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Biomedical subjects

T E Wagner

Publications and source records attributed to T E Wagner.

At least 37 records · Page 2Linked to original sources
PubMed1997

Human growth hormone antagonist (G120R) delivered by a murine yolk sac cell-derived mini-organ decreases the growth rate of mice.

Long-term cultured murine embryonic yolk sac cells that are capable of forming capillary structures when cultured on base membrane proteins (Matrigel) were successfully transfected with a human growth hormone antagonist (G120R) gene. Cells that stably express relatively high levels of G120R were co-implanted s.c. with Matrigel into BALB/c mice. G120R can be detected in the sera of those implanted mice for more than 14 days at levels from 4 ng/ml to 28 ng/ml. The insulin-like growth factor-1 levels in the sera of those implanted mice were significantly affected by the delivered G120R. One of the physiological effects of G120R delivered by this murine embryonic yolk sac cell-derived mini-organ system is to decrease the growth rate of the implanted mice. This gene delivery system can also be used as an alternative to transgenic animals to study protein function in vivo.

Animals↗
PubMed1997

Cardiomyopathy induced by cardiac Gs alpha overexpression.

The goal of this study was to determine whether chronic endogenous sympathetic stimulation resulting from the overexpression of cardiac stimulatory G protein alpha subunit (Gs alpha) in transgenic mice (15.3 +/- 0.1 mo old) resulted in a clinical picture of cardiomyopathy. The left ventricular ejection fraction, measured by echocardiography, was reduced in older mice with Gs alpha overexpression (50.4 +/- 5.4%) compared with age-matched control mice (70.9 +/- 1.6%; P < 0.05). When ejection fractions were compared at similar heart rates, the Gs alpha mice exhibited a greater left ventricular end-diastolic dimension than control mice (4.3 +/- 0.2 vs. 3.7 +/- 0.1 mm; P < 0.05). Baseline heart rates were elevated in conscious Gs alpha mice (722 +/- 27 beats/min; n = 5) compared with control mice (656 +/- 28 beats/min; n = 5). Moreover, electrocardiographic monitoring demonstrated a high incidence of arrhythmias. Increased mortality compared with control mice (31.6 vs. 3.0%; P < 0.01) was also observed. Thus older mice with Gs alpha overexpression exhibit many of the features of dilated cardiomyopathy. This study supports the concept that chronic sympathetic stimulation over an extended period of time, i.e., over the life of an animal, is deleterious and actually may result in cardiomyopathy.

Animals↗
PubMed1996

Long-term expression of human growth hormone (hGH) in mice containing allogeneic yolk sac cell derived neovascular implants expressing hGH.

We have established a systemic gene delivery animal model system by using cultured murine embryonic yolk sac cells, which can be easily genetically modified in vitro and participate in angiogenesis in vivo when basement membrane proteins (Matrigel) are provided in syngeneic mice. In the present study, we successfully applied this system to allogeneic mice. In order to suppress donor cell-specific immune responses, the costimulatory signal transduction pathway of T cell activation was blocked by treating the recipient allogeneic C57BL/6 mice with rat-antimouse B7.2 antibody. As a result of this suppression, human growth hormone, the therapeutic gene product, could be detected for over 340 days, while it could only be detected in mice treated with rat-IgG2a, the iso-type control of anti-B7.2, for fewer than 50 days. This is the first ex vivo gene delivery system that can express a therapeutic gene product, long-term, in an allogeneic host.

Animals↗
PubMed1996

A long-term culture system for the expansion of hematopoietic stem cells from embryonic yolk sac with the capacity to seed erythroid and lymphoid development in vitro and to reconstitute the lymphoid compartment in severe combined immunodeficient mice.

We have established a long-term culture system under which hematopoietic stem cells derived from the embryonic yolk sac may be maintained for long periods. Evidence for the persistence of stem cells in this culture is provided by three experimental observations. First, erythroid progenitors, as evidenced by morphological, functional, and phenotypical analysis, may be generated from these yolk sac cultures after more than 7 months in culture. The yolk sac derived erythroid progenitors are distinct from those of bone marrow derived cells both qualitatively and quantitatively. This is evidenced by the high colony plating efficiency, large colony size, different growth factor requirements, increased sensitivity to Epo and other cytokines, as well as significant and prolonged expansion capability, suggesting that the yolk sac derived progenitors are both more proliferative and more primitive than their bone marrow derived analogs. Second, under different conditions, lymphoid progenitors may also be derived from these long term yolk sac cultures in the presence of the appropriate cytokines. Third, preliminary data suggest the engraftment of these yolk sac cells and reconstitution of at least some compartments of the hematopoietic system of host animals. This long-term culture system will provide a useful model for the study of early embryonic hematopoiesis, and the cells derived from this culture system may also have the potential of serving as donor cells for hematopoietic cell transplantation.

Animals↗
PubMed1996

Adverse effects of chronic endogenous sympathetic drive induced by cardiac GS alpha overexpression.

To study the physiological effect of the overexpression of myocardial Gsalpha (protein levels increased by approximately threefold in transgenic mice), we examined the responsiveness to sympathomimetic amines by echocardiography (9 MHz) in five transgenic mice and five control mice (both 10.3 +/- 0.2 months old). Myocardial contractility in transgenic mice, as assessed by left ventricular (LV) fractional shortening (LVFS) and LV ejection fraction (LVEF) was not different from that of control mice at baseline (LVFS, 40 +/- 3% versus 36 +/- 2%; LVEF, 78 +/- 3% versus 74 +/- 3%). LVFS and LVEF values in transgenic mice during isoproterenol (ISO, 0.02 micrograms/kg per minute) infusion were higher than the values in control mice (LVFS, 68 +/- 4% versus 48 +/- 3%; LVEF, 96 +/- 1% versus 86 +/- 3%; P < .05). Norepinephrine (NE, 0.2 micrograms/kg per minute) infusion also increased LVFS and LVEF in transgenic mice more than in control mice (LVFS, 59 +/- 4% versus 47 +/- 3%; LVEF, 93 +/- 2% versus 85 +/- 3%; P < .05). Heart rates of transgenic mice were higher than those of control mice during ISO and NE infusion. In three transgenic mice with heart rates held constant, LV dP/dt rose by 33 +/- 2% with ISO (0.02 micrograms/kg per minute) and by only 13 +/- 2% in three wild-type control mice (P < .01). NE (0.1 micrograms/kg per minute) also induced a greater effect on LV dP/dt in the three transgenic mice with heart rates held constant compared with three wild-type control mice (65 +/ 8% versus 28 +/- 4%, P < .05). Pathological and histological analyses of older transgenic mouse hearts (16.0 +/- 0.8 months old) revealed hypertrophy, degeneration, atrophy of cells, and replacement fibrosis reflected by significant increases in collagen volume in the subendocardium (5.2 +/- 1.4% versus 1.2 +/- 0.3%, P < .05) and in the cross-sectional area of myocytes (298 +/- 29 versus 187 +/- 12 micron2, P < .05) compared with control mouse hearts. These results suggest that Gsalpha overexpression enhances the efficacy of the beta-adrenergic receptor-Gs-adenylyl cyclase signaling pathway. This in turn leads to augmented inotropic and chronotropic responses to endogenous sympathetic stimulation. This action over the life of the animal results in myocardial damage characterized by cellular degeneration, necrosis, and replacement fibrosis, with the remaining cells undergoing compensatory hypertrophy. As a model, this transgenic mouse offers new insights into the mechanisms of cardiomyopathy and heart failure and provides a new tool for their study.

Animals↗
PubMed1995

Directed endothelial differentiation of cultured embryonic yolk sac cells in vivo provides a novel cell-based system for gene therapy.

Cultured murine yolk sac cells transfected with the cytomegalovirus immediate early promoter/human growth hormone (CMVIE-hGH) fusion gene, expressing high levels of hGH in culture, and suspended in Matrigel were subcutaneously (s.c.) injected into experimental mice. The injected cells were shown to form discrete vesicular structures within the Matrigel implant, suggesting directed differentiation of the embryonic yolk sac cells into endothelial tissue. Human growth hormone radioimmune assay of these mice showed sustained physiologically significant levels of hGH in their serum for beyond four months. These results confirmed that long-term cultured murine embryonic yolk sac cells can be induced to differentiate into endothelial cells both in vivo and in vitro and suggested a novel approach to the delivery to the circulation of therapeutic proteins for the treatment of inherited and acquired diseases.

Animals↗
PubMed1995

Overexpression of Gs alpha protein in the hearts of transgenic mice.

Alterations in beta-adrenergic receptor-Gs-adenylyl cyclase coupling underlie the reduced catecholamine responsiveness that is a hallmark of human and animal models of heart failure. To study the effect of altered expression of Gs alpha, we overexpressed the short isoform of Gs alpha in the hearts of transgenic mice, using a rat alpha-myosin heavy chain promoter. Gs alpha mRNA levels were increased selectively in the hearts of transgenic mice, with a level 38 times the control. Despite this marked increase in mRNA, Western blotting identified only a 2.8-fold increase in the content of the Gs alpha short isoform, whereas Gs activity was increased by 88%. The discrepancy between Gs alpha mRNA and Gs alpha protein levels suggests that the membrane content of Gs alpha is posttranscriptionally regulated. The steady-state adenylyl cyclase catalytic activity was not altered under either basal or stimulated conditions (GTP + isoproterenol, GTP gamma S, NaF, or forskolin). However, progress curve studies did show a significant decrease in the lag period necessary for GppNHp to stimulate adenylyl cyclase activity. Furthermore, the relative number of beta-adrenergic receptors binding agonist with high affinity was significantly increased. Our data demonstrate that a relatively small increase in the amount of the coupling protein Gs alpha can modify the rate of catalyst activation and the formation of agonist high affinity receptors.

Adenylyl Cyclases↗
PubMed1995

Amino acid residues in the third alpha-helix of growth hormone involved in growth promoting activity.

The third amphiphilic alpha-helix of GH has been found to be an important motif in the biological activities of the molecule. To further characterize this growth-promoting domain, three bovine (b) GH analogs were designed: one contained a scrambled third amphiphilic alpha-helix (SAH); a second contained a scrambled hydrophilic region of the helix (SAP); and a third contained a scrambled hydrophobic region of the helix (SAB). Transgenic mice that expressed these mutated bGH genes were produced. SAH transgenic mice displayed a phenotype identical to nontransgenic littermates. SAB transgenic mice grew slightly larger than nontransgenic littermates but remained smaller than bGH transgenic mice. On the other hand, SAP transgenic mice exhibited a dwarf phenotype. We subsequently generated individual amino acid substitutions in the hydrophilic region of the helix. The results from the growth rates of corresponding transgenic mice demonstrated that most bGH analogs with individual amino acid substitution within the third alpha-helix retained wild type-like growth-promoting activity except those with alterations at positions 115, 119, 122, and 123. Together these residues are predicted to form a cleft in the helix. To further substantiate the importance of the cleft, we deleted Gly 119 (delta 119). This resulting bGH analog was inactive in vivo as well as in in vitro assays. These results indicated that the primary structure of the third alpha-helix is critical for GH's growth-promoting activity and Gly 119 is a crucial amino acid in this region. Three adjacent amino acids, Asp 115, Ala 122, and Leu 123, also contribute to the growth-enhancing ability of the molecule.

Amino Acid Sequence↗
PubMed1995

A novel nonviral cytoplasmic gene expression system and its implications in cancer gene therapy.

We recently have developed a unique cytoplasmic transient gene expression system based on cotransfection of target cells with bacteriophage T7 RNA polymerase (RNAP) and plasmid DNA vectors containing a T7 autogene. Because this T7 system is self-initiating, self-maintaining, and requires no cellular factors for transcription, it is therefore likely to function in any mammalian cell with any gene both in vitro and, more importantly, in vivo. In this study we demonstrate that the T7 DNA vector and T7 RNAP could be efficiently codelivered to cultured cells by lipofection. Different target genes were expressed by the T7 system in a wide variety of mammalian cells including several tumor cell lines. Gene expression could be detected in more than 30% of the cells of some tumor cell lines transiently transfected by the T7 vector. Average activity of the reporter enzyme (luciferase) expressed by a transfected cell was relatively constant regardless of the cell line used. When a T7-luciferase vector was directly injected into various tissues of mice without the use of liposomes, luciferase activity could be found in the injected liver, muscle, brain and tail connective tissues. The luciferase levels expressed by the T7 system were found to be up to 200-fold higher, depending upon the injected tissues, than levels achieved with a traditional nuclear gene expression vector. Direct tumor injection with a T7-beta-galactosidase (beta-gal) construct resulted in beta-gal gene expression in tumor cells near the injection sites. In addition, direct injection of the T7 system in mice did not generate detectable quantities of antibodies against the T7 RNAP. These results suggest that this gene expression system may be useful in many different medical applications such as cancer gene therapies and DNA vaccination, where transient but rapid and efficient gene expression is required.

Animals↗
PubMed1994

A self-initiating eukaryotic transient gene expression system based on contransfection of bacteriophage T7 RNA polymerase and DNA vectors containing a T7 autogene.

A novel cytoplasmic gene expression system has been developed. This system differs from other expression systems in that it relies on the co-delivery of plasmid DNA and T7 RNA polymerase (RNAP) during transfection. The plasmid contains a T7 RNAP gene driven by the T7 promoter (T7 autogene) and a functional/reporter gene driven by another T7 promoter (T7T7/T7-gene construct). Once this DNA-enzyme complex is introduced into eukaryotic cells, the transcription of the T7 RNAP and the functional/reporter genes is initiated by the co-delivered T7 RNAP. The T7 RNAP, which is responsible for the initiation and maintenance of expression of both T7 and functional/reporter genes, is replenished by translation of newly synthesized T7 mRNA. This T7 system was designed in such a manner that the expression of the functional/reporter genes can occur in the cytoplasm and does not require any nuclear involvement. When transfected by either a pT7T7/T7Luc or a pT7T7/T7hGH plasmids with the cointroduced T7 RNAP, mouse L cells were found to express high levels of luciferase immediately after transfection, apparently due to the cytoplasmic gene expression; the expression of human growth hormone (hGH) could be sustained for at least 6 days. Both T7 and hGH mRNA were expressed by the cells transfected with pT7T7/T7hGH. These results suggest that this cytoplasmic expression system may be used for certain targets of somatic gene therapy.

Animals↗
PubMed1994

In vitro and in vivo studies of antagonistic effects of human growth hormone analogs.

A bovine growth hormone (bGH) analog, bGH-G119R, has been shown to act as a functional antagonist of GH activity both in vitro and in vivo. In the present study, human (h) GH analogs with alterations in the third alpha-helix (G120A, G120R) and N terminus (I4A) were generated. These two regions have been reported to form binding site 2 in hGH, which is involved in in vitro dimerization of the GH receptor (GHR). The biological activities of these hGH analogs were tested in vitro by a radioreceptor assay and an assay to test the ability of the molecules to induce tyrosine phosphorylation of a 93-kDa protein in a human lymphocyte line, IM-9. The growth rate of transgenic mice that express different hGH analogs was used as an in vivo test of the activity of the molecules. The results demonstrated that G120R is a potent hGH antagonist both in vitro and in vivo. Transgenic mice expressing G120R demonstrated a growth-suppressed phenotype. However, I4A, which has been demonstrated to be a potent inhibitor of in vitro GHR dimerization, exhibited full growth promoting activity in transgenic mice. Thus, the ability of hGH analogs to induce GHR dimerization in vitro and the ability to promote growth in vivo are not directly correlated.

Amino Acid Sequence↗
PubMed1994

Transgenic mice: a decade of progress in technology and research.

In little more than a decade, the techniques developed for altering the genetic makeup of laboratory and livestock animals and plants have changed the landscape of biological research. It is now possible to introduce virtually any cloned gene into the germ line and study the expression pattern and effects of the introduced gene, or transgene. This has allowed the extension of in vitro and in vivo cell-culture studies into whole animal systems in which the introduced gene is subject to all normal regulatory processes from the onset of development. Although there have been reports of foreign gene expression resulting from direct injection of DNA in animals (e.g., Wolff et al., 1990; Zhu et al., 1993), transgenic animals are the primary model system for examining molecular genetic phenomena in vivo.

Animals↗
PubMed1994

In vitro and in vivo development of bovine embryos from zygotes and 2-cell embryos microinjected with exogenous DNA.

The objectives of these experiments were: 1) to determine an effective culture method for production of transferable bovine embryos following exogenous DNA microinjection; 2) to determine the effect of these methods on the ability of the injected zygotes and 2-cell embryos to develop in vivo; and, 3) to compare development of embryos microinjected as zygotes or 2-cell embryos. DNA fragments encoding bovine growth hormone (bGH), bGH-10Delta6, and a bGH antagonist, bGH-M8 (5) were used. A total of 639 zygotes and 153 2-cell embryos were injected. Zygotes and 2-cell embryos microinjected with bGH-M8 were incubated for 6 days in oviducts of intermediate recipients (rabbits or sheep) or co-cultured in vitro with bovine oviduct epithelial cells. Zygotes and 2-cell embryos microinjected with bGH-10Delta6 were co-cultured in vitro only. The most effective method for the production of transferable bovine embryos following exogenous DNA microinjection was via in vitro co-culturing with bovine epithelial cells. For example, 32.3% of the bGH-M8 and 33.5% of the bGH-10Delta6 microinjected zygotes reached the morula/blastocyst stage while 48.4% and 63.0% of the 2-cell embryos injected with bGH-M8 and bGH-10Delta6, respectively, developed to the morula/blastocyst stage. The percentage of blastocysts obtained for control, non-injected zygotes and 2-cell embryos was 34.5% and 69.6%, respectively. The developmental rate to the morula/blastocyst stage was approximately 20% greater for embryos obtained from microinjected 2-cell embryos relative to microinjected zygotes. However, there was no significant difference in pregnancy rates following transfer of these blastocysts to cow uteri.

Journal Article↗
PubMed1994

Increased hypothalamic somatostatin expression in mice transgenic for bovine or human GH.

Acute studies of GH removal by hypophysectomy or GH replacement in adult rats have shown that GH has a positive influence on its hypothalamic inhibitory hormone somatostatin (SRIH). The present study was undertaken to assess the effect of lifelong exposure to elevated GH on the development and differentiation of SRIH-producing hypothalamic neurons, including comparison of differing GH levels and heterologous species of GH. Expression of somatostatin peptide and mRNA was evaluated using respective immunocytochemistry and in situ hybridization in brains of transgenic mice bearing constructs of either human (hGH) or bovine (bGH) linked to metallothionein (MT) promoter or bGH linked to phosphoenolpyruvate carboxykinase (PEPCK) promoter. Nontransgenic littermates served as controls. All transgenic constructs resulted in high levels of circulating heterologous GH and significantly elevated body weights. Both bGH levels and body weights were higher in PEPCK-bGH than in MT-bGH mice; mean weights were not different between MT-bGH and MT-hGH mice. Numbers of SRIH-immunoreactive neurons in the hypophysiotropic periventricular nucleus (PeN) of transgenic mice showed a two-fold increase (P < 0.01) relative to control animals; the number of SRIH-positive cells in the medial basal hypothalamus (MBH) was comparable for transgenic and control mice. Total SRIH mRNA in situ hybridization intensity also showed a two-fold increase (P < 0.05) in the PeN of all transgenic mice compared with controls, and was not elevated in the MBH. The higher levels of GH produced in PEPCK-bGH transgenic mice led to greater weight gain, but not to greater SRIH expression than in other GH-transgenic mice, suggesting that the increased SRIH cell number and mRNA in the PeN of MT-GH-transgenic mice may represent a plateau of maximal feedback stimulation. The results indicate that lifelong elevated heterologous GH in mice stimulates hypothalamic SRIH expression markedly. It is not known whether this mechanism is direct or indirect via a mediator of GH such as IGF, but the heterologous GH appears to be specific to these hypophysiotropic neurons.

Animals↗
PubMed1993

Application of biotechnology and transgenic animals toward the study of growth hormone.

Biotechnology has played a key role in our understanding of the molecular biology and physiology of growth hormone. The application of recombinant-DNA techniques and transgenic animals has resulted in the production of unlimited quantities of purified growth hormone and the development of growth hormone analogues. Ultimately, these analogues will be used to enhance growth and production characteristics in domestic livestock and treat certain growth and metabolic disorders in humans.

Animals↗
PubMed1993

Gonadotropin secretion, synthesis, and gene expression in two types of bovine growth hormone transgenic mice.

Expression of the mouse metallothionein-I (MT) promoter/bovine growth hormone (bGH) or the phosphoenolpyruvate carboxykinase (Pepck) promoter/bGH fusion genes in male transgenic mice is associated with alternations in adenohypophyseal function and fertility. To determine the effects of these gene constructs on gonadotropin synthesis and secretion, we have examined basal and GnRH-stimulated LH and FSH release in vitro using static incubations and perifusions of the pituitary; we have also examined pituitary content of LH, FSH, LH beta mRNA, and FSH beta mRNA in MT/bGH and Pepck/bGH transgenic mice as well as in normal mice. In addition, we have measured LH and FSH release from normal pituitaries transplanted under the kidney capsules of Pepck/bGH transgenic or normal mice. We found that in Pepck/bGH transgenic mice, pituitary contents of FSH and FSH beta mRNA were reduced, while FSH release in vitro in pituitary incubations and perifusions was increased. Steady-state levels of LH beta mRNA as well as LH responses to GnRH in perifusions were reduced; LH release in incubations and pituitary LH content were not changed; and basal LH secretion in perifusions was increased. In MT/bGH transgenic mice, in which peripheral bGH levels are much lower than in Pepck/bGH mice, similar trends were observed, but most of the apparent differences between transgenic and normal animals were not statistically significant. When normal pituitaries were transplanted under the kidney capsules of Pepck/bGH transgenic mice, the expected decrease in LH and FSH secretion was attenuated and the response to GnRH stimulation was lost.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Gonadotropin secretion, synthesis, and gene expression in human growth hormone transgenic mice and in Ames dwarf mice.

The expression of the mouse metallothionein-I (MT) promoter/human GH (hGH) fusion gene leads to reduced fertility and increased plasma LH levels in male MT/hGH transgenic mice. To determine the effects of hGH on gonadotropin synthesis and release, we have examined basal and GnRH stimulated LH and FSH release in pituitary incubations and perifusions; and pituitary content of LH, FSH, LH-beta messenger RNA (mRNA), and FSH-beta mRNA in MT/hGH transgenic males and in their normal littermates. For comparison, similar studies were performed in GH and PRL deficient Ames dwarf mice in which plasma gonadotropin levels are known to be reduced. We have also measured the LH and FSH release from normal pituitaries transplanted under the kidney capsule of MT/hGH transgenic or normal mice. We found that in MT/hGH transgenic mice, there were parallel increases in unstimulated and GnRH stimulated LH release from pituitary incubation, in pituitary LH content and in LH-beta mRNA levels. In pituitary perifusion, the basal LH secretion was elevated, whereas LH responses to GnRH pulses were not altered. In transgenic males, FSH-beta mRNA was increased, whereas basal and GnRH-stimulated FSH release and pituitary FSH content did not differ from their normal controls. After normal pituitaries were transplanted to kidney capsules of MT/hGH transgenic mice, the expected decrease in LH and FSH secretion was attenuated and the responsiveness to GnRH stimulation was maintained. In Ames dwarf mice, all gonadotropin content and release, as well as pituitary beta-mRNA contents were decreased. We conclude that in MT/hGH transgenic mice, the expression of LH-beta and FSH-beta gene is increased. In addition, there is a translational or posttranslational inhibitory influence on FSH synthesis. Although our previous studies suggest that the effects of hGH gene expression on LH and FSH release are exerted primarily at the hypothalamic level, the present results suggest existence of GnRH unrelated peripheral factors which can directly stimulate pituitary gonadotropin synthesis and release. In Ames dwarf mice, the deficiency of GH and PRL, as well as TSH, is associated with decreased LH-beta and FSH-beta gene expression which may account for the reduction in plasma gonadotropin levels.

Animals↗