgc tgt gaa gaa
ggc tgg acc ggc ccg gcg tgt 2170Met Gly Gly Ser Cys Arg Cys Glu Glu
Gly Trp Thr Gly Pro Ala Cys695 700 705aat
cag aga gct tgc cac cct cgc tgt gct gag cac ggg acg tgc aag 2218Asn
Gln Arg Ala Cys His Pro Arg Cys Ala Glu His Gly Thr Cys Lys710
715 720gac ggc aag tgc gag tgc agc caa gga tgg aac
gga gag cac tgc aca 2266Asp Gly Lys Cys Glu Cys Ser Gln Gly Trp Asn
Gly Glu His Cys Thr725 730 735att gct cac
tat ttg gat aag ata gtt aaa gag ggt tgc ccc ggc ttg 2314Ile Ala His
Tyr Leu Asp Lys Ile Val Lys Glu Gly Cys Pro Gly Leu740
745 750 755tgc aac agc aat ggg aga tgc
aca ctg gac caa aac ggc tgg cac tgc 2362Cys Asn Ser Asn Gly Arg Cys
Thr Leu Asp Gln Asn Gly Trp His Cys760 765
770gtt tgc cag cca ggg tgg aga gga gca ggc tgt gac gta gcc atg gag
2410Val Cys Gln Pro Gly Trp Arg Gly Ala Gly Cys Asp Val Ala Met Glu775
780 785acc ctc tgt aca gac agc aaa gac aac
gaa gga gac gga ctc att gac 2458Thr Leu Cys Thr Asp Ser Lys Asp Asn
Glu Gly Asp Gly Leu Ile Asp790 795 800tgc
atg gat cct gat tgc tgc ctc cag agc tcc tgc caa aac cag ccc 2506Cys
Met Asp Pro Asp Cys Cys Leu Gln Ser Ser Cys Gln Asn Gln Pro805
810 815tac tgt cgt ggc ttg cct gat cct cag gat atc
att agc caa agc ctt 2554Tyr Cys Arg Gly Leu Pro Asp Pro Gln Asp Ile
Ile Ser Gln Ser Leu820 825 830
835cag aca cca tct cag caa gct gcc aag tcc ttc tat gac cga atc agt
2602Gln Thr Pro Ser Gln Gln Ala Ala Lys Ser Phe Tyr Asp Arg Ile Ser840
845 850ttc ctg att gga tcg gat agc acc cac
gtg ctc cct gga gaa agt ccg 2650Phe Leu Ile Gly Ser Asp Ser Thr His
Val Leu Pro Gly Glu Ser Pro855 860 865ttc
aat aag agt ctt gcg tcc gtc atc aga ggc caa gta cta aca gct 2698Phe
Asn Lys Ser Leu Ala Ser Val Ile Arg Gly Gln Val Leu Thr Ala870
875 880gat gga acc cca ctt att ggc gtc aac gtg tcg
ttt tta cac tac tcg 2746Asp Gly Thr Pro Leu Ile Gly Val Asn Val Ser
Phe Leu His Tyr Ser885 890 895gaa tat gga
tat acc att acc cgc cag gat gga atg ttt gac ttg gtg 2794Glu Tyr Gly
Tyr Thr Ile Thr Arg Gln Asp Gly Met Phe Asp Leu Val900
905 910 915gca aat ggt ggc gct tct ctg
act ttg gta ttt gag cgt tcc cca ttc 2842Ala Asn Gly Gly Ala Ser Leu
Thr Leu Val Phe Glu Arg Ser Pro Phe920 925
930ctc act cag tac cac act gtg tgg att ccc tgg aat gtc ttt tat gtg
2890Leu Thr Gln Tyr His Thr Val Trp Ile Pro Trp Asn Val Phe Tyr Val935
940 945atg gat acc ctt gtc atg aag aaa gag
gag aac gac att ccc agc tgt 2938Met Asp Thr Leu Val Met Lys Lys Glu
Glu Asn Asp Ile Pro Ser Cys950 955 960gac
ctc agt ggc ttt gtg agg cca agt ccc atc att gtg tct tca ccg 2986Asp
Leu Ser Gly Phe Val Arg Pro Ser Pro Ile Ile Val Ser Ser Pro965
970 975tta tcc acc ttc ttc agg tct tcc cct gag gac
agc ccc atc atc ccc 3034Leu Ser Thr Phe Phe Arg Ser Ser Pro Glu Asp
Ser Pro Ile Ile Pro980 985 990
995gag aca cag gtc ctg cat gaa gaa acc aca att cca gga aca gat
3079Glu Thr Gln Val Leu His Glu Glu Thr Thr Ile Pro Gly Thr Asp1000
1005 1010ttg aaa ctt tcc tac ctg agt tcc
aga gcg gca ggg tac aag tca 3124Leu Lys Leu Ser Tyr Leu Ser Ser
Arg Ala Ala Gly Tyr Lys Ser1015 1020
1025gtt ctt aag att acc atg acc cag gcc gtc ata ccg ttt aac ctc
3169Val Leu Lys Ile Thr Met Thr Gln Ala Val Ile Pro Phe Asn Leu1030
1035 1040atg aag gtc cat ctg atg gtg gcc gtg
gtt ggg aga ctc ttc cag 3214Met Lys Val His Leu Met Val Ala Val
Val Gly Arg Leu Phe Gln1045 1050 1055aag
tgg ttt cct gcc tcg cca aac ttg gcc tac acg ttc atc tgg 3259Lys
Trp Phe Pro Ala Ser Pro Asn Leu Ala Tyr Thr Phe Ile Trp1060
1065 1070gat aag acg gac gca tat aat cag aaa gtc
tac ggc ttg tca gag 3304Asp Lys Thr Asp Ala Tyr Asn Gln Lys Val
Tyr Gly Leu Ser Glu1075 1080 1085gca gtt
gtg tcc gtc gga tac gag tac gag tcg tgc ttg gac ctg 3349Ala Val
Val Ser Val Gly Tyr Glu Tyr Glu Ser Cys Leu Asp Leu1090
1095 1100act ctc tgg gaa aag agg act gcc gtt ttg caa
ggc tat gag ttg 3394Thr Leu Trp Glu Lys Arg Thr Ala Val Leu Gln
Gly Tyr Glu Leu1105 1110 1115gat gct tcg
aac atg ggc ggc tgg acg ttg gac aag cac cat gta 3439Asp Ala Ser
Asn Met Gly Gly Trp Thr Leu Asp Lys His His Val1120
1125 1130ctg gac gtt cag aac ggt ata cta tac aaa gga
aat gga gaa aat 3484Leu Asp Val Gln Asn Gly Ile Leu Tyr Lys Gly
Asn Gly Glu Asn1135 1140 1145cag ttc atc
tct cag cag cct ccg gtg gtc agc agc atc atg ggt 3529Gln Phe Ile
Ser Gln Gln Pro Pro Val Val Ser Ser Ile Met Gly1150
1155 1160aat ggt cgg agg cgt agc atc tca tgc cca agt
tgc aat ggt caa 3574Asn Gly Arg Arg Arg Ser Ile Ser Cys Pro Ser
Cys Asn Gly Gln1165 1170 1175gct gac ggg
aac aaa ctc ctg gca ccc gtg gcg ctt gcc tgt ggg 3619Ala Asp Gly
Asn Lys Leu Leu Ala Pro Val Ala Leu Ala Cys Gly1180
1185 1190atc gac ggc agt cta tac gta ggg gat ttc aat
tac gtc cgg cgg 3664Ile Asp Gly Ser Leu Tyr Val Gly Asp Phe Asn
Tyr Val Arg Arg1195 1200 1205ata ttc ccg
tct ggg aat gtg aca agt gtt tta gaa cta aga aat 3709Ile Phe Pro
Ser Gly Asn Val Thr Ser Val Leu Glu Leu Arg Asn1210
1215 1220aaa gat ttt aga cat agt agc aac cca gct cac
aga tac tac ctg 3754Lys Asp Phe Arg His Ser Ser Asn Pro Ala His
Arg Tyr Tyr Leu1225 1230 1235gct acg gac
cca gtc acc gga gat ttg tac gtc tct gat act aac 3799Ala Thr Asp
Pro Val Thr Gly Asp Leu Tyr Val Ser Asp Thr Asn1240
1245 1250acc cgc aga atc tat cgg ccg aaa tca ctc acg
gga gcc aaa gac 3844Thr Arg Arg Ile Tyr Arg Pro Lys Ser Leu Thr
Gly Ala Lys Asp1255 1260 1265ctg act aaa
aac gct gaa gtg gtg gca ggg acc ggg gaa cag tgc 3889Leu Thr Lys
Asn Ala Glu Val Val Ala Gly Thr Gly Glu Gln Cys1270
1275 1280ctt ccc ttt gac gag gcc agg tgt ggg gat gga
ggc aag gct gtg 3934Leu Pro Phe Asp Glu Ala Arg Cys Gly Asp Gly
Gly Lys Ala Val1285 1290 1295gaa gca acg
ctc atg agt ccc aaa gga atg gca atc gat aag aac 3979Glu Ala Thr
Leu Met Ser Pro Lys Gly Met Ala Ile Asp Lys Asn1300
1305 1310gga ctg atc tac ttt gtt gat gga acc atg atc
aga aag gtt gat 4024Gly Leu Ile Tyr Phe Val Asp Gly Thr Met Ile
Arg Lys Val Asp1315 1320 1325caa aat gga
atc ata tca act ctc ctg ggc tcc aac gac ctc acg 4069Gln Asn Gly
Ile Ile Ser Thr Leu Leu Gly Ser Asn Asp Leu Thr1330
1335 1340tca gct cga cct tta acc tgt gat act agc atg
cat atc agc cag 4114Ser Ala Arg Pro Leu Thr Cys Asp Thr Ser Met
His Ile Ser Gln1345 1350 1355gtg cgt ctg
gaa tgg ccc act gac ctc gcg atc aac ccc atg gat 4159Val Arg Leu
Glu Trp Pro Thr Asp Leu Ala Ile Asn Pro Met Asp1360
1365 1370aac tcc atc tac gtc ctg gat aat aac gta gtt
tta cag atc act 4204Asn Ser Ile Tyr Val Leu Asp Asn Asn Val Val
Leu Gln Ile Thr1375 1380 1385gaa aac cgt
cag gtc cgc atc gct gcc ggg cgg ccc atg cac tgt 4249Glu Asn Arg
Gln Val Arg Ile Ala Ala Gly Arg Pro Met His Cys1390
1395 1400cag gtc cct gga gtg gaa tac ccg gtg ggg aag
cac gcg gtt cag 4294Gln Val Pro Gly Val Glu Tyr Pro Val Gly Lys
His Ala Val Gln1405 1410 1415acc acc ctg
gag tca gcc acg gcc att gct gtg tcc tac agc ggg 4339Thr Thr Leu
Glu Ser Ala Thr Ala Ile Ala Val Ser Tyr Ser Gly1420
1425 1430gtc ctt tac atc acg gaa act gat gag aag aag
atc aac cga ata 4384Val Leu Tyr Ile Thr Glu Thr Asp Glu Lys Lys
Ile Asn Arg Ile1435 1440 1445agg cag gtc
acg aca gac ggg gag atc tcc tta gtg gct ggg ata 4429Arg Gln Val
Thr Thr Asp Gly Glu Ile Ser Leu Val Ala Gly Ile1450
1455 1460cct tcg gaa tgt gac tgc aag aac gac gcc aac
tgt gac tgc tac 4474Pro Ser Glu Cys Asp Cys Lys Asn Asp Ala Asn
Cys Asp Cys Tyr1465 1470 1475caa agc gga
gac ggc tac gcc aaa gat gcc aaa ctc aat gcg ccg 4519Gln Ser Gly
Asp Gly Tyr Ala Lys Asp Ala Lys Leu Asn Ala Pro1480
1485 1490tcc tcc ctg gcc gcc tcg cca gat ggc act ctg
tac att gca gat 4564Ser Ser Leu Ala Ala Ser Pro Asp Gly Thr Leu
Tyr Ile Ala Asp1495 1500 1505ctg gga aat
atc agg atc cgg gcc gtt tcg aag aat aaa cct tta 4609Leu Gly Asn
Ile Arg Ile Arg Ala Val Ser Lys Asn Lys Pro Leu1510
1515 1520ctg aac tca atg aac ttt tac gaa gtt gcc tct
cca act gat caa 4654Leu Asn Ser Met Asn Phe Tyr Glu Val Ala Ser
Pro Thr Asp Gln1525 1530 1535gag ctc tac
atc ttt gac atc aac ggt act cac cag tac acc gtg 4699Glu Leu Tyr
Ile Phe Asp Ile Asn Gly Thr His Gln Tyr Thr Val1540
1545 1550agc ctg gtc acg ggt gac tac cta tat aat ttt
agt tac agc aat 4744Ser Leu Val Thr Gly Asp Tyr Leu Tyr Asn Phe
Ser Tyr Ser Asn1555 1560 1565gac aat gac
gtc acc gct gta act gac agc aat ggc aac acc ctc 4789Asp Asn Asp
Val Thr Ala Val Thr Asp Ser Asn Gly Asn Thr Leu1570
1575 1580cga atc cga agg gat ccg aat cgg atg ccg gtg
cgg gtg gtg tct 4834Arg Ile Arg Arg Asp Pro Asn Arg Met Pro Val
Arg Val Val Ser1585 1590 1595cct gat aac
cag gtg ata tgg ttg acc ata ggc acc aac ggg tgt 4879Pro Asp Asn
Gln Val Ile Trp Leu Thr Ile Gly Thr Asn Gly Cys1600
1605 1610ctg aaa agc atg acc gct cag ggc ctg gaa ctg
gtt ttg ttt act 4924Leu Lys Ser Met Thr Ala Gln Gly Leu Glu Leu
Val Leu Phe Thr1615 1620 1625tac cat ggc
aac agt ggg ctt tta gcc acc aaa agt gac gaa act 4969Tyr His Gly
Asn Ser Gly Leu Leu Ala Thr Lys Ser Asp Glu Thr1630
1635 1640gga tgg aca aca ttt ttt gac tat gac agt gaa
ggt cgc ctg acg 5014Gly Trp Thr Thr Phe Phe Asp Tyr Asp Ser Glu
Gly Arg Leu Thr1645 1650 1655aat gtt acc
ttc ccc act ggg gtg gtt aca aac ctg cac ggg gac 5059Asn Val Thr
Phe Pro Thr Gly Val Val Thr Asn Leu His Gly Asp1660
1665 1670atg gac aag gct atc acg gtg gac atc gag tca
tcc agc aga gag 5104Met Asp Lys Ala Ile Thr Val Asp Ile Glu Ser
Ser Ser Arg Glu1675 1680 1685gaa gat gtc
agc atc act tcg aac ttg tcc tcc atc gat tcc ttc 5149Glu Asp Val
Ser Ile Thr Ser Asn Leu Ser Ser Ile Asp Ser Phe1690
1695 1700tac acc atg gtc caa gac cag tta aga aac agt
tac cag att ggg 5194Tyr Thr Met Val Gln Asp Gln Leu Arg Asn Ser
Tyr Gln Ile Gly1705 1710 1715tat gat ggc
tcc ctt aga atc ttc tat gcc agt ggt ctg gac tct 5239Tyr Asp Gly
Ser Leu Arg Ile Phe Tyr Ala Ser Gly Leu Asp Ser1720
1725 1730cac tac cag aca gag ccc cac gtt ctg gct ggc
acg gcg aat ccc 5284His Tyr Gln Thr Glu Pro His Val Leu Ala Gly
Thr Ala Asn Pro1735 1740 1745aca gta gcc
aaa aga aac atg act ctt ccc ggt gag aac ggg cag 5329Thr Val Ala
Lys Arg Asn Met Thr Leu Pro Gly Glu Asn Gly Gln1750
1755 1760aat ctg gtg gag tgg aga ttc cga aaa gaa caa
gcc cag ggc aaa 5374Asn Leu Val Glu Trp Arg Phe Arg Lys Glu Gln
Ala Gln Gly Lys1765 1770 1775gtc aac gta
ttc ggc cgg aag ctc agg gtc aat ggg cgc aac cta 5419Val Asn Val
Phe Gly Arg Lys Leu Arg Val Asn Gly Arg Asn Leu1780
1785 1790ctc tca gtg gac ttt gat cgg acc acc aag acg
gaa aag atc tat 5464Leu Ser Val Asp Phe Asp Arg Thr Thr Lys Thr
Glu Lys Ile Tyr1795 1800 1805gat gac cac
cgg aaa ttt ctc ctg agg atc gct tac gac acg tcg 5509Asp Asp His
Arg Lys Phe Leu Leu Arg Ile Ala Tyr Asp Thr Ser1810
1815 1820ggg cac ccg act ctc tgg ctg ccg agt agc aag
cta atg gca gtg 5554Gly His Pro Thr Leu Trp Leu Pro Ser Ser Lys
Leu Met Ala Val1825 1830 1835aac gtc acc
tac tca tcc acc ggt caa att gcc agc atc cag aga 5599Asn Val Thr
Tyr Ser Ser Thr Gly Gln Ile Ala Ser Ile Gln Arg1840
1845 1850ggg acc acg agc gaa aag gtg gac tat gac agc
cag ggg agg atc 5644Gly Thr Thr Ser Glu Lys Val Asp Tyr Asp Ser
Gln Gly Arg Ile1855 1860 1865gta tct cgg
gtc ttt gcc gat ggg aaa aca tgg agt tac acg tac 5689Val Ser Arg
Val Phe Ala Asp Gly Lys Thr Trp Ser Tyr Thr Tyr1870
1875 1880ttg gaa aag tcc atg gtt ctt ctg ctc cat agc
cag cgg cag tac 5734Leu Glu Lys Ser Met Val Leu Leu Leu His Ser
Gln Arg Gln Tyr1885 1890 1895atc ttc gaa
tac gac atg tgg gac cgc ctg tcc gcc atc acc atg 5779Ile Phe Glu
Tyr Asp Met Trp Asp Arg Leu Ser Ala Ile Thr Met1900
1905 1910ccc agt gtg gct cgc cac acc atg cag acc atc
cgg tcc att ggc 5824Pro Ser Val Ala Arg His Thr Met Gln Thr Ile
Arg Ser Ile Gly1915 1920 1925tac tac cgc
aac atc tac aat ccc cca gaa agc aat gcc tct atc 5869Tyr Tyr Arg
Asn Ile Tyr Asn Pro Pro Glu Ser Asn Ala Ser Ile1930
1935 1940atc acc gac tac aac gag gaa ggg ctg ctt ctg
caa aca gct ttc 5914Ile Thr Asp Tyr Asn Glu Glu Gly Leu Leu Leu
Gln Thr Ala Phe1945 1950 1955ctg gga acg
agt cgg agg gtc tta ttc aag tat aga agg cag acc 5959Leu Gly Thr
Ser Arg Arg Val Leu Phe Lys Tyr Arg Arg Gln Thr1960
1965 1970agg cta tca gaa att tta tac gac agc aca aga
gtc agt ttt acc 6004Arg Leu Ser Glu Ile Leu Tyr Asp Ser Thr Arg
Val Ser Phe Thr1975 1980 1985tac gac gaa
aca gcg gga gtc ctg aaa aca gta aac ctt cag agt 6049Tyr Asp Glu
Thr Ala Gly Val Leu Lys Thr Val Asn Leu Gln Ser1990
1995 2000gat ggt ttt att tgc acc att aga tac agg caa
att ggt ccc ctg 6094Asp Gly Phe Ile Cys Thr Ile Arg Tyr Arg Gln
Ile Gly Pro Leu2005 2010 2015att gac aga
cag att ttc cgc ttc agc gag gat gga atg gta aat 6139Ile Asp Arg
Gln Ile Phe Arg Phe Ser Glu Asp Gly Met Val Asn2020
2025 2030gcg aga ttt gac tat agc tac gac aac agc ttt
cga gtg acc agc 6184Ala Arg Phe Asp Tyr Ser Tyr Asp Asn Ser Phe
Arg Val Thr Ser2035 2040 2045atg cag ggt
gtc atc aat gaa aca cca ctg ccc att gat cta tac 6229Met Gln Gly
Val Ile Asn Glu Thr Pro Leu Pro Ile Asp Leu Tyr2050
2055 2060cag ttt gat gac atc tct ggc aaa gtc gag cag
ttt gga aaa ttc 6274Gln Phe Asp Asp Ile Ser Gly Lys Val Glu Gln
Phe Gly Lys Phe2065 2070 2075gga gtg ata
tac tac gac atc aac caa atc att tcc acg gcc gtg 6319Gly Val Ile
Tyr Tyr Asp Ile Asn Gln Ile Ile Ser Thr Ala Val2080
2085 2090atg act tat aca aag cac ttt gat gct cat ggg
cgc atc aag gag 6364Met Thr Tyr Thr Lys His Phe Asp Ala His Gly
Arg Ile Lys Glu2095 2100 2105atc caa tat
gag ata ttt agg tca ctc atg tac tgg att aca att 6409Ile Gln Tyr
Glu Ile Phe Arg Ser Leu Met Tyr Trp Ile Thr Ile2110
2115 2120caa tat gat aat atg ggc cgg gta acc aag aga
gag att aaa att 6454Gln Tyr Asp Asn Met Gly Arg Val Thr Lys Arg
Glu Ile Lys Ile2125 2130 2135ggg cct ttt
gcc aac act acc aaa tac gcg tac gag tac gac gtc 6499Gly Pro Phe
Ala Asn Thr Thr Lys Tyr Ala Tyr Glu Tyr Asp Val2140
2145 2150gat gga cag ctc caa aca gtt tac cta aac gaa
aag atc atg tgg 6544Asp Gly Gln Leu Gln Thr Val Tyr Leu Asn Glu
Lys Ile Met Trp2155 2160 2165cgg tac aac
tac gac cta aat gga aac ctc cac ttg ctc aac ccc 6589Arg Tyr Asn
Tyr Asp Leu Asn Gly Asn Leu His Leu Leu Asn Pro2170
2175 2180agc agc agc gcc cgc ctg acc cct ctg cgc tat
gac ctg cgc gac 6634Ser Ser Ser Ala Arg Leu Thr Pro Leu Arg Tyr
Asp Leu Arg Asp2185 2190 2195aga atc acc
cgc ctg ggc gat gtt cag tac cgg ctg gat gaa gat 6679Arg Ile Thr
Arg Leu Gly Asp Val Gln Tyr Arg Leu Asp Glu Asp2200
2205 2210ggt ttc ctg cgt cag agg ggc act gaa att ttt
gaa tac agc tcc 6724Gly Phe Leu Arg Gln Arg Gly Thr Glu Ile Phe
Glu Tyr Ser Ser2215 2220 2225aaa ggg ctt
ctg act cga gtc tac agt aaa ggc agt ggc tgg aca 6769Lys Gly Leu
Leu Thr Arg Val Tyr Ser Lys Gly Ser Gly Trp Thr2230
2235 2240gtg atc tat cgg tac gac ggc ctg gga aga cgt
gtt tct agc aaa 6814Val Ile Tyr Arg Tyr Asp Gly Leu Gly Arg Arg
Val Ser Ser Lys2245 2250 2255acc agc ctg
gga cag cac ctt cag ttt ttc tac gcc gac ctg aca 6859Thr Ser Leu
Gly Gln His Leu Gln Phe Phe Tyr Ala Asp Leu Thr2260
2265 2270tac ccc acg aga att act cac gtc tac aac cat
tcc agt tca gaa 6904Tyr Pro Thr Arg Ile Thr His Val Tyr Asn His
Ser Ser Ser Glu2275 2280 2285atc acc tcc
ctg tac tat gac ctc caa gga cat ctc ttc gcc atg 6949Ile Thr Ser
Leu Tyr Tyr Asp Leu Gln Gly His Leu Phe Ala Met2290
2295 2300gag atc agc agt ggg gat gag ttc tac atc gcc
tcg gac aac acg 6994Glu Ile Ser Ser Gly Asp Glu Phe Tyr Ile Ala
Ser Asp Asn Thr2305 2310 2315ggg aca ccg
ctg gct gtt ttc agc agc aac ggg ctc atg ctg aaa 7039Gly Thr Pro
Leu Ala Val Phe Ser Ser Asn Gly Leu Met Leu Lys2320
2325 2330cag acc cag tac act gcc tat ggt gag atc tac
ttt gac tcc aac 7084Gln Thr Gln Tyr Thr Ala Tyr Gly Glu Ile Tyr
Phe Asp Ser Asn2335 2340 2345gtc gac ttt
cag ctg gta att gga ttc cac ggg ggc ttg tat gac 7129Val Asp Phe
Gln Leu Val Ile Gly Phe His Gly Gly Leu Tyr Asp2350
2355 2360ccg ctc acc aaa cta atc cac ttt gga gaa aga
gat tat gac att 7174Pro Leu Thr Lys Leu Ile His Phe Gly Glu Arg
Asp Tyr Asp Ile2365 2370 2375ttg gcg gga
aga tgg acc aca ccg gac att gaa atc tgg aaa agg 7219Leu Ala Gly
Arg Trp Thr Thr Pro Asp Ile Glu Ile Trp Lys Arg2380
2385 2390atc gga aag gac cct gct cct ttt aac ctg tat
atg ttt cgg aat 7264Ile Gly Lys Asp Pro Ala Pro Phe Asn Leu Tyr
Met Phe Arg Asn2395 2400 2405aac aac ccc
gcg agc aaa atc cat gat gtg aaa gat tac atc acg 7309Asn Asn Pro
Ala Ser Lys Ile His Asp Val Lys Asp Tyr Ile Thr2410
2415 2420gat gtt aac agc tgg ctg gtg acg ttt ggc ttc
cat ctg cac aat 7354Asp Val Asn Ser Trp Leu Val Thr Phe Gly Phe
His Leu His Asn2425 2430 2435gct att cct
gga ttc cct gtt ccc aaa ttt gat tta act gag cct 7399Ala Ile Pro
Gly Phe Pro Val Pro Lys Phe Asp Leu Thr Glu Pro2440
2445 2450tcc tat gag ctt gtg aag agt caa cag tgg gaa
gat gtg ccg ccc 7444Ser Tyr Glu Leu Val Lys Ser Gln Gln Trp Glu
Asp Val Pro Pro2455 2460 2465atc ttt gga
gtt cag cag caa gtg gca agg caa gcc aag gcc ttc 7489Ile Phe Gly
Val Gln Gln Gln Val Ala Arg Gln Ala Lys Ala Phe2470
2475 2480ttg tcc ctg ggg aag atg gcc gag gtg cag gtg
agc cga cgc aaa 7534Leu Ser Leu Gly Lys Met Ala Glu Val Gln Val
Ser Arg Arg Lys2485 2490 2495gct ggc gcc
gag cag tcg tgg ctg tgg ttc gcc acg gtc aag tcg 7579Ala Gly Ala
Glu Gln Ser Trp Leu Trp Phe Ala Thr Val Lys Ser2500
2505 2510ctc atc ggc aag ggc gtc atg ctg gcc gtg agc
caa ggc cgc gtg 7624Leu Ile Gly Lys Gly Val Met Leu Ala Val Ser
Gln Gly Arg Val2515 2520 2525cag acc aac
gtg ctc aac atc gcc aac gag gac tgc atc aag gtg 7669Gln Thr Asn
Val Leu Asn Ile Ala Asn Glu Asp Cys Ile Lys Val2530
2535 2540gcg gcg gtg ctc aac aac gcc ttc tac ctg gag
aac ctg cac ttc 7714Ala Ala Val Leu Asn Asn Ala Phe Tyr Leu Glu
Asn Leu His Phe2545 2550 2555acc atc gag
ggc aag gac aca cac tac ttc atc aag acc acc aca 7759Thr Ile Glu
Gly Lys Asp Thr His Tyr Phe Ile Lys Thr Thr Thr2560
2565 2570ccc gag agc gac ctg ggc aca ctg cgg ctg acg
agc ggt cgc aag 7804Pro Glu Ser Asp Leu Gly Thr Leu Arg Leu Thr
Ser Gly Arg Lys2575 2580 2585gcc ctg gag
aac ggg atc aac gtg acc gtg tct cag tcc acc acg 7849Ala Leu Glu
Asn Gly Ile Asn Val Thr Val Ser Gln Ser Thr Thr2590
2595 2600gtg gtg aac ggc agg act cgc agg ttc gcc gac
gtg gag atg cag 7894Val Val Asn Gly Arg Thr Arg Arg Phe Ala Asp
Val Glu Met Gln2605 2610 2615ttc ggt gcc
ctg gca ctg cat gtg cgc tat ggc atg acg ctg gac 7939Phe Gly Ala
Leu Ala Leu His Val Arg Tyr Gly Met Thr Leu Asp2620
2625 2630gag gag aag gcg cgc att ctg gag cag gcg cgc
cag cgc gcg ctc 7984Glu Glu Lys Ala Arg Ile Leu Glu Gln Ala Arg
Gln Arg Ala Leu2635 2640 2645gcc cgg gcg
tgg gca cgg gag cag cag cgc gtg cgc gac ggc gag 8029Ala Arg Ala
Trp Ala Arg Glu Gln Gln Arg Val Arg Asp Gly Glu2650
2655 2660gag ggt gcg cgc ctc tgg acg gag ggt gag aaa
cgg cag ctg ctg 8074Glu Gly Ala Arg Leu Trp Thr Glu Gly Glu Lys
Arg Gln Leu Leu2665 2670 2675agc gct ggc
aag gtg cag ggc tac gat ggg tac tac gta ctg tcg 8119Ser Ala Gly
Lys Val Gln Gly Tyr Asp Gly Tyr Tyr Val Leu Ser2680
2685 2690gtg gag cag tac ccc gag ctg gct gac agt gcc
aac aac atc cag 8164Val Glu Gln Tyr Pro Glu Leu Ala Asp Ser Ala
Asn Asn Ile Gln2695 2700 2705ttc ttg cga
caa agt gag atc ggc aag agg taa cccccgggcc 8207Phe Leu Arg
Gln Ser Glu Ile Gly Lys Arg2710 2715acccctgtgc
agattctcct gtagcacaat ccaaaccgga ctctccaaag agccttccaa 8267aatgacactg
ctctgcagac agacacatcg cagatacaca cgcaacacaa accagaaaca 8327aagacaactt
tttttttttt ctgaatgacc ttaaaggtga tcggctttaa agaatatgtt 8387tacatacgca
tatcgctgca ctcaattgga ctggaagtat gagaaaggaa aaaaaagcat 8447taaaaaaggc
aacgttttgc catgacccct ctgtaccttc gaggcactgt atttaacaaa 8507ggttttaaaa
aggaaaaaaa aatgcgtaca atgtttccag atattactga attgtcgacc 8567tttgcttaca
ggaagtaatc tctacttagg atgtgatata tatagatctg ttcattttaa 8627aatgtggggc
aaagttactg tttatagaac ccaactgctt tcccgtgctg ctttgtaaaa 8687ggacactggc
acaagggacg tctgcttcgg cggggattta ataatggatt ttactaacat 8747ggcttgccct
gggagggaaa aactgacgaa tagaatcctt gtcactgata agcaaaggaa 8807accctgattt
ttttgtaaat tatgtgagac aagttgttta tggattttta tatgaattac 8867aatttactgt
acatcaaata ttagtctcag aggagttaat ttatgtaaag tgtttaaaaa 8927gtttatactt
aaaaataaaa tgataaaaac aaaaaaa
89641332253DNAHomo sapiensexon(107)..(1090) 133gtgccccgga tgtgcccagc
tggctcctgg ccccacccct cgggcctttg ggctggacca 60gccacctctg cctgagacct
ccggtcgccg caagaagctg gagagg atg tac agc 115Met Tyr Ser1gtt gac cgt
gtg tct gac gac atc cct att cgt acc tgg ttc ccc aag 163Val Asp Arg
Val Ser Asp Asp Ile Pro Ile Arg Thr Trp Phe Pro Lys5 10
15gaa aat ctt ttc agc ttc cag aca gca acc aca act atg
caa gcg gtg 211Glu Asn Leu Phe Ser Phe Gln Thr Ala Thr Thr Thr Met
Gln Ala Val20 25 30
35ttc agg ggc tac gcg gag agg aag cgc cgg aaa cgg gag aat gat tcc
259Phe Arg Gly Tyr Ala Glu Arg Lys Arg Arg Lys Arg Glu Asn Asp Ser40
45 50gcg tct gta atc cag agg aac ttc cgc aaa
cac ctg cgc atg gtc ggc 307Ala Ser Val Ile Gln Arg Asn Phe Arg Lys
His Leu Arg Met Val Gly55 60 65agc cgg
agg gtg aag gcc cag acg ttc gct gag cgg cgc gag cgg agc 355Ser Arg
Arg Val Lys Ala Gln Thr Phe Ala Glu Arg Arg Glu Arg Ser70
75 80ttc agc cgg tcc tgg agc gac ccc acc ccc atg aaa
gcc gac act tcc 403Phe Ser Arg Ser Trp Ser Asp Pro Thr Pro Met Lys
Ala Asp Thr Ser85 90 95cac gac tcc cga
gac agc agt gac ctg cag agc tcc cac tgc acg ctg 451His Asp Ser Arg
Asp Ser Ser Asp Leu Gln Ser Ser His Cys Thr Leu100 105
110 115gac gag gcc ttc gag gac ctg gac tgg
gac act gag aag ggc ctg gag 499Asp Glu Ala Phe Glu Asp Leu Asp Trp
Asp Thr Glu Lys Gly Leu Glu120 125 130gct
gtg gcc tgc gac acc gaa ggc ttc gtg cca cca aag gtc atg ctc 547Ala
Val Ala Cys Asp Thr Glu Gly Phe Val Pro Pro Lys Val Met Leu135
140 145att tcc tcc aag gtg ccc aag gct gag tac atc
ccc act atc atc cgc 595Ile Ser Ser Lys Val Pro Lys Ala Glu Tyr Ile
Pro Thr Ile Ile Arg150 155 160cgg gat gac
ccc tcc atc atc ccc atc ctc tac gac cat gag cac gca 643Arg Asp Asp
Pro Ser Ile Ile Pro Ile Leu Tyr Asp His Glu His Ala165
170 175acc ttc gag gac atc ctt gag gag ata gag agg aag
ctg aac gtc tac 691Thr Phe Glu Asp Ile Leu Glu Glu Ile Glu Arg Lys
Leu Asn Val Tyr180 185 190
195cac aag gga gcc aag atc tgg aaa atg ctg att ttc tgc cag gga ggt
739His Lys Gly Ala Lys Ile Trp Lys Met Leu Ile Phe Cys Gln Gly Gly200
205 210cct gga cac ctc tat ctc ctc aag aac
aag gtg gcc acc ttt gcc aaa 787Pro Gly His Leu Tyr Leu Leu Lys Asn
Lys Val Ala Thr Phe Ala Lys215 220 225gtg
gag aag gaa gag gac atg att cac ttc tgg aag cgg ctg agc cgc 835Val
Glu Lys Glu Glu Asp Met Ile His Phe Trp Lys Arg Leu Ser Arg230
235 240ctg atg agc aaa gtg aac cca gag ccg aac gtc
atc cac atc atg ggc 883Leu Met Ser Lys Val Asn Pro Glu Pro Asn Val
Ile His Ile Met Gly245 250 255tgc tac att
ctg ggg aac ccc aat gga gag aag ctg ttc cag aac ctc 931Cys Tyr Ile
Leu Gly Asn Pro Asn Gly Glu Lys Leu Phe Gln Asn Leu260
265 270 275agg acc ctc atg act cct tat
agg gtc acc ttc gag tca ccc ctg gag 979Arg Thr Leu Met Thr Pro Tyr
Arg Val Thr Phe Glu Ser Pro Leu Glu280 285
290ctc tca gcc caa ggg aag cag atg atc gag acg tac ttt gac ttc cgg
1027Leu Ser Ala Gln Gly Lys Gln Met Ile Glu Thr Tyr Phe Asp Phe Arg295
300 305ttg tat cgc ctg tgg aag agc cgc cag
cac tcg aag ctg ctg gac ttt 1075Leu Tyr Arg Leu Trp Lys Ser Arg Gln
His Ser Lys Leu Leu Asp Phe310 315 320gac
gac gtc ctg tga ggggcagagg cctccgccca gtcaccatca ggccactccc 1130Asp
Asp Val Leu325tctgcaccgg gacctggggc tgggccgcct cgtgctcccc gggactgtgt
agctccggtc 1190tcgcctggag ccacttcagg gcacctcaga cgttgctcag gttccccctg
tgggttccgg 1250tcctcgctgc acccgtggcc gcagaggctg cagtccctgg gggccgggag
gatcccgccc 1310tgtggcccgt ggatgctcag cggccaggca ctgacctgcc atgcctcgcc
tggaggctca 1370gctgtgggca tccctccatg gggttcatag aaataagtgc aatttctaca
cccccgaaac 1430aattcaaagg gaagcagcat ttcttgttaa ctagttaagc actatgctgc
tagttacagt 1490gtaggcaccc cggcccagca gcccagcagc ccacatgtgt tcaggaccct
ccctgcccac 1550cccctccctg ccgtatcgat caccagcacc agggtggccc gtgtgcgtgg
ggccagcgtc 1610gccgggctgc ccagcctggc tctgtctaca ctggccgagt ctctgggtct
gtctacactg 1670gccgagtctc cgactgtctg tgctttcact tacactcctc ttgccacccc
ccatccctgc 1730ttacttagac ctcagccggc gccggacccg gtaggggcag tctgggcagc
aggaaggaag 1790ggcgcagcgt cccctccttc agaggaggct ctgggtgggg cctgctcctc
atccccccaa 1850gcccacccag cactctcatt gctgctgttg agttcagctt ttaccagcct
cagtgtggag 1910gctccatccc agcacacagg cctggggctt ggcaggggcc cagctggggc
tgggccctgg 1970gttttgagaa actcgctggc accacagtgg gcccctggac ccggccgcgc
agctggtgga 2030ctgtaggggc tcctgactgg gcacaggagc tcccagcttt tgtccacggc
cagcaggatg 2090ggctgtcgtg tatatagctg gggcgagggg gcaggccccc cttgtgcaga
gccaggggtc 2150tgagggcacc tggctgtgtt cccagctgag ggagggctgg ggcgggggcc
gggcttggaa 2210cgatgtacga taccctcata gtgaccatta aacctgatcc tcc
22531342253DNADanio rerioexon(1)..(298) 134gtg ccc cgg atg tgc
cca gct ggc tcc tgg ccc cac ccc tcg ggc ctt 48Val Pro Arg Met Cys
Pro Ala Gly Ser Trp Pro His Pro Ser Gly Leu1 5
10 15tgg gct gga cca gcc acc tct gcc tga gac ctc
cgg tcg ccg caa gaa 96Trp Ala Gly Pro Ala Thr Ser Ala Asp Leu
Arg Ser Pro Gln Glu20 25 30gct gga
gag gat gta cag cgt tga ccg tgt gtc tga cga cat ccc tat 144Ala Gly
Glu Asp Val Gln Arg Pro Cys Val Arg His Pro Tyr35
40 45tcg tac ctg gtt ccc caa gga aaa tct ttt
cag ctt cca gac agc aac 192Ser Tyr Leu Val Pro Gln Gly Lys Ser Phe
Gln Leu Pro Asp Ser Asn50 55 60cac aac
tat gca agc ggt gtt cag ggg cta cgc gga gag gaa gcg ccg 240His Asn
Tyr Ala Ser Gly Val Gln Gly Leu Arg Gly Glu Glu Ala Pro65
70 75gaa acg gga gaa tga ttc cgc gtc tgt aat cca gag
gaa ctt ccg caa 288Glu Thr Gly Glu Phe Arg Val Cys Asn Pro Glu
Glu Leu Pro Gln80 85 90aca cct gcg
c atggtcggca gccggagggt gaaggcccag acgttcgctg 338Thr Pro
Ala95agcggcgcga gcggagcttc agccggtcct ggagcgaccc cacccccatg aaagccgaca
398cttcccacga ctcccgagac agcagtgacc tgcagagctc ccactgcacg ctggacgagg
458ccttcgagga cctggactgg gacactgaga agggcctgga ggctgtggcc tgcgacaccg
518aaggcttcgt gccaccaaag gtcatgctca tttcctccaa ggtgcccaag gctgagtaca
578tccccactat catccgccgg gatgacccct ccatcatccc catcctctac gaccatgagc
638acgcaacctt cgaggacatc cttgaggaga tagagaggaa gctgaacgtc taccacaagg
698gagccaagat ctggaaaatg ctgattttct gccagggagg tcctggacac ctctatctcc
758tcaagaacaa ggtggccacc tttgccaaag tggagaagga agaggacatg attcacttct
818ggaagcggct gagccgcctg atgagcaaag tgaacccaga gccgaacgtc atccacatca
878tgggctgcta cattctgggg aaccccaatg gagagaagct gttccagaac ctcaggaccc
938tcatgactcc ttatagggtc accttcgagt cacccctgga gctctcagcc caagggaagc
998agatgatcga gacgtacttt gacttccggt tgtatcgcct gtggaagagc cgccagcact
1058cgaagctgct ggactttgac gacgtcctgt gaggggcaga ggcctccgcc cagtcaccat
1118caggccactc cctctgcacc gggacctggg gctgggccgc ctcgtgctcc ccgggactgt
1178gtagctccgg tctcgcctgg agccacttca gggcacctca gacgttgctc aggttccccc
1238tgtgggttcc ggtcctcgct gcacccgtgg ccgcagaggc tgcagtccct gggggccggg
1298aggatcccgc cctgtggccc gtggatgctc agcggccagg cactgacctg ccatgcctcg
1358cctggaggct cagctgtggg catccctcca tggggttcat agaaataagt gcaatttcta
1418cacccccgaa acaattcaaa gggaagcagc atttcttgtt aactagttaa gcactatgct
1478gctagttaca gtgtaggcac cccggcccag cagcccagca gcccacatgt gttcaggacc
1538ctccctgccc accccctccc tgccgtatcg atcaccagca ccagggtggc ccgtgtgcgt
1598ggggccagcg tcgccgggct gcccagcctg gctctgtcta cactggccga gtctctgggt
1658ctgtctacac tggccgagtc tccgactgtc tgtgctttca cttacactcc tcttgccacc
1718ccccatccct gcttacttag acctcagccg gcgccggacc cggtaggggc agtctgggca
1778gcaggaagga agggcgcagc gtcccctcct tcagaggagg ctctgggtgg ggcctgctcc
1838tcatcccccc aagcccaccc agcactctca ttgctgctgt tgagttcagc ttttaccagc
1898ctcagtgtgg aggctccatc ccagcacaca ggcctggggc ttggcagggg cccagctggg
1958gctgggccct gggttttgag aaactcgctg gcaccacagt gggcccctgg acccggccgc
2018gcagctggtg gactgtaggg gctcctgact gggcacagga gctcccagct tttgtccacg
2078gccagcagga tgggctgtcg tgtatatagc tggggcgagg gggcaggccc cccttgtgca
2138gagccagggg tctgagggca cctggctgtg ttcccagctg agggagggct ggggcggggg
2198ccgggcttgg aacgatgtac gataccctca tagtgaccat taaacctgat cctcc
225313540PRTArtificial SequenceTCAP 3 General Motif 135Gln Leu Leu Ser
Xaa Xaa Lys Val Xaa Gly Tyr Asp Gly Tyr Tyr Val1 5
10 15Leu Ser Xaa Glu Gln Tyr Pro Glu Leu Ala
Asp Ser Ala Asn Asn Xaa20 25 30Gln Phe
Leu Arg Gln Ser Glu Ile35 4013636PRTArtificial
SequenceG. gallus TCAP2 136Thr Gly Arg Val Gln Gly Tyr Glu Gly Tyr Tyr
Val Leu Pro Val Glu1 5 10
15Gln Tyr Pro Glu Leu Ala Asp Ser Ser Ser Asn Ile Gln Phe Leu Arg20
25 30Gln Asn Glu Met35
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