scholarly journals Effects of Mutant Ubiquitin on ts1 Retrovirus-Mediated Neuropathology

2003 ◽  
Vol 77 (13) ◽  
pp. 7193-7201 ◽  
Author(s):  
Mei Zhang ◽  
Sherry Thurig ◽  
Maria Tsirigotis ◽  
Paul K. Y. Wong ◽  
Kenneth R. Reuhl ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Therapeutic Potential ◽  
Leukemia Virus ◽  
Lewy Bodies ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Spongiform Encephalopathy ◽  
Temperature Sensitive ◽  
Transgenic Mouse Line

ABSTRACT ts1 is a temperature-sensitive mutant of Moloney murine leukemia virus that induces a rapid spongiform encephalopathy in mice infected as newborns. The pathological features include the formation of ubiquitinated inclusions resembling Lewy bodies. To determine how perturbation of the ubiquitin-proteasome pathway might affect ts1-mediated neurodegeneration, the virus was introduced into transgenic mice in which the assembly of ubiquitin chains was compromised by the expression of dominant-negative mutant ubiquitin. The onset of symptoms was greatly delayed in a transgenic mouse line expressing K48R mutant ubiquitin; no such delay was observed in mice expressing a wild-type ubiquitin transgene or K63R mutant ubiquitin. The extended latency was found to correlate with a delayed increase in viral titers. Pathological findings in K48R transgenic mice at 60 days were found to be similar to those in the other strains at 30 days, suggesting that while delayed, the neurodegenerative process in K48R mice was otherwise similar. These data demonstrate the sensitivity of retroviral replication to the partial disruption of ubiquitin-mediated proteolysis in vivo, a finding that may have therapeutic potential.

Overexpression of sphingosine kinase 1 is an oncogenic event in erythroleukemic progression

Blood ◽  
2005 ◽  
Vol 106 (5) ◽  
pp. 1808-1816 ◽  
Author(s):  
Erwan Le Scolan ◽  
Dimitri Pchejetski ◽  
Yoshiko Banno ◽  
Nicole Denis ◽  
Patrick Mayeux ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Molecular Mechanisms ◽  
Sphingosine Kinase ◽  
Dominant Negative ◽  
Recurrent Event ◽  
Dominant Negative Mutant ◽  
Apoptosis Resistance ◽  
Kinase Gene ◽  
Extracellular Signal

Abstract The erythroleukemia developed by spi-1/PU.1-transgenic mice is a model of multistage oncogenic process. Isolation of tumor cells representing discrete stages of leukemic progression enables the dissection of some of the critical events required for malignant transformation. To elucidate the molecular mechanisms of multistage leukemogenesis, we developed a microarray transcriptome analysis of nontumorigenic (HS1) and tumorigenic (HS2) proerythroblasts from spi-1-transgenic mice. The data show that transcriptional up-regulation of the sphingosine kinase gene (SPHK1) is a recurrent event associated with the tumorigenic phenotype of these transgenic proerythroblasts. SPHK1 is an enzyme of the metabolism of sphingolipids, which are essential in several biologic processes, including cell proliferation and apoptosis. HS1 erythroleukemic cells engineered to overexpress the SPHK1 protein exhibited growth proliferative advantage, increased clonogenicity, and resistance to apoptosis in reduced serum level by a mechanism involving activation of the extracellular signal-related kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. In addition, SPHK1-overexpressing HS1 cells acquired tumorigenicity when engrafted in vivo. Finally, enforced expression of a dominant-negative mutant of SPHK1 in HS2 tumorigenic cells or treatment with a pharmacologic inhibitor reduced both cell growth and apoptosis resistance. Altogether, these data suggest that overexpression of the sphingosine kinase may represent an oncogenic event during the multistep progression of an erythroleukemia. (Blood. 2005;106:1808-1816)

scholarly journals Expression of a Dominant Negative Mutant of Interleukin-1β Converting Enzyme in Transgenic Mice Prevents Neuronal Cell Death Induced by Trophic Factor Withdrawal and Ischemic Brain Injury

1997 ◽  
Vol 185 (5) ◽  
pp. 933-940 ◽  
Author(s):  
Robert M. Friedlander ◽  
Valeria Gagliardini ◽  
Hideaki Hara ◽  
Klaus B. Fink ◽  
Weiwei Li ◽  
...  
Keyword(s):  
Brain Injury ◽  
Transgenic Mice ◽  
Dorsal Root ◽  
Neuron Specific Enolase ◽  
Dominant Negative ◽  
Trophic Factor ◽  
Dominant Negative Mutant ◽  
Lacz Gene ◽  
Converting Enzyme ◽  
Induced Apoptosis

To explore the role of the interleukin (IL)-1β converting enzyme (ICE) in neuronal apoptosis, we designed a mutant ICE gene (C285G) that acts as a dominant negative ICE inhibitor. Microinjection of the mutant ICE gene into embryonal chicken dorsal root ganglial neurons inhibits trophic factor withdrawal–induced apoptosis. Transgenic mice expressing the fused mutant ICE-lacZ gene under the control of the neuron specific enolase promoter appeared neurologically normal. These mice are deficient in processing pro–IL-1β, indicating that mutant ICEC285G blocks ICE function. Dorsal root ganglial neurons isolated from transgenic mice were resistant to trophic factor withdrawal–induced apoptosis. In addition, the neurons isolated from newborn ICE knockout mice are similarly resistant to trophic factor withdrawal–induced apoptosis. After permanent focal ischemia by middle cerebral artery occlusion, the mutant ICEC285G transgenic mice show significantly reduced brain injury as well as less behavioral deficits when compared to the wild-type controls. Since ICE is the only enzyme with IL-1β convertase activity in mice, our data indicates that the mutant ICEC285G inhibits ICE, and hence mature IL-1β production, and through this mechanism, at least in part, inhibits apoptosis. Our data suggest that genetic manipulation using ICE family dominant negative inhibitors can ameliorate the extent of ischemia-induced brain injury and preserve neurological function.

scholarly journals Nuclear Import of IκBα Is Accomplished by a Ran-Independent Transport Pathway

2000 ◽  
Vol 20 (5) ◽  
pp. 1571-1582 ◽  
Author(s):  
Shrikesh Sachdev ◽  
Sriparna Bagchi ◽  
Donna D. Zhang ◽  
Angela C. Mings ◽  
Mark Hannink
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Nuclear Pore ◽  
Transport Pathway ◽  
Repeat Domain ◽  
Nuclear Export Receptor

ABSTRACT The inhibitor of kappa B alpha (IκBα) protein is able to shuttle between the cytoplasm and the nucleus. We have utilized a combination of in vivo and in vitro approaches to provide mechanistic insight into nucleocytoplasmic shuttling by IκBα. IκBα contains multiple functional domains that contribute to shuttling of IκBα between the cytoplasm and the nucleus. Nuclear import of IκBα is mediated by the central ankyrin repeat domain. Similar to previously described nuclear import pathways, nuclear import of IκBα is temperature and ATP dependent and is blocked by a dominant-negative mutant of importin β. However, in contrast to classical nuclear import pathways, nuclear import of IκBα is independent of soluble cytosolic factors and is not blocked by the dominant-negative RanQ69L protein. Nuclear export of IκBα is mediated by an N-terminal nuclear export sequence. Nuclear export of IκBα requires the CRM1 nuclear export receptor and is blocked by the dominant-negative RanQ69L protein. Our results are consistent with a model in which nuclear import of IκBα is mediated through direct interactions with components of the nuclear pore complex, while nuclear export of IκBα is mediated via a CRM1-dependent pathway.

Abstract 331: HCN Overexpression in Failing Heart Possibly Contributes to Ventricular Arrhythmias

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Yoshihiro Kuwabara ◽  
Koichiro Kuwabara ◽  
Makoto Takano ◽  
Hideyuki Kinoshita ◽  
Yasuaki Nakagawa ◽  
...  
Keyword(s):  
Heart Failure ◽  
Transgenic Mice ◽  
Ventricular Arrhythmias ◽  
Ventricular Myocytes ◽  
Effective Means ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Hcn Channels ◽  
Arrhythmic Death ◽  
Significant Difference

Accumulating evidence suggests increased ventricular expression of hyperpolarization-activated cation channels (HCNs) in hypertrophied and failing hearts contributes to the induction of arrhythmias. In this study, we addressed the capacity of HCNs blockade to prevent lethal arrhythmias associated with heart failure. Transgenic mice expressing a dominant-negative mutant of neuron-restrictive silencer factor in a cardiac-specific manner (dnNRSF-Tg) exhibited dilated cardiomyopathy and sudden arrythmic death with an increase in ventricular HCNs expression, which are potentially responsible for the observed lethal arrhythmias. Ivabradine (Iva), a specific HCN channel inhibitor, significantly improved the survival among dnNRSF-Tg mice. Though echocardiographic, hemodynamic, and histological analyses showed no significant difference between Iva and control, ECG telemetric monitoring showed the significant reduction of arrhythmias in dnNRSF-Tg mice treated with Iva (VT; Iva 19/h, control 92/h ; p<0.05), suggesting that Iva improved the survival by preventing lethal arrhythmias. We also found that the transgenic mice overexpressing HCN2 specifically in the heart (HCN2-Tg) are susceptible to ventricular arrhythmias induced by chronic isoproterenol infusion. In isolated ventricular myocytes from HCN2-Tg, but not in those from wild type mice, isoproterenol induced abnormal spontaneous action potentials, which were suppressed with Iva. Our findings suggest that increased ventricular expression of HCN channels possibly contributes to the ventricular arrhythmias, and HCN channels blockade may represent a new and effective means of preventing sudden arrhythmic death in patients with heart failure.

Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation

Development ◽  
1996 ◽  
Vol 122 (10) ◽  
pp. 3173-3183 ◽  
Author(s):  
K.L. Kroll ◽  
E. Amaya
Keyword(s):  
Large Scale ◽  
Neural Induction ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mesoderm Induction ◽  
Fgf Signaling ◽  
Fgf Receptor ◽  
Transgenic Expression

We have developed a simple approach for large-scale transgenesis in Xenopus laevis embryos and have used this method to identify in vivo requirements for FGF signaling during gastrulation. Plasmids are introduced into decondensed sperm nuclei in vitro using restriction enzyme-mediated integration (REMI). Transplantation of these nuclei into unfertilized eggs yields hundreds of normal, diploid embryos per day which develop to advanced stages and express integrated plasmids nonmosaically. Transgenic expression of a dominant negative mutant of the FGF receptor (XFD) after the mid-blastula stage uncouples mesoderm induction, which is normal, from maintenance of mesodermal markers, which is lost during gastrulation. By contrast, embryos expressing XFD contain well-patterned nervous systems despite a putative role for FGF in neural induction.

scholarly journals Keratin 18 is an integral part of the intermediate filament network in murine skeletal muscle

2020 ◽  
Vol 318 (1) ◽  
pp. C215-C224 ◽  
Author(s):  
Joaquin M. Muriel ◽  
Andrea O’Neill ◽  
Jaclyn P. Kerr ◽  
Emily Kleinhans-Welte ◽  
Richard M. Lovering ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Isometric Force ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Type I ◽  
Mrna Levels ◽  
Force Transmission ◽  
Keratin 18 ◽  
Murine Skeletal Muscle

Intermediate filaments (IFs) contribute to force transmission, cellular integrity, and signaling in skeletal muscle. We previously identified keratin 19 (Krt19) as a muscle IF protein. We now report the presence of a second type I muscle keratin, Krt18. Krt18 mRNA levels are about half those for Krt19 and only 1:1,000th those for desmin; the protein was nevertheless detectable in immunoblots. Muscle function, measured by maximal isometric force in vivo, was moderately compromised in Krt18-knockout ( Krt18-KO) or dominant-negative mutant mice ( Krt18 DN), but structure was unaltered. Exogenous Krt18, introduced by electroporation, was localized in a reticulum around the contractile apparatus in wild-type muscle and to a lesser extent in muscle lacking Krt19 or desmin or both proteins. Exogenous Krt19, which was either reticular or aggregated in controls, became reticular more frequently in Krt19-null than in Krt18-null, desmin-null, or double-null muscles. Desmin was assembled into the reticulum normally in all genotypes. Notably, all three IF proteins appeared in overlapping reticular structures. We assessed the effect of Krt18 on susceptibility to injury in vivo by electroporating siRNA into tibialis anterior (TA) muscles of control and Krt19-KO mice and testing 2 wk later. Results showed a 33% strength deficit (reduction in maximal torque after injury) compared with siRNA-treated controls. Conversely, electroporation of siRNA to Krt19 into Krt18-null TA yielded a strength deficit of 18% after injury compared with controls. Our results suggest that Krt18 plays a complementary role to Krt19 in skeletal muscle in both assembling keratin-based filaments and transducing contractile force.

scholarly journals Gap Junction–mediated Cell–Cell Communication Modulates Mouse Neural Crest Migration

1998 ◽  
Vol 143 (6) ◽  
pp. 1725-1734 ◽  
Author(s):  
G.Y. Huang ◽  
E.S. Cooper ◽  
K. Waldo ◽  
M.L. Kirby ◽  
N.B. Gilula ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Neural Crest ◽  
Gap Junction ◽  
Heart Defects ◽  
Neural Crest Cells ◽  
Dominant Negative ◽  
Cardiac Neural Crest ◽  
Gap Junction Communication ◽  
Neural Crest Migration

Previous studies showed that conotruncal heart malformations can arise with the increase or decrease in α1 connexin function in neural crest cells. To elucidate the possible basis for the quantitative requirement for α1 connexin gap junctions in cardiac development, a neural crest outgrowth culture system was used to examine migration of neural crest cells derived from CMV43 transgenic embryos overexpressing α1 connexins, and from α1 connexin knockout (KO) mice and FC transgenic mice expressing a dominant-negative α1 connexin fusion protein. These studies showed that the migration rate of cardiac neural crest was increased in the CMV43 embryos, but decreased in the FC transgenic and α1 connexin KO embryos. Migration changes occurred in step with connexin gene or transgene dosage in the homozygous vs. hemizygous α1 connexin KO and CMV43 embryos, respectively. Dye coupling analysis in neural crest cells in the outgrowth cultures and also in the living embryos showed an elevation of gap junction communication in the CMV43 transgenic mice, while a reduction was observed in the FC transgenic and α1 connexin KO mice. Further analysis using oleamide to downregulate gap junction communication in nontransgenic outgrowth cultures showed that this independent method of reducing gap junction communication in cardiac crest cells also resulted in a reduction in the rate of crest migration. To determine the possible relevance of these findings to neural crest migration in vivo, a lacZ transgene was used to visualize the distribution of cardiac neural crest cells in the outflow tract. These studies showed more lacZ-positive cells in the outflow septum in the CMV43 transgenic mice, while a reduction was observed in the α1 connexin KO mice. Surprisingly, this was accompanied by cell proliferation changes, not in the cardiac neural crest cells, but in the myocardium— an elevation in the CMV43 mice vs. a reduction in the α1 connexin KO mice. The latter observation suggests that cardiac neural crest cells may have a role in modulating growth and development of non–neural crest– derived tissues. Overall, these findings suggest that gap junction communication mediated by α1 connexins plays an important role in cardiac neural crest migration. Furthermore, they indicate that cardiac neural crest perturbation is the likely underlying cause for heart defects in mice with the gain or loss of α1 connexin function.

scholarly journals Therapeutic Effect of a Gag-Nuclease Fusion Protein against Retroviral Infection In Vivo

2001 ◽  
Vol 75 (15) ◽  
pp. 7030-7041 ◽  
Author(s):  
Gerald Schumann ◽  
Monika Hermankova ◽  
Keith Cannon ◽  
Joseph L. Mankowski ◽  
Jef D. Boeke
Keyword(s):  
Transgenic Mice ◽  
Fusion Protein ◽  
Leukemia Virus ◽  
Coat Proteins ◽  
Antiviral Protein ◽  
Combination Drug ◽  
Retroviral Infection ◽  
Gag Protein ◽  
Therapeutic Benefits

ABSTRACT Recently, remarkable progress has been made in developing effective combination drug therapies that can control but not cure retroviral replication. Even when effective, these drug regimens are toxic, they require demanding administration schedules, and resistant viruses can emerge. Thus the need for new gene-based therapies continues. In one such approach, capsid-targeted viral inactivation (CTVI), nucleases fused to viral coat proteins are expressed in infected cells and become incorporated during virion assembly. CTVI can eliminate infectious murine retrovirus titer in tissue culture. Here we describe transgenic mice expressing fusions of the Moloney murine leukemia virus (Mo-MuLV) Gag protein to staphylococcal nuclease. This work tests the protective effect and demonstrates in vivo proof-of-principle of CTVI in transgenic mice expressing endogenous proviral copies of Mo-MuLV. The antiviral protein-expressing mice are phenotypically normal, attesting to the lack of toxicity of the fusion protein. The Mo-MuLV infection was much less virulent in transgenic littermates than in nontransgenic littermates. Gag-nuclease expression reduced infectious titers in blood up to 10-fold, decreased splenomegaly and leukemic infiltration, and increased life spans up to 2.5-fold in transgenic relative to nontransgenic infected animals. These results suggest that gene therapies based on similar fusion proteins, designed to attack human immunodeficiency virus or other retroviruses, could provide substantial therapeutic benefits.

scholarly journals La Autoantigen Is Necessary for Optimal Function of the Poliovirus and Hepatitis C Virus Internal Ribosome Entry Site In Vivo and In Vitro

2004 ◽  
Vol 24 (15) ◽  
pp. 6861-6870 ◽  
Author(s):  
Mauro Costa-Mattioli ◽  
Yuri Svitkin ◽  
Nahum Sonenberg
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Ribosomal Subunit ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Entry Site ◽  
Internal Ribosome Entry

ABSTRACT Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5′ untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.

scholarly journals Perturbation of β1-Integrin Function in Involuting Mammary Gland Results in Premature Dedifferentiation of Secretory Epithelial Cells

2002 ◽  
Vol 13 (10) ◽  
pp. 3521-3531 ◽  
Author(s):  
Marisa M. Faraldo ◽  
Marie-Ange Deugnier ◽  
Sylvie Tlouzeau ◽  
Jean Paul Thiery ◽  
Marina A. Glukhova
Keyword(s):  
Mammary Gland ◽  
Epithelial Cells ◽  
Nuclear Factor Κb ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Dominant Negative Mutant ◽  
Β1 Integrin ◽  
Mrna Levels ◽  
Pregnancy And Lactation

To study the mechanism of β1-integrin function in vivo, we have generated transgenic mouse expressing a dominant negative mutant of β1-integrin under the control of mouse mammary tumor virus (MMTV) promoter (MMTV-β1-cyto). Mammary glands from MMTV-β1-cyto transgenic females present significant growth defects during pregnancy and lactation and impaired differentiation of secretory epithelial cells at the onset of lactation. We report herein that perturbation of β1-integrin function in involuting mammary gland induced precocious dedifferentiation of the secretory epithelium, as shown by the premature decrease in β-casein and whey acidic protein mRNA levels, accompanied by inactivation of STAT5, a transcription factor essential for mammary gland development and up-regulation of nuclear factor-κB, a negative regulator of STAT5 signaling. This is the first study demonstrating in vivo that cell–extracellular matrix interactions involving β1-integrins play an important role in the control of milk gene transcription and in the maintenance of the mammary epithelial cell differentiated state.

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