Biomedical subjects
Publications and source records attributed to J D Engel.
This case illustrates how a vascular malformation can turn an ordinary tooth extraction into an emergency situation. Arteriovenous malformations are rare and difficult to detect on radiographs. Early warning signs are discussed and surgical treatment is described.
Explore the source record for details and available documents.
To understand the functional roles that the GATA factors may play during hematopoietic cell differentiation, we examined the expression of GATA factor mRNAs and protein products in various human cell lines. Blot hybridization analyses demonstrated that GATA-1 and GATA-2 mRNAs are expressed abundantly in a set of cell lines established from human myelogenous leukemia cells, but the expression pattern of each factor is distinct. GATA-2 mRNA is expressed in all cell lines tested that express erythroid markers, and, in addition, the mRNA is also expressed in three CD34+ cell lines and two early myeloid cell lines. In contrast, the expression of GATA-1 mRNA showed tight correlation to that of the erythroid/megakaryocytic lineage markers. We also found that the GATA-2 probe identifies two types of mRNA. Structural analysis of genomic DNA clones encoding human GATA-2 coupled with RNA blot analysis demonstrated that there exists an alternative use of polyadenylation consensus sequences in a single exon and this causes the molecular heterogeneity among GATA-2 mRNAs. Through immunochemical and immunohistochemical analyses using anti-GATA-1- and anti-GATA-2-specific antibodies, GATA-2 protein was clearly shown to be present in the nuclei of leukemia-derived early myeloid and CD34+ cell lines, whereas both GATA-1 and GATA-2 proteins are expressed in erythroid/megakaryocytic cell lines. Thus, the expression profile of GATA-2 is consistent with the hypothesis that GATA-2 plays unique roles for the transcriptional activation of genes in cells at an early stage of hematopoietic differentiation and in developing cells of the erythroid and myeloid lineages.
In transiently transfected chicken erythroid cells, beta-like globin gene switching is mediated through differential activation of the cis-linked embryonic epsilon- and adult beta-globin genes by a shared enhancer. Two underlying mechanisms have been proposed: (i) tissue- and stage-specific factors activate the beta-globin promoter in adult erythroid cells (autonomous regulation); and (ii) the epsilon-globin promoter, although transcriptionally competent in both embryonic and adult cells, is suppressed at the adult stage through competition with the beta-globin promoter for interaction with the enhancer (competitive regulation). Analyses of transgenic mice carrying the chicken beta/epsilon-globin locus demonstrated that both genes depended on the enhancer for erythroid expression, but only the epsilon-globin gene exhibited developmentally appropriate transcription at levels comparable to the endogenous mouse globin genes. Surprisingly, the chicken epsilon-globin gene also appeared to be autonomously regulated, as has been observed for human embryonic and fetal beta-like globin genes in transgenic mice. These results suggest that the chicken beta/epsilon-globin enhancer possesses either embryonic stage or epsilon-globin gene specificity when incorporated into the murine germ line.
Erythroid cells regulate heme biosynthesis in a manner that is distinct from all other cell types. While heme negatively regulates the synthesis of the housekeeping delta-aminolevulinate synthase (ALAS-N) in all non-erythroid cells, the expression of an erythroid-specific isozyme (ALAS-E) is developmentally regulated in red blood cells. As a first step towards understanding the molecular basis for the transcriptional regulation of ALAS-E during erythropoiesis, we cloned and characterized the chicken ALAS-E locus. This gene spans 18 kbp and is composed of eleven exons. The intron/exon structure of erythroid ALAS was found to be conserved among several vertebrate species. Direct RNA sequencing identified a 5' untranslated region that is derived from two continuous exons and is predicted to form a very stable stem-loop structure that bears resemblance to the ferritin iron-responsive element. Tissue-specific expression of the ALAS-E gene was analyzed by transient transfection assays in hematopoietic cells of both erythroid and non-erythroid origins. These experiments identified distal (-784 to -505 bp) and proximal (-155 to +21 bp) promoter elements which are required for high level, erythroid-specific transcription.
Analysis of the nuclear factors responsible for erythroid gene regulation has led to the cloning of several transcriptional regulatory proteins with clear lines of evidence suggesting or demonstrating their roles in tissue-specific gene expression. However, the erythroid cell lineage-restricted proteins that have been characterized to date do not differ significantly in activity during the distinct developmental stages of erythropoiesis, a characteristic that might be expected for a nuclear factor that actively participates in the differential regulation of various alpha- or beta-type globin genes (called globin gene switching) at distinct stages of embryonic development. We reported previously the identification of such a differentially expressed DNA binding activity, which appeared to be present in mature definitive (adult) erythroid cells but absent in immature definitive or primitive (embryonic) erythroid cells. Here we detail the biochemical properties of this erythroid tissue- and developmental stage-specific DNA binding activity (called nuclear factor-erythroid 4; NF-E4), which contains characteristics of a molecule that may be intimately involved in globin gene switching.
Explore the source record for details and available documents.
The molecular mechanisms specifying patterns of gene expression in the vertebrate brain, which in turn determine the developmental fates of specific neurons, are yet to be clearly defined. Individual members of a recently identified family of transcriptional regulatory proteins, the GATA factors, are required for the differentiation of certain hematopoietic cell lineages. We show here that two of the members of this gene family, GATA-2 and GATA-3, are expressed within discrete cell populations of the chicken optic tectum during embryogenesis, and that they have highly restricted patterns of expression in the developing chicken brain. Furthermore, the induction of GATA factor expression within specific cell layers parallels the well established spatial (rostral to caudal) and temporal pattern of optic tectum development. The observation that both the timing of appearance and the localization of expression of GATA-2 and GATA-3 are correlated with optic tectum development suggest that these transcription factors may be associated with the initiation of gene transcription required for the determination of specific neuronal fates within visual areas of the vertebrate brain.
Pluripotent hematopoietic stem cells can differentiate into a number of distinct specialized cell types; however, no single lineage-specific master regulators have been identified that can activate individual patterns of gene expression. Recent evidence suggests that such lineage determination is regulated by a combinatorial matrix of regulatory proteins with overlapping tissue specificities which cooperate to define individual cell types.
GATA-3 is a zinc finger transcription factor which is expressed in a highly restricted and strongly conserved tissue distribution pattern in vertebrate organisms, specifically, in a subset of hematopoietic cells, in cells within the central and peripheral nervous systems, in the kidney, and in placental trophoblasts. Tissue-specific cellular genes regulated by GATA-3 have been identified in T lymphocytes and the placenta, while GATA-3-regulated genes in the nervous system and kidney have not yet been defined. We prepared monoclonal antibodies with which we could dissect the biochemical and functional properties of human GATA-3. The results of these experiments show some anticipated phenotypes, for example, the definition of discrete domains required for specific DNA-binding site recognition (amino acids 303 to 348) and trans activation (amino acids 30 to 74). The signaling sequence for nuclear localization of human GATA-3 is a property conferred by sequences within and surrounding the amino finger (amino acids 249 to 311) of the protein, thereby assigning a function to this domain and thus explaining the curious observation that this zinc finger is dispensable for DNA binding by the GATA family of transcription factors.
This article presents a critique of the social and political context in which the idea of construct validation developed. It outlines the rhetorical as well as the empirical and philosophical dimensions of the concept. Finally, the essay offers an alternative viewpoint for the conceptualization of construct validation and links it to current treatments of the topic by members of the measurement community.
The molecular determinants governing tissue-specific gene expression in the placenta are at present only poorly defined, particularly with respect to the regulation of specific hormone genes whose products are vital to embryonic development and the maintenance of a nurturing maternal environment. In continuing our analysis of the trophoblast-specific expression of the mouse placental lactogen I gene, we now demonstrate that the transcription factors GATA-2 and GATA-3 regulate the activity of this gene promoter. These factors are expressed in placental trophoblast cells, with peak levels of the GATA-2, GATA-3 and placental lactogen I mRNAs each accumulating at midgestation. Analysis of a region of the placental lactogen I gene promoter, previously shown to be sufficient for directing trophoblast-specific transcription, revealed the presence of three consensus binding sites for GATA-2 or GATA-3. Both GATA-2 and GATA-3 bind to these sites in vitro and mutation of these sites results in a significant decrease in promoter activity as assayed by transient transfection into the choriocarcinoma-derived cell line Rcho-1, which expresses endogenous GATA-2 and GATA-3. Furthermore, overexpression of GATA factors in Rcho-1 cells stimulates transcription from a co-transfected placental lactogen I gene promoter. Most significantly, expression of GATA-2 or GATA-3 was found to induce transcription from this promoter in transfected non-trophoblast (fibroblast) cells. These data indicate that GATA factors are both limiting and required transcriptional regulatory molecules in placental trophoblasts, and that the tissue specificity of the placental lactogen I gene is determined, at least in part, by GATA-2 and/or GATA-3.
GATA-1 is an essential factor for the transcriptional activation of erythroid-specific genes, and is also abundantly expressed in a discrete subset of cells bordering the seminiferous epithelium in tubules of the murine testis. In examining normal and germ-line defective mutant mice, we show here that GATA-1 is expressed only in the Sertoli cell lineage in mouse testis. GATA-1 expression in Sertoli cells is induced concomitantly with the first wave of spermatogenesis, and GATA-1-positive cells are uniformly distributed among all tubules during prepubertal testis development. However, the number of GATA-1-positive cells declines thereafter and were found only in the peripheral zone of seminiferous tubules in stages VII, VIII and IX of spermatogenesis in the adult mouse testis. In contrast, virtually every Sertoli cell in mutant W/Wv, jsd/jsd or cryptorchid mice (all of which lack significant numbers of germ cells) expresses GATA-1, thus showing that the expression of this transcription factor is negatively controlled by the maturing germ cells. These observations suggest that transcription factor GATA-1 is a developmental stage- and spermatogenic cycle-specific regulator of gene expression in Sertoli cells.
We describe the embryonic expression pattern as well as the cloning and initial transcriptional regulatory analysis of the murine (m) GATA-3 gene. In situ hybridization shows that mGATA-3 mRNA accumulation is temporally and spatially regulated during early development: although found most abundantly in the placenta prior to 10 days of embryogenesis, mGATA-3 expression becomes restricted to specific cells within the embryonic central nervous system (in the mesencephalon, diencephalon, pons and inner ear) later in gestation. GATA-3 also shows a restricted expression pattern in the peripheral nervous system, including terminally differentiating cells in the cranial and sympathetic ganglia. In addition to this distinct pattern in the nervous system, mGATA-3 is also expressed in the embryonic kidney and the thymic rudiment, and further analysis showed that it is expressed throughout T lymphocyte differentiation. To begin to investigate how this complex gene expression pattern is elicited, cloning and transcriptional regulatory analyses of the mGATA-3 gene were initiated. At least two regulatory elements (one positive and one negative) appear to be required for appropriate tissue-restricted regulation after transfection of mGATA-3-directed reporter genes into cells that naturally express GATA-3 (T lymphocytes and neuroblastoma cells). Furthermore, this same region of the locus confers developmentally appropriate expression in transgenic mice, but only in a subset of the tissues that naturally express the gene.
Family medicine investigators are beginning to use qualitative approaches to research questions. This paper reviews guidelines for qualitative research from several social sciences, and summarizes discussions of "standards" for qualitative research among health professionals. From these sources, the authors propose desirable characteristics of qualitative research. These include values of empathy, collaboration, service, and moral sensitivity; characteristics of clarity and coherence; techniques of participatory dialogue, triangulation, purposeful sampling, and immersion in context; and outcomes of useful knowledge and behavior effectively shared with readers.
Erythropoietin (Epo) is a cytokine which specifically regulates differentiation and proliferation of erythroid progenitor cells. We report here that Epo receptor expressed in interleukin 3-dependent lymphoid Ba/F3 cells transmits both differentiation and growth signals. Epo stimulation of these cells leads to activation of transcription and/or translation of the erythroid-specific transcription factors GATA-1 and SCL, followed by the accumulation of both alpha- and beta-globin chains. These results suggest that expression and activation of the Epo receptor regulates erythroid-specific gene expression and might play a role in determining a cell lineage in vivo and that GATA-1 and SCL may exert their effects after Epo binds to its receptor. It was further found that chimeric receptors composed of extracellular domains of Epo receptor and cytoplasmic domains of interleukin 2 or interleukin 3 receptors could also induce erythroid-specific gene expression in Ba/F3 cells. Taking these data together with previous observations, we conclude that interaction of the extracellular domains of the Epo receptor with other membrane components is essential for transmission of both the erythroid differentiation and the growth signals.
Genes encoding the accessory molecules CD8 and CD4 are activated early in thymocyte development, generating CD4+8+ double positive intermediates, which give rise to two functionally distinct mature T cell subsets that express either CD4 or CD8. The mechanisms that govern the activation or suppression of the CD8 gene are likely to be central to the T cell development program. To identify the key regulatory factors, we have initiated an analysis of the transcriptional regulation of the murine CD8 alpha gene. We have identified three CD8+ cell-specific DNAase I hypersensitive sites (HSS) located upstream of the murine CD8 alpha gene. In vitro mobility shift analysis of the -4.0-kb HSS region has revealed multiple binding sites for the T cell-restricted transcription factor GATA-3. In vitro translated murine GATA-3 binds specifically to both CD8 GATA sites, and coexpression of this factor in transient transfection assays transactivates a reporter construct containing these sequences. These results provide the first evidence for the role of a T cell-restricted factor in the regulation of either CD8 or CD4 genes.
Although the formation of terminally differentiated erythroid cells has been shown to require the presence of a functional GATA-1 gene in vivo, the role of this transcription factor and other members of the GATA family at earlier stages of erythroid differentiation is unclear. In this report, the expression of GATA-1, GATA-2, and GATA-3 has been examined in enriched peripheral blood progenitors before and after culture in a well-characterized liquid culture system. In addition primary leukemic cells as well as several erythroleukemic and nonerythroid cell lines were analyzed for GATA factor expression. The results show that the profile of GATA factor expression in erythroid cells is distinct from that of myeloid or lymphoid lineages. Erythroleukemic cell lines express little or no GATA-3, but high levels of GATA-1 and GATA-2. When they are induced to display the terminal erythroid phenotype, little change in the level of GATA-1 is detected but a significant decline in the levels of GATA-2 is observed commensurate with the degree of maturation achieved by the cells. Enrichment of erythroid progenitors from peripheral blood leads to selection of cells that express both GATA-1 and GATA-2. As the enriched populations are cultured in suspension in the presence of multiple cytokines, the levels of both GATA-1 and GATA-2 initially increase. However, in cultures containing only erythropoietin, which show exclusive erythroid differentiation, the levels of GATA-1 continue to increase, whereas GATA-2 expression declines as erythroid maturation progresses. In contrast, cultures lacking Epo (ie, with interleukin-3 and kit ligand) display limited progression towards both the myeloid and erythroid pathways, and high levels of expression of both GATA-1 and GATA-2 are maintained. Despite the initial upregulation of GATA-1 expression in the latter cultures, terminal erythroid differentiation does not occur in the absence of erythropoietin. These results indicate that GATA-1 upregulation is associated with both the initiation and the maintenance of the erythroid program, but that these two processes appear to be under separate regulatory control. Thus, the dynamic changes in the levels of different GATA factors that occur during primary erythroid differentiation suggest that the levels of these factors may influence the progression to specific hematopoietic pathways.