| Program : Biomedical Research and Countermeasures | Ground Research | ||||
| Element : Physiology | |||||
GH/IGF-I Transgene Expression on Muscle Homeostasis |
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| Principal Investigator: | |||||
Robert J. Schwartz, Ph.D. Department of Molecular and Cellular Biology 145 EA Baylor College of Medicine One Baylor Plaza Houston, TX 77030 |
Phone: (713) 798-6649 Email: schwartz@bcm.tmc.edu Fax: (713) 798-7799 Congressional District: TX-25 | ||||
| Co-Investigator(s): | |||||
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| Monitoring Center: NSBRI | Solicitation: NSBRI | ||||
| Initial Funding Date: 1997 | Expiration: 2000 | ||||
| Students Funded Under Research: 3 | Post-Doctoral Associates: 1 | ||||
| Task Description: | |||||
| A current goal of the National Space Biomedical Research Institute is to develop countermeasures that allow humans to live and work in microgravity for duration over a year and to minimize readapting to Earth's gravity, and optimize crew safety, well-being, and performance. We propose to test the hypothesis that the GH/IGF-I axis through autocrine/paracrine mechanisms may provide long term muscle homeostasis under conditions of prolonged weightlessness. As a key alternative to hormone replacement therapy, ectopic production of hGH, growth hormone releasing hormone (GHRH), and IGF-I will be studied for its potential on muscle mass impact in transgenic mice under simulated microgravity. Transgenic mice will be used in hind-limb suspension models to determine the role of GH/IGF-I on maintenance of muscle mass and whether concentric exercises might act in synergy with hormone treatment. As a means to engineer and ensure long-term protein production that would be workable in humans, gene therapy technology will be used by to monitor muscle mass preservation during hind-limb suspension, after direct intramuscular injection of a genetically engineered muscle-specific vector expressing GHRH. Effects of this gene-based therapy will be assessed in both fast twitch (medial gastrocnemius) and slow twitch muscle (soleus). End-points include muscle size, ultrastructure, fiber type, and contractile function, in normal animals, hind limb suspension, and reambulation. The following aims are: Aim I: Does IGF-I and the IGFI receptor provide signaling for muscle mass homeostasis in response to unloading, overloading and exercise? Aim II: Does exercise and GH/IGF-I axis synergize in alleviating muscle atrophy? To determine the role of overexpression of hGH, and IGF-I in transgenic mice on muscle mass accretion under condition of hindlimb underloading? | |||||
| Role of IGF-I as a countermeasure: Previously Dr. Richard Grindeland has shown that a combination of IGF-I administraion and ladder climbing was more effective than either alone in attenuating atrophy of unloaded skeletal muscles in hypophysectomized rats. These animals without pituitary glands would have no growth hormone or glucocorticoid hormones. Since astronauts have pituitary glands, it is important to confirm these findings in animals with intact pituitary glands. The effects seen by these investigators may have been a result of using hypophysectomized rats. We hypothesized that a combination of IGF-I overexpression and stretching exercise would be more effective than either alone in attenuating muscle atrophy from unloading in unloaded muscles of hindlimbs of mice with intact pituitaries. We tested localized IGF-I transgene expression in underloaded muscle by using hind limb suspension model. Twenty male transgenic mice (about 6-mo-old) harboring the human IGF-I gene driven by regulatory regions from the chicken skeletal alpha actin promoter extending to -424 bp upstream of the transcription initiation start site, the first intron, and the 3' untranslated region (SK733IGF-I3'SK) were used to asses the potential of locally produced IGF-I to prevent unloading induced atrophy. In our recent study with Dr. Frank Booth, we observed that the local over expression of IGF-I was associated with a higher absolute muscle mass in the weight bearing, transgenic mice (GAST:+21%,TA:+15%) compared to the FVB, nontransgenic mice, even though tibial lengths differed by only 2.7%. Following 14 days of suspension, however, the percentage loss of mass in the GAST and TA of the transgenic mice(about 20%) did not differ from the HU- induced atrophy in the FVB wild type mice, resulting in an absolute mass of these two muscles in the HU transgenics equivalent to that of weight bearing FVB mice. We also found that endogenous IGF-I mRNA level was unaltered in atrophying GAST muscle of nontransgenic mice, suggesting that a down regulation of IGF-I may not be involved in unloading-induced skeletal muscle atrophy as we had hypothesized. Conversely, we reported that the expression of the skeletal a-actin driven IGF-I transgene is down regulated following 14 days of HU and that this expression also occurs in a fiber type-specific manner. Yet, despite this down regulation, the GAST and TA of the transgenic mice still expressed hIGF-ImRNA and peptide levels that were magnitudes higher than the corresponding muscle in the nontransgenic FVB mice. Therefore, our hypothesis that locally produced IGF-I would be more effective in preventing unloading-induced atrophy of skeletal muscle was not supported. We also conclude that elevated IGF-I expression alone is ineffective in preventing unloading-induced muscle atrophy within fast-twitch skeletal muscle. We observed no facilitative interaction by IGF-I for the attenuation of unloading atrophy by stretching unloaded muscles (See Criswell, D.A., F.W. Booth, F. DeMayo, R.J. Schwartz, S.E. Gordon, and M.L. Fiorotto. Over-expression of IGF-I in skeletal muscle of transgenic mice does not prevent unloading-induced atrophy. Am. J. Physiol. 275:E373-E379, 1998) Although this was a surprise, the following information indicates that it is very premature to conclude that this approach will not work . First, after this study was completed, Vandenburgh et al.(Hum Gene Ther 9:2555?64, 1998) published a paper in which they reported the manufacture of human growth hormone from proliferating murine C2C12 skeletal myoblasts that had been stably transduced with the recombinant human growth hormone gene and that had been tissue engineered in vitro into bioartificial muscles (C2?BAMs) containing organized postmitotic myofibers secrete 3?5 microgram of recombinant human growth hormone/day in vitro. When implanted subcutaneously into syngeneic mice, C2?BAMs delivered a sustained physiologic dose of 2.5 to 11.3 ng of recombinant human growth hormone per milliliter of serum. Recombinant human growth hormone synthesized and secreted by the myofibers was in the 22?kDa monomeric form and was biologically active, based on down regulation of a growth hormone?sensitive protein synthesized in the liver. Skeletal muscle disuse atrophy was induced in mice by hindlimb unloading, causing the fast plantaris and slow soleus muscles to atrophy by 21 to 35% (P< 0.02). This atrophy was significantly attenuated 41 to 55% (p < 0.02) in animals that received C2?BAM implants, but not in animals receiving daily injections of purified recombinant human growth hormone (1 mg/kg/day). This data show that the mode of presentation of growth factors to atrophying skeletal muscle is important. Currently we have developed a gene therapeutic method via GHRH secretion from muscle to increase blood growth hormone levels (see below). Role of Growth Hormone Releasing Hormone Gene Therapy as Counter Measure: Regulated expression of the growth hormone/insulin-like growth factor axis is essential for optimal linear growth, as well as homeostasis of carbohydrate, protein, and fat metabolism. GH synthesis and secretion from the anterior pituitary is stimulated by the natural GH secretagogue growth hormone releasing hormone (GHRH) and inhibited by somatostatin, both hypothalamic hormones. GH increases production of insulin-like growth factor I (IGF-I), primarily in the liver, and possibly other target organs. Increased levels of serum IGF-I, in turn, feeds back on the hypothalamus and anterior pituitary to inhibit GHRH release and GH secretion. The pulsaile pattern is thought to arise from alternating episodes of stimulation by GHRH and inhibition by somatostatin. The endogenous rhythm of GH secretion becomes entrained to the imposed rhythm of exogenous GH administration. It is well established that ectopically secreted GHRH, as mature peptide or truncated molecules (as seen with pancreatic islet cell tumors and various located carcinoids) are often biologically active and can even produce acromegaly. Administration of recombinant GHRH to GH-deficient children or adult humans augment IGF-1 levels, increases GH pulsatile secretion proportionally to GHRH dose, with preserve response to bolus doses of GHRH. Thus, the GHRH administration represent a more physiological alternative of increasing subnormal GH and IGF-1 levels. Most importantly, however, is that GH secretion is effected at vanishingly low levels of GHRH (10 pg/ml) in the blood supply. Thus, by employing a gene therapy approach, the human GHRH cDNA could be targeted into peripheral organs and expressed by the transfected cells and the peptide processed, secreted, transported to the anterior pituitary, where it could stimulate GH release. To establish the baseline expression of the human GHRH cDNA, driven by skeletal actin promoter in a DNA plasmid after in vivo administration directly into mouse muscle. These experiments will create the foundation for demonstrating reproducibility, dose response, and duration of expressed product; persistence and state of DNA; and, initial safety. The route and method of vector DNA administration to muscle are critical steps towards controlling the expression of a therapeutic product in somatic gene therapy. Conventional intramuscular injections have been shown to be an effective means of introducing genes into muscle and achieving detectable levels of gene expression. Studies have shown that DNA introduced into expression from muscle has been reported to persist for several months to even up to a year in vivo. In a gene therapy approach, the human GHRH cDNA could be targeted into peripheral organs and expressed by the transfected cells and the peptide processed, secreted, transported to the anterior pituitary, where it could stimulate GH release. Skeletal muscle can be transfected in vivo by direct plasmid DNA injection, which can be expressed at significant levels for different periods of time, up to 19 month. A 228bp fragment of hGHRH, which encode for the 31 amino acid signal peptide and the entire mature peptide hGHRH(1-44)OH (Tyr1-Leu44), was cloned into a skeletal muscle actin promoter followed by the 3' untranslated region of human growth hormone cDNA. Role of Growth Hormone Releasing Hormone: Regulated expression of the growth hormone/insulin-like growth factor axis is essential for optimal linear growth, as well as homeostasis of carbohydrate, protein, and fat metabolism. GH synthesis and secretion from the anterior pituitary is stimulated by the natural GH secretagogue growth hormone releasing hormone (GHRH) and inhibited by somatostatin, both hypothalamic hormones. GH increases production of insulin- like growth factor I (IGF-I), primarily in the liver, and possibly other target organs. Increased levels of serum IGF-I, in turn, feeds back on the hypothalamus and anterior pituitary to inhibit GHRH release and GH secretion. The pulsaile pattern is thought to arise from alternating episodes of stimulation by GHRH and inhibition by somatostatin. The endogenous rhythm of GH secretion becomes entrained to the imposed rhythm of exogenous GH administration. It is well established that ectopically secreted GHRH, as mature peptide or truncated molecules (as seen with pancreatic islet cell tumors and various located carcinoids) are often biologically active and can even produce acromegaly. Administration of recombinant GHRH to GH-deficient children or adult humans augment IGF-1 levels, increases GH pulsatile secretion proportionally to GHRH dose, with preserve response to bolus doses of GHRH. Thus, the GHRH administration represent a more physiological alternative of increasing subnormal GH and IGF-1 levels. Most importantly, however, is that GH secretion is effected at vanishingly low levels of GHRH (10 pg/ml) in the blood supply. Thus, by employing a gene therapy approach, the human GHRH cDNA could be targeted into peripheral organs and expressed by the transfected cells and the peptide processed, secreted, transported to the anterior pituitary, where it could stimulate GH release. Growth hormone releasing hormone gene therapy in large animals, testing of an early stage countermeasure: Although GHRH protein therapy entrains and stimulates normal cyclical GH secretion with virtually no side effects; the short half-life of GHRH in vivo, requires frequent (one to three times a day) intravenous, subcutaneous or intranasal (requiring 300-fold higher dose) administration. Thus, as a chronic treatment, GHRH administration is not practical. However, extracranial secreted GHRH, as processed protein species (Tyr1-40 or Tyr1-Leu44) or even as shorter truncated molecules are biologically active. Importantly, a low level of GHRH (100 pg/ml) in the blood supply stimulates GH secretion and makes GHRH an excellent candidate for gene therapeutic expression. Direct plasmid DNA gene transfer is currently the basis of many emerging gene therapy strategies, which does not require viral genes or lipid particles. Skeletal muscle is a preferred target tissue, because muscle fiber has a long life span and can be transduced by circular DNA plasmids that express over months or years in an immunocompetent host. Previously, we reported that human GHRH cDNA could be delivered to muscle, by an injectable myogenic expression vector in mice, where it transiently stimulated GH secretion barely over a period of two weeks. We have now optimized this injectable myogenic vector system by incorporating a powerful synthetic muscle promoter coupled with a novel protease resistant GHRH molecule (pSP-GHRH-HV), possessing substantially longer half-life and greater GH secretory activity, together with improved muscle delivery via highly efficient electropration technology. Intramuscular injection of plasmid DNA in pigs: Three groups of five, 3-4 weeks old hybrid cross barrows (Yorkshire, Landrace, Hampshire and Duroc), were used in the GHRH studies. The animals were individually housed with ad lib access to water, and 6% of their body weight diet (24% protein pig meal, Producers Cooperative Association, Bryan, TX). The animals were weighted every other day, at 8:30 am, and the feed was subsequently added. Animals were maintained in accordance with NIH Guide, USDA and Animal Welfare Act guidelines. The plasmid pSPc5-12 contains a 360bp SacI/BamHI fragment of the SPc5-12 synthetic promoter 23 in the SacI/BamHI sites of pSK-GHRH backbone 24. The wild type and mutated porcine GHRH cDNAs were obtained by site directed mutagenesis of human GHRH cDNA (Altered Sites II in vitro Mutagenesis System, Promega, Madison, WI), and cloned into the BamHI/ Hind III sites of pSK-GHRH. The GHRH cDNA is followed by the 3' untranslated region of human growth hormone. Endotoxin-free plasmid (Qiagen Inc., Chatsworth, CA, USA) preparation of pSPc5- 12-HV-GHRH, pSPc5-12-wt-GHRH and pSPc5-12?gal were diluted in PBS pH=7.4 to 1mg/ml. The animals were assigned equally to one of treatments. The pigs were anesthetized with isoflurane (concentration of 2-6 % for induction and 1-3 % for maintenance). By surgical procedure, we implanted jugular catheters, to drawn blood from these animals at day 3, 7, 14, 21, 28, 45 and 65 post-injection. While anesthetized, 10mg of plasmid was injected directly into the semitendinosus muscle of pigs. Two minutes after injection, the injected muscle was placed in between a set of calipers and electroporated, as described 16. At 65 days post-injection, animals were killed and internal organs and injected muscle collected, weighted, frozen in liquid nitrogen, and stored at -80C. Porcine GHRH was measured by a heterologous human assay system (Peninsula Laboratories, Belmont, CA). Sensitivity of the assay is 1 pg/tube. Porcine GH in plasma was measured a specific double antibody procedure RIA (The Pennsylvania State University). The sensitivity of the assay is 4ng/tube. Porcine IGF-1 was measured by heterologous human assay (Diagnostic System Lab., Webster, TX). Body composition measurements were performed either in vivo, at day 30 and 65 post- injection (densitometry, K40) or post-mortem (organ, carcass, body fat, direct dissection followed by neutron activation chamber). Data are analyzed using Microsoft Excel statistics analysis package. Values shown in the figures are the mean s.e.m. Specific p values will be obtained by comparison using Students t test. A p < 0.05 will be set as the level of statistical significance Ectopic expression of a novel serum protease resistant porcine growth hormone releasing hormone, directed by an injectable muscle synthetic promoter plasmid vector, pSP-GHRH-HV, elicits profound growth in pigs. Single intramuscular injections of 10 mg of pSP-GHRH-HV plasmid DNA, followed by electroporation in three-week-old piglets elevates serum GHRH levels by 2-4 fold, increases growth hormone secretion and enhances serum IGF-1 levels up to 3-6 fold over control pigs injected with a placebo myogenic vector. Average body weight increased approximately 37%; over 65 days in the pSP-GHRH-HV injected pigs resulted in a significant reduction of serum urea, indicating decreased amino acid catabolism. Evaluation of body composition indicates uniform increase in mass, with no organomegaly or associated pathology. This novel gene therapeutic vector enhances long term growth and performance in domestic. Employing electrogene therapy, which does not require viral genes or particles, allows genes to be transferred and expressed in desired organs or tissues, and it may lead to the development of a new type of highly effective gene therapy. Although, the exact mechanism for enhanced DNA uptake in muscle is not yet known, it is thought that change in polarity increases the plasmid DNA uptake probably by opening of membrane pores and that repetitive depolarizations uniformly spreads DNA throughout the muscle by forming transient protein DNA complexes. The electroporation system has been previously used in rodents and small animals and does cause any serious discomfort. In combination with improved muscle expression vectors electrogene therapy increased vector activity over one hundred fold and allowed for prolonged GHRH-HV expression over 60 days in pigs Enhancing biological potency reduces the theoretical quantity of GHRH plasmid needed to achieve physiological levels of GH that are necessary for animal gene therapy. The treated pigs did not experience any side effects from the therapy, have normal biochemical profiles, with no associated pathology and no organomegaly. The profound increases in IGF-I levels and resulting growth enhancement in growth over two months indicates that ectopic expression of myogenic GHRH-HV vectors via electrogene treatment may replace classical GH protein regimens and has the potential to stimulate GH axis in a more natural fashion. We predict that the GHRH-HV species which display a high degree of stability and GH secretory activity in pigs, might also be employed in human clinical medicine, since the serum proteases that turn-over GHRH are similar in most mammals. | |||||
| Our study will provide a framework against which other types of therapies can be compared, both in clinical studies on GH deficient children or in elderly, where our therapy could be combined with an adequate diet for optimized results in growth, muscle strength and/or bone mineralization. As a key hormone replacement therapy, induced secretion of GH by GH secretogoues, has been studied for its potential on improving nutrition, skeletal growth and maintenance of muscle homeostatsis. Second, when adult rats were treated with dexamethasone, IGF-I was still able to stimulate protein synthesis in the epitrochlearis muscle, but only by 41.9% of the increase found in a pair-fed control group (Dardevet et al. J. Endocr.. 156:83, 1998). As hindlimb unloaded mice should have increases in plasma glucocorticoids, it is possible that this would suppress the action of IGF-I. Furthermore, Dr. Fred Goldberg showed three decades ago that inactive skeletal muscle becomes more sensitive to glucocorticoids (J. Physiol. 200:667, 1969). ). In addition, space flight conditions include several human factors that induce stress to some degree including: heavy work loads, lack of sleep, impaired circadian daylight cycles and nutrition. Hormones secreted in greater amounts as part of the stress response include ACTH and cortisol which increased greater than 100% during Skylab missions (41). Cortisol is one of the primary muscle catabolic factors that rapidly elicits muscle protein break down and impairs GH secretion and reduces IGF-I levels, increases bone loss and damages the immune system (41 Ectopic expression of a novel serum protease resistant porcine growth hormone releasing hormone, directed by an injectable muscle synthetic promoter plasmid vector, pSP-GHRH-HV, elicits profound growth in pigs. Single intramuscular injections of 10 mg of pSP-GHRH-HV plasmid DNA, followed by electroporation in three-week-old piglets elevates serum GHRH levels by 2-4 fold, increases growth hormone secretion and enhances serum IGF-1 levels up to 3-6 fold over control pigs injected with a placebo myogenic vector. Average body weight increased approximately 37%; over 65 days in the pSP-GHRH-HV injected pigs resulted in a significant reduction of serum urea, indicating decreased amino acid catabolism. Evaluation of body composition indicates uniform increase in mass, with no organomegaly or associated pathology. This novel gene therapeutic vector enhances long term growth and performance in large mammals.. Employing electrogene therapy, which does not require viral genes or particles, allows genes to be transferred and expressed in desired organs or tissues, and it may lead to the development of a new type of highly effective gene therapy. Although, the exact mechanism for enhanced DNA uptake in muscle is not yet known, it is thought that change in polarity increases the plasmid DNA uptake probably by opening of membrane pores and that repetitive depolarizations uniformly spreads DNA throughout the muscle by forming transient protein DNA complexes. The electroporation system has been previously used in rodents and small animals and does cause any serious discomfort. In combination with improved muscle expression vectors electrogene therapy increased vector activity over one hundred fold and allowed for prolonged GHRH-HV expression over 60 days in pigs Enhancing biological potency reduces the theoretical quantity of GHRH plasmid needed to achieve physiological levels of GH that are necessary for animal gene therapy. The treated pigs did not experience any side effects from the therapy, have normal biochemical profiles, with no associated pathology and no organomegaly. The profound increases in IGF-I levels and resulting growth enhancement in growth over two months indicates that ectopic expression of myogenic GHRH-HV vectors via electrogene treatment may replace classical GH protein regimens and has the potential to stimulate GH axis in a more natural fashion. We predict that the GHRH-HV species which display a high degree of stability and GH secretory activity in pigs, might also be employed in human clinical medicine, since the serum proteases that turn-over GHRH are similar in most mammals. Our study will provide a framework against which other types of therapies can be compared, both in clinical studies on GH deficient children or in elderly, where our therapy could be combined with an adequate diet for optimized results in growth, muscle strength and/or bone mineralization. As a key hormone replacement therapy, induced secretion of GH by GH secretogoues, has been studied for its potential on improving nutrition, skeletal growth and maintenance of muscle homeostatsis. Second, when adult rats were treated with dexamethasone, IGF-I was still able to stimulate protein synthesis in the epitrochlearis muscle, but only by 41.9% of the increase found in a pair-fed control group (Dardevet et al. J. Endocr.. 156:83, 1998). As hindlimb unloaded mice should have increases in plasma glucocorticoids, it is possible that this would suppress the action of IGF-I. Furthermore, Dr. Fred Goldberg showed three decades ago that inactive skeletal muscle becomes more sensitive to glucocorticoids (J. Physiol. 200:667, 1969). ). In addition, space flight conditions include several human factors that induce stress to some degree including: heavy work loads, lack of sleep, impaired circadian daylight cycles and nutrition. Hormones secreted in greater amounts as part of the stress response include ACTH and cortisol which increased greater than 100% during Skylab missions. Cortisol is one of the primary muscle catabolic factors that rapidly elicits muscle protein break down and impairs GH secretion and reduces IGF-I levels, increases bone loss and damages the immune system. | |||||
| FY00 Publications, Presentations, and Other Accomplishments: | |||||
| Wei, L., Zhou, W. and Schwartz, R.J. ''Beta-Integrin and PI 3'- kinase regulate RhoA-dependent activation of skeletal-actin promoter in myoblasts.'' Am. J. Physiol. Heart Circ. Physiol., 278: 278: H1736-H1743, (2000). Newman C.S., Reecy J., Grow M.W., Ni K., Boettger T., Kessel M., Schwartz R.J., and Krieg P.A. ''Transient cardiac expression of the tinman-family homeobox gene, XNkx2-10.'' Mech. Dev., 91(1-2):369-73, (2000). Zhang D., Gaussin V., Taffet G.E., Belaguli N.S., Yamada M., Schwartz R.J., Michael L.H., Overbeek P.A, Schneider, M.D. ''TAK1 is activated in the myocardium after pressure overload and is sufficient to provoke heart failure in transgenic mice..'' Nat Med., 5:556-63, (2000). Belaguli, N.S., Sepulveda, J.L., Nigam,V., Charron,F., Mona NemerM., Schwartz, R.J. ''Cardiac Tissue Enriched Factors, SRF and GATA-4 are Mutual Coregulators.'' Mol. Cell. Biol., 20, 7550-7558, (2000). Schildmeyer,L.A., Braun, R., Taffet, G., DeBiasi, M., Burns, A.E., Bradley, A. Schwartz, R.J. ''Impaired vascular contractility and blood pressure homeostasis in the smooth muscle alpha actin null mouse.'' FASEB J. , 14, 2213- 2220, (2000). Yamada, M., Revelli, J.P., Eichele, G., Barron, M., Eichele, G., Schwartz, R.J. ''Expression Of Chick Tbx-2, Tbx-3 and Tbx-5 genes during early heart development: evidence for BMP2 induction of Tbx2.'' Dev. Biol., 228, 95-105, (2000). Chakravarthy M.V., Abraha T.W., Schwartz R.J., Fiorotto M.L., Booth F.W ''IGF-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of PI3'-kinase/Akt signaling pathway.'' J Biol Chem, 275, 35942-35952, (2000). Moore M.L., Wang G.L., Belaguli N.S., Schwartz R.J., McMillin J.B. ''GATA-4 and Serum Response Factor Regulate Transcription of the Muscle-Specific Carnitine Palmitoyltransferase I {beta} in Rat Heart.'' J Biol Chem, 2276, 1026- 1033, (2001). Carson J.A., Fillmore R.A., Schwartz R.J., Zimmer W.E. ''The smooth muscle gamma-actin gene promoter is a molecular target for mNkx 3-1, the mouse Bagpipe homologue, and serum response factor.'' J Biol Chem, 275, 39061-39072, (2000). | |||||
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