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Biomedical subjects

E F Wagner

Publications and source records attributed to E F Wagner.

At least 91 records · Page 5Linked to original sources
PubMed1995

Activation of HIV transcription by human foamy virus in transgenic mice.

BACKGROUND: Although infection by HIV-1 is the primary cause of AIDS, cofactorial agents of an infectious nature may be involved in the pathogenesis of the disease. The present work addresses the cofactorial potential of human foamy virus (HFV) in AIDS. It has been suggested that HFV seroprevalence reaches 5% in East Africa, and HFV seroprevalence in East African patients suffering from AIDS and AIDS-related complex may be as high as 20%. Although the pathogenic potential of HFV in humans has not yet been investigated in detail, HFV transgenic mice develop an encephalopathy reminiscent of some of the features of HIV-associated brain diseases. EXPERIMENTAL DESIGN: We set out to investigate the possibility that the regulatory genes of HFV may act as transcriptional cofactors of HIV. To study the effects of bel1, the transcriptional activator of HFV, on the HIV-1 LTR, we generated double transgenic mice for bel1 and for the HIV-1 LTR linked to a lacZ reporter gene. Moreover, to identify the cis-acting elements mediating bel1 action on the HIV LTR, we analyzed the consequences of deletions in the negative regulatory element or in the NF-kappa B binding sites. RESULTS: We demonstrate that bel1 is capable of activating the transcription of HIV-1 LTR in vivo. Such transactivational activity, however, was observed exclusively in a subset of hippocampal neurons, whereas cortical neurons expressing bel1 did not show transactivation. Transactivation was completely abolished by the deletion of the NF-kappa B binding sites. In contrast, deletion of the negative regulatory element region seems to enhance transactivation of bel1 on HIV-1 LTR over a prolonged period of time. CONCLUSIONS: Our study indicates that transcriptional transactivation of HIV-1 by HFV can be accomplished in vivo and is dependent on NF-kappa B binding sequences. Therefore, transcriptional transactivation is a potential mechanism of cooperation between HFV and HIV. It is conceivable that this phenomenon attains clinical significance in a situation of coinfection with both viruses.

Animals↗
PubMed1995

Expression of interstitial collagenase during skeletal development of the mouse is restricted to osteoblast-like cells and hypertrophic chondrocytes.

We determined the expression pattern of the matrix metalloproteinase interstitial collagenase (MMP-1) during mouse embryo development using in situ hybridization and immunohistochemistry. Localized MMP-1 mRNA was first detected at 14.5 days postconceptus. The spatial and temporal expression was restricted to areas of endochondral and intramembranous bone formation, such as in the mandibula, maxilla, clavicle, scapula, in the vertebrae, and in the dorsal, but not the ventral part of the ribs. The highest levels of MMP-1 transcripts and MMP-1 protein were found in the metaphyses and diaphyses of the long bones. MMP-1 was expressed by hypertrophic chondrocytes and by osteoblastic cells localized along the newly formed bone trabeculae. No expression was detected in osteoclasts. Two other related members of the MMP family, stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10), were not expressed during days 7.5 and 16.5 of mouse embryogenesis. The tissue-specific expression of MMP-1 and the exclusive ability of interstitial collagenase to digest native collagen of types I, II, III, and X, the major components of bone, cartilage, and tendon, strongly suggests an important and specific function of this enzyme in bone development and remodeling.

Animals↗
PubMed1994

Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP.

The Pax5 gene, coding for the transcription factor BSAP, was mutated in the mouse germline by targeted disruption. Homozygous mutant mice were born alive, became growth retarded, and usually died within three weeks. About 5% of mutants survived to adulthood and were fertile, but severely runted. Morphogenesis of the posterior midbrain was affected as early as embryonic day 16.5, leading to a reduction of the inferior colliculus near the midline and to altered foliation of the anterior cerebellum. Moreover, all mutants failed to produce small pre-B, B, and plasma cells owing to a complete arrest of B cell development at an early precursor stage. These data define a key role for Pax5 in early B lymphopoiesis and midbrain patterning.

Animals↗
PubMed1994

Regulation of embryonic growth and lysosomal targeting by the imprinted Igf2/Mpr gene.

The receptor for insulin-like growth factor type 2, also known as the cation-independent mannose-6-phosphate receptor (Igf2/Mpr), is a multifunctional receptor thought to play a role in lysosomal targeting, cell growth and signal transduction. Igf2/Mpr has been mapped to the mouse Tme locus and shown to be an imprinted gene, which further suggests a role in embryonic growth regulation. To define the functions of Igf2/Mpr, we have generated mice lacking this gene. We report here that maternal inheritance of an Igf2/Mpr null allele (-/+) as well as homozygosity for the inactive allele (-/-) is generally lethal at birth and mutants are about 30% larger, indicating that maternal expression of Igf2/Mpr is essential for late embryonic development and growth regulation. The phenotype is probably caused by an excess of Igf2 because the introduction of an Igf2 null allele rescued the Igf2/Mpr mutant mice. Mutant mice also have organ and skeletal abnormalities and missort mannose-6-phosphate-tagged proteins. A few (-/+) mice reactivated their paternal Igf2/Mpr allele in some tissues and survived to adults. But no (-/-) mice survived, indicating a role for the reactivated paternal allele in postnatal survival.

Alleles↗
PubMed1994

c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling.

Mice lacking the proto-oncogene c-fos develop the bone disease osteopetrosis. Fos mutant mice were found to have a block in the differentiation of bone-resorbing osteoclasts that was intrinsic to hematopoietic cells. Bone marrow transplantation rescued the osteopetrosis, and ectopic c-fos expression overcame this differentiation block. The lack of Fos also caused a lineage shift between osteoclasts and macrophages that resulted in increased numbers of bone marrow macrophages. These results identify Fos as a key regulator of osteoclast-macrophage lineage determination in vivo and provide insights into the molecular mechanisms underlying metabolic bone diseases.

Animals↗
PubMed1994

Down-regulation of wild-type p53 activity interferes with apoptosis of IL-3-dependent hematopoietic cells following IL-3 withdrawal.

Overexpression of wild-type p53 in p53-deficient leukemic cells induces apoptosis, which can be inhibited by hematopoietic survival factors. This suggests that p53 may contribute to survival factor dependence. To assess the role of wild-type p53 in mediating apoptosis following survival factor withdrawal, we interfered with endogenous p53 activity in interleukin-3 (IL-3)-dependent cells. Extended survival without IL-3 was conferred by recombinant retroviruses encoding either a full-length p53 mutant or a C-terminal p53 miniprotein, both of which can act as negative-dominant inhibitors of wild-type p53. On the other hand, excess wild-type p53 activity failed to elicit apoptosis as long as IL-3 was present. We propose that p53 is a positive, though not exclusive, mediator of survival factor dependence in hematopoietic cells.

Animals↗
PubMed1994

Endothelial cell transformation by polyomavirus middle T antigen in mice lacking Src-related kinases.

BACKGROUND: Expression of polyomavirus middle T antigen (PymT) rapidly induces endothelial tumors (hemangiomas) in mice, with an apparent single rate-limiting step. Because activation of Src-like kinases is thought to be an important component of PymT-induced transformation, we have analyzed the functional requirement for individual kinases in this process. This type of analysis has only recently become possible, with the generation of 'gene knock-out' mice lacking each of the kinase genes src, fyn and yes. RESULTS: Hemangiomas develop efficiently in newborn mice lacking either src, fyn or yes after inoculation with a PymT-transducing retrovirus. In src- and fyn-deficient mice, the kinetics of induction and the histological properties of the tumors were indistinguishable from those in wild-type mice. In contrast, a reduced number of tumors arose in yes-deficient mice, with a significantly longer latency period. Transformed endothelial cell lines derived from the induced hemangiomas, however, did not differ in their morphological and tumorigenic properties from cell lines established previously from wild-type mice. Biochemical analysis of complexes between PymT and the Src-related kinases in these cell lines suggests that the Yes kinase is responsible for a significant amount of the PymT-associated kinase activity in transformed endothelial cells. CONCLUSION: We have demonstrated that inactivation of a single tyrosine kinase of the Src family in endothelial cells does not abrogate PymT-induced hemangioma formation. As the remaining kinases do not compensate for the absence of a family member by elevated kinase activity, the loss--which affects the transformation process to varying degrees--can be studied in this model system. Our studies suggest that the PymT-Yes kinase complex plays a major role in the tumor-initiating action of PymT.

Animals↗
PubMed1994

Generation of normal T and B lymphocytes by c-jun deficient embryonic stem cells.

To determine the potential roles of c-jun in lymphocyte development, we generated somatic chimeric mice by injecting homozygous c-jun mutant embryonic stem (ES) cells into blastocysts from recombination activating gene-2 (RAG-2)-deficient mice. Chimeric mice had poor restoration of thymocytes, but contained substantial numbers of mature T and B lymphocytes in the periphery. Stimulation of c-jun-/- B cells resulted in normal levels of proliferation and immunoglobulin secretion. Likewise, stimulation of c-jun-/- T cells resulted in essentially normal levels of IL-2R alpha expression, IL-2 secretion, and proliferation. We further showed that the relatively normal activation responses of the c-jun-/- T cells probably results from the fact that other members of the Jun family contribute to the bulk of the activator protein-1 (AP-1) complexes in normal T cells and, as a result, AP-1 complexes are found at relatively normal levels in c-jun-/- T cells.

Animals↗
PubMed1994

Degeneration of neural cells in the central nervous system of mice deficient in the gene for the adhesion molecule on Glia, the beta 2 subunit of murine Na,K-ATPase.

We generated mice, null mutant in the adhesion molecule on glia (AMOG), the beta 2 subunit of the murine Na,K-ATPase gene. These mice exhibit motor incoordination at 15 d of age, subsequently tremor and paralysis of extremities, and die at 17-18 d after birth. At these ages, the mutants have enlarged ventricles, degenerating photoreceptor cells, and swelling and degeneration of astrocytic endfeet, leading to vacuoles adjoining capillaries of brain stem, thalamus, striatum, and spinal cord. In tissue homogenates from entire brains of 16-17-d-old mutants, Na,K-ATPase activity and expression of the beta 1 subunit of the Na,K-ATPase and of the neural adhesion molecules L1, N-CAM, and MAG appear normal. We suggest that the mutant phenotype can be related primarily to reduced pump activity, with neural degeneration as a possible consequence of osmotic imbalance.

Adenosine Triphosphatases↗
PubMed1994

Mice lacking c-fos have normal hematopoietic stem cells but exhibit altered B-cell differentiation due to an impaired bone marrow environment.

Mice lacking c-fos develop severe osteopetrosis with deficiencies in bone remodeling and exhibit extramedullary hematopoiesis, thymic atrophy, and altered B-cell development. In this study, we have used these mice to characterize in detail the developmental potential of hematopoietic stem cells lacking c-fos and to analyze how the lymphoid differentiation is altered. In c-fos -/- mice, B-cell numbers are reduced in the spleen, lymph nodes, and the peripheral blood as a result of a marked reduction (> 90%) in the number of clonogenic B-cell precursors. In contrast, the number and lineage distribution of myeloid progenitor cells are not affected. The thymic defects observed in a large number of these mice correlate with their health status, suggesting that this may be an indirect effect of the c-fos mutation. In vitro differentiation and bone marrow reconstitution experiments demonstrated that hematopoietic stem cells lacking c-fos can give rise to all mature myeloid as well as lymphoid cells, suggesting that the observed B lymphopenia in the mutant mice is due to an altered environment. Transplantation of wild-type bone marrow cells into newborn mutant mice resulted in the establishment of a bone marrow space and subsequent correction of the B-cell defect. These results demonstrate that hematopoietic stem cells lacking Fos have full developmental potential and that the observed defect in B-cell development is most likely due to the impaired bone marrow environment as a consequence of osteopetrosis.

Animals↗
PubMed1994

Oncogenes in the study of endothelial cell growth and differentiation.

During embryogenesis endothelial cells differentiate from mesodermal blood islands, proliferate and form new blood vessels throughout embryonic and early postnatal life by the processes of vasculogenesis and angiogenesis. Proliferation then ceases and is very low in the adult, although it resumes under certain physiological and pathological conditions, such as wound healing, tumor growth and hemangiomatous diseases. Expression of the polyoma middle T (PymT) oncogene in mouse endothelial cells leads to their rapid transformation and to the development of hemangiomas. These endothelial tumors allow the establishment of endothelioma (End) cell lines, which resemble normal endothelial cells yet exhibit a drastically altered proteolytic activity. The specific effects of PymT on the growth of endothelial cells appear in part to be mediated through the activation of cellular tyrosine kinases.

Animals↗
PubMed1994

Decreased rhodopsin regeneration in diabetic mouse eyes.

PURPOSE: To evaluate the effect of diabetes on rhodopsin regeneration in the excised mouse eye. METHODS: A superfused excised mouse eye preparation that exhibits rhodopsin regeneration after moderate bleaches and that is responsive to the composition of the perfusate was used. Diabetes was induced in albino mice (BALB/c) with the diabetogenic agent streptozotocin. Absorption spectrophotometry of the excised eye was used to monitor rhodopsin concentrations. RESULTS: Significant reductions in rhodopsin regeneration were observed in diabetic mice. Severely diabetic mice exhibited only 64% and 55% regeneration (at perfusate glucose levels of 5.1 mM and 10 mM, respectively), and moderately diabetic mice exhibited 74% and 73% regeneration, compared to the greater than 100% regeneration observed in nondiabetic mice. Glucose perfusate concentration has a major effect on rhodopsin regeneration. Lower concentrations of perfusate glucose (3 mM) reduced the amount of rhodopsin regeneration in both nondiabetic mice and diabetic mice. The diabetic mice seemed to tolerate higher concentrations of perfusate glucose (20 mM) better than the nondiabetic mice. Neither correction for osmolarity nor substitution with a nonglycolytic substrate increased the amount of rhodopsin regeneration in the diabetic mice. CONCLUSIONS: Diabetes reduced the amount of rhodopsin regeneration that followed moderate bleaches in excised mouse eyes. The data suggest that some process or processes associated with rhodopsin regeneration have been affected in the diabetic.

Animals↗
PubMed1993

c-jun is essential for normal mouse development and hepatogenesis.

The proto-oncogene c-jun is the cellular homologue of v-jun, the transforming oncogene of the avian sarcoma virus 17 (ref. 1). c-jun encodes one major component of the AP-1 transcription factor complex and is expressed in many organs during mouse development and in the adult. Because of its rapid induction in cells following growth stimulation and the presence of AP-1 binding sites in the promoter regions of many genes, the c-Jun protein is thought to have important functions in cell proliferation and differentiation. But embryonic stem (ES) cells lacking c-Jun are viable and have a normal in vitro differentiation capacity, although c-Jun appears to be important for growth of teratocarcinomas in vivo. To define the function of c-jun better, targeted ES cells were used to generate mice lacking c-Jun. Here we report that heterozygous mutant mice appear normal, but embryos lacking c-Jun die at mid- to late-gestation and exhibit impaired hepatogenesis, altered fetal liver erythropoiesis and generalized oedema. Interestingly, c-jun-/- ES cells can participate efficiently in the development of all somatic cells in chimaeric mice except liver cells, further suggesting an essential function of c-Jun in hepatogenesis.

Animals↗
PubMed1993

Rescue of W-associated mast cell defects in W/Wv bone marrow cells by ectopic expression of normal and mutant epidermal growth factor receptors.

The normal human epidermal growth factor receptor (EGF-R) (HERc), a chimeric EGF-R/v-erbB (HERerbB) receptor, and the ligand-independent oncogenic EGF-R variant (v-erbB) were used to correct the mast cell defects in W/Wv bone marrow (BM) cells. In culture, all three receptor molecules transduced functional mitogenic signals in infected interleukin-3 (IL-3)-dependent bone marrow-derived mast cells (BMMCs) and enabled their differentiation into safranin-positive mast cells resembling connective tissue-type mast cells (CTMCs). Furthermore, expression of these receptors restored the capacity of W/Wv BMMCs to colonize the peritoneal cavity of mast cell-deficient W/Wv mice where they differentiated to safranin-positive cells with similar frequencies as wild-type BMMCs. These experiments show that expression of normal and mutant EGF-Rs in W/Wv BM cells is able to complement the function of the c-kit-encoded Steel factor receptor (SLF-R) in mast cell development. We conclude that signal transduction by normal and mutant EGF-Rs in murine hematopoietic cells apparently involves components also used by the SLF-R, which suggests that these receptors use overlapping pathways for signal transduction.

Animals↗
PubMed1993

Stable murine chondrogenic cell lines derived from c-fos-induced cartilage tumors.

This study describes the detailed characterization of four murine chondrogenic cell lines (wT2-1, wT2-7, wT2-8, and wT2-9) that were isolated from a cartilage tumor induced by the protooncogene c-fos in chimeric mice. All cell lines are clonal and display a fibroblastic morphology with a doubling time of 1-2 days. Northern blot analysis demonstrated that in addition to expressing high levels of exogenous c-fos, all clones express varying levels of the cartilage marker gene type II collagen in addition to type I collagen. The clones also expressed high levels of the AP-1 genes c-jun and fra-1. The doubling times of these clones did not change over a period of 14 months in culture. Most importantly, however, expression of type II collagen was maintained in all cell lines for 8 months in culture, and two cell lines maintained type II collagen expression when analyzed after 14 months. Interestingly, type I collagen expression was lost after long-term culture. Following injection into syngeneic and nude mice, all cell lines formed tumors containing areas with the morphologic appearance of hyaline cartilage, indicating that these cell lines are chondrogenic. Thus, these stable murine chondrogenic cell lines provide a useful tool for studying the transcriptional control of cartilage-specific gene expression, as well as the growth control of chondrogenic cells.

Alkaline Phosphatase↗
PubMed1993

Osteoblasts are target cells for transformation in c-fos transgenic mice.

We have generated transgenic mice expressing the proto-oncogene c-fos from an H-2Kb class I MHC promoter as a tool to identify and isolate cell populations which are sensitive to altered levels of Fos protein. All homozygous H2-c-fosLTR mice develop osteosarcomas with a short latency period. This phenotype is specific for c-fos as transgenic mice expressing the fos- and jun-related genes, fosB and c-jun, from the same regulatory elements do not develop any pathology despite high expression in bone tissues. The c-fos transgene is not expressed during embryogenesis but is expressed after birth in bone tissues before the onset of tumor formation, specifically in putative preosteoblasts, bone-forming osteoblasts, osteocytes, as well as in osteoblastic cells present within the tumors. Primary and clonal cell lines established from c-fos-induced tumors expressed high levels of exogenous c-fos as well as the bone cell marker genes, type I collagen, alkaline phosphatase, and osteopontin/2ar. In contrast, osteocalcin/BGP expression was either low or absent. All cell lines were tumorigenic in vivo, some of which gave rise to osteosarcomas, expressing exogenous c-fos mRNA, and Fos protein in osteoblastic cells. Detailed analysis of one osteogenic cell line, P1, and several P1-derived clonal cell lines indicated that bone-forming osteoblastic cells were transformed by Fos. The regulation of osteocalcin/BGP and alkaline phosphatase gene expression by 1,25-dihydroxyvitamin D3 was abrogated in P1-derived clonal cells, whereas glucocorticoid responsiveness was unaltered. These results suggest that high levels of Fos perturb the normal growth control of osteoblastic cells and exert specific effects on the expression of the osteoblast phenotype.

Animals↗
PubMed1993

Induction of tumor formation and cell transformation by polyoma middle T antigen in the absence of Src.

In polyomavirus-transformed cells, middle T antigen binds to and activates the protein tyrosine kinase, Src. To determine whether this interaction is critical for middle T transformation, we examined the ability of middle T to transform cells that lack endogenous Src (because of a targeted disruption of both Src alleles). Infection of newborn or 2-week-old Src-negative mice with a retrovirus encoding middle T led to the induction of visceral hemangiomas that were indistinguishable from tumors in wild-type mice with respect to their morphology, frequency or latency period. In addition, middle T was able to induce foci formation on cell monolayers and colony formation in soft agar in Src-negative immortalized fibroblasts. These results indicated that Src is not essential for middle T-induced transformation of the cells targeted in these assays. To examine the protein tyrosine kinases that interact with middle T in the absence of Src, we compared the level of middle T phosphorylation in immune complex kinase assays from Src-negative and Src-positive cell lysates, and identified the middle T-associated kinases in these cells. In Src-positive cell lysates, there was a similar level of middle T phosphorylation in Src and Yes immunoprecipitates, suggesting that middle T can bind to Src and Yes to a similar extent in this cell type. Fyn immunoprecipitates displayed fourfold lower levels of middle T phosphorylation than that detected in the Src and Yes immunoprecipitates. In Src-negative cells, the level of middle T phosphorylation in Yes and Fyn immunoprecipitates was not significantly different from that detected in the Src-positive cells, suggesting that the absence of Src does not lead to a compensating increase in the proportion of middle T associated with these kinases. The level of middle T-associated phosphatidylinositol 3'-kinase was also examined since this kinase is known to interact with middle T-kinase complexes. Phosphatidylinositol 3'-kinase activity associated with middle T was reduced 30-60% in Src-negative cells, suggesting that Src contributes at least one-third of the total middle T associated in wild-type cells. Taken together, these results indicate that Src is not required for middle T-induced hemangiomas in mice or for focus induction in immortalized fibroblasts, and that the residual level of Yes, Fyn and phosphatidylinositol kinase activity associated with middle T in Src-negative cells may compensate for the absence of Src.

Animals↗