Biomedical subjects

P E Funk

Publications and source records attributed to P E Funk.

10 recordsLinked to original sources

PubMed1999

Regulation of avian fibroblast growth factor receptor 1 (FGFR-1) gene expression during skeletal muscle differentiation.

Myogenic cell proliferation and differentiation are regulated by a fibroblast growth factor (FGF) signal transduction cascade mediated by a high-affinity fibroblast growth factor receptor (FGFR). Exogenous FGF added to myogenic cultures has a mitogenic effect promoting myoblast proliferation while repressing differentiation. We have examined the regulation of the FGFR-1 gene (cek-1) in avian myogenic cultures by immunocytochemistry and Northern blot analysis. FGFR-1 protein was readily detected in undifferentiated myoblast cultures and was significantly reduced in differentiated muscle fiber cultures. Similarly, FGFR-1 mRNA was 2.5-fold more abundant in myoblast cultures than in differentiated cultures. To define the molecular mechanism regulating FGFR-1 gene expression in proliferating myoblasts and post-mitotic muscle fibers, we have isolated and partially characterized the avian FGFR-1 gene promoter. Transfection of FGFR-1 promoter-chloramphenicol acetyltransferase gene constructs into myogenic cultures identified two regions regulating expression of this gene in myoblasts. A distal region of 2226 bp conferred a high level of expression in myoblasts. This region functioned in an orientation-dependent manner and interacted with a promoter element(s) in a proximal 1058 bp promoter region to direct transcription. Deletion analysis revealed a 78 bp region that confers a high level of cek1 promoter activity in myoblasts. This DNA segment also contains Spl binding sites and interacts with a component in myoblast nuclear protein extracts. The proximal promoter region alone demonstrated no activity in directing transcription in either myoblasts or muscle fibers. Using the full-length promoter, gene expression was significantly decreased in differentiated muscle fibers relative to undifferentiated myoblasts indicating that the promoter-reporter gene constructs contain elements regulating expression of the endogenous FGFR-1 gene in both myoblasts and muscle fibers.

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PubMed1999

Protein kinase C activity regulates slow myosin heavy chain 2 gene expression in slow lineage skeletal muscle fibers.

Expression of the slow myosin heavy chain (MyHC) 2 gene defines slow versus fast avian skeletal muscle fiber types. Fetal, or secondary, skeletal muscle fibers express slow MyHC isoform genes in developmentally regulated patterns within the embryo, and this patterning is at least partly dependent on innervation in vivo. We have previously shown that slow MyHC 2 gene expression in vitro is regulated by a combination of innervation and cell lineage. This pattern of gene expression was indistinguishable from the pattern observed in vivo in that it was restricted to innervated muscle fibers of slow muscle origin. We show here that slow MyHC 2 gene expression in the slow muscle fiber lineage is regulated by protein kinase C (PKC) activity. Inhibition of PKC activity induced slow MyHC 2 gene expression, and the capacity to express the slow MyHC 2 gene was restricted to muscle fibers of slow muscle (medial adductor) origin. Fast muscle fibers derived from the pectoralis major did not express significant levels of slow MyHC 2 with or without inhibitors of PKC activity. This differential expression pattern coincided with different inherent PKC activities in fast versus slow muscle fiber types. Furthermore, over-expression of an unregulated PKCalpha mutant suppressed slow MyHC 2 gene expression in muscle fibers of the slow lineage. Lastly, denervation of skeletal muscles caused an increase in PKC activity, particularly in the slow medial adductor muscle. This increase in PKC activity was associated with lack of slow MyHC 2 gene expression in vivo. These results provide a mechanistic link between innervation, an intracellular signaling pathway mediated by PKC, and expression of a muscle fiber type-specific contractile protein gene. Dev Dyn 1999;216:177-189.

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PubMed1998

Identification of a lectin that induces cell death in developing chicken B cells.

The bursa of Fabricius is required for the development of a diverse B cell repertoire in chickens. Bursal B cells are dependent on survival signals within the bursa and their removal from the bursa results in death by apoptosis. To find molecules that regulate B cell survival, a panel of mAb and lectins was screened for the ability to either accelerate or prevent B cell death in culture. The fucose-specific lectin Aleuria aurantia agglutinin (AAA) rapidly rendered B cells permeable to propidium iodide. Incubation with the lectin also accelerated the appearance of internucleosomal DNA fragmentation and nuclear condensation, characteristics of apoptotic cell death. On Western blots the lectin detects a single protein band of approximately 48-50 kDa molecular weight. AAA detects fucose in an alpha 1-6 linkage and the restriction of this fucose linkage to a single protein suggests that it may be functionally important in the regulation of cell survival.

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PubMed1997

The avian chB6 (Bu-1) alloantigen can mediate rapid cell death.

The control of cell death is critical in the immune system. T and B lymphocytes must be censored during their development to remove nonfunctional or self-reactive lymphocytes. However, the molecules controlling cell deletion during lymphopoiesis have not been defined. B cells removed from the avian bursa of Fabricius rapidly undergo cell death in culture. We screened bursal B cells with a panel of Abs and lectins to identify molecules affecting their viability. Abs to the chB6 alloantigen caused a rapid loss of cell viability as measured by staining with propidium iodide. ChB6 Abs also cause adhesion between B cells. Transfection of cDNA encoding chB6 reconstituted the allele-specific cell death and adhesion effects in avian cell lines. These effects can be separated by binding cells onto Ab-coated plastic dishes. In these experiments, cells were killed in the absence of cell:cell contact. The ability of chB6 cross-linking to evoke cell aggregation and cell death is also observed when chB6 is expressed in growth factor-dependent mammalian cells. In these cells growth factor can almost completely prevent cell death but not cell aggregation. This suggests that known cell survival stimuli can suppress the cell death brought about by chB6 cross-linking. These results show that chB6 may have an important role in controlling cell survival and/or adhesion during avian B cell development.

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PubMed1997

Avian B cell development.

Development of B cells in chickens proceeds via a series of discrete developmental stages that includes the maturation of committed B cell progenitors in the specialized microenvironment of the bursa of Fabricius. The bursa has been shown to be required for the amplification of the B cell pool and selects for cells with productive immunoglobulin rearrangement events. Other events regulating chicken B cell development such as lymphocyte trafficking and apoptosis are just beginning to be elucidated. Within the bursa, the variable regions of immunoglobulin genes of B cell progenitors are diversified by a process of intrachromosomal gene conversion, where blocks of sequence information are transferred from pseudo-V regions to the recombined variable regions of the immunoglobulin genes. Recently gene conversion has been determined to play a role in the diversification of the immune repertoire in other species. In this review we focus on the current understanding and recent advances of B cell development in the chicken.

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PubMed1996

Current concepts in chicken B cell development.

The chicken has provided fundamental insights into the workings of vertebrate immunity. In particular, the development of B cells in a unique organ, the bursa of Fabricius, has provided a novel opportunity to study B cell development. Although chickens generate their Ig repertoire in a different way than mice and humans, there are many striking similarities in the developmental process. In particular, the control of lymphocyte migration and survival is key to the development of an immune system. The evolutionary distance of chickens and mammals underscore how common the problems are as well as how the solutions are often similar. Such commonalities serve to maintain the chicken as a compelling animal in which to study B cell development.

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PubMed1995

Vascular cell adhesion molecule 1-positive reticular cells express interleukin-7 and stem cell factor in the bone marrow.

In vitro studies have defined an essential role for stromal cells in supporting B-cell development, including production of lymphopoietic cytokines. It has been suggested that stromal cells are equivalent to adventitial reticular cells in the marrow; however, evidence of reticular cells producing cytokines has been difficult to obtain. Staining of bone marrow (BM) sections with antibodies to interleukin-7 (IL-7) showed a reticular pattern, mimicking that obtained using antibodies to vascular cell adhesion molecule 1 (VCAM-1), a molecule present on both stromal cells in vitro and reticular cells. To more closely examine cytokine production within normal marrow, an immunomagnetic separation scheme was devised to directly enrich VCAM-1+ stromal cells. Twenty to thirty percent of cells isolated in the VCAM-1+ fraction shared characteristics with stromal cells from long term BM cultures, including cellular morphology and expression of alkaline phosphatase and alpha actin. These were termed "reticular stromal" cells. Immunohistochemical staining showed that virtually all of the latter cells possessed cytoplasmic IL-7 protein, and about half expressed stem cell factor. In contrast with cultured stromal cells, very few had detectable macrophage-colony-stimulating factor. These data constitute the first report of cytokine expression by marrow reticular cells in vivo. The implications of this data with respect to the existence of stromal cell subsets and their regulation of lymphopoiesis is discussed.

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PubMed1994

Native associations of early hematopoietic stem cells and stromal cells isolated in bone marrow cell aggregates.

In suspensions of murine bone marrow, many stromal cells are tightly entwined with hematopoietic cells. These cellular aggregations appear to exist normally within the marrow. Previous studies showed that lymphocytes and stem cells adhered to stromal cells via vascular cell adhesion molecule 1 (VCAM1). Injection of anti-VCAM1 antibody into mice disrupts the aggregates, showing the importance of VCAM1 in the adhesion between stromal cells and hematopoietic cells in vivo. Early hematopoietic stem cells were shown to be enriched in aggregates by using a limiting-dilution culture assay. Myeloid progenitors responsive to WEHI-3CM in combination with stem cell factor (c-kit ligand) and B220- B-cell progenitors responsive to insulin-like growth factor-1 in combination with interleukin-7 are not enriched. We propose a scheme of stromal cell-hematopoietic cell interactions based on the cell types selectively retained within the aggregates. The existence of these aggregates as native elements of bone marrow organization presents a novel means to study in vivo stem cell-stromal cell interaction.

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PubMed1993

Activity of stem cell factor and IL-7 in combination on normal bone marrow B lineage cells.

The production of B cells is regulated by soluble and cell contact signals presumably provided by bone marrow stromal cells. Among these is IL-7, a well characterized proliferative stimulus for a subset of pre-B cells. Stem cell factor (SCF), a stromal cell-derived cytokine with broad hemopoietic effects, has been reported to synergize with IL-7 to drive the proliferation and differentiation of B220- bone marrow cells into B220+ B cell precursors in long term culture. A subsequent report has cast doubt on this result by showing that SCF and IL-7 were incapable of producing mu+ pre-B cells after short term culture. Here, using the cell sorter to assure discrete separation of B220+ and B220- cells followed by soft agar culture to prevent interaction with accessory cells, we demonstrate that the combination of SCF and IL-7 does not stimulate the expansion or differentiation of B220- lymphoid precursors but can act synergistically in the clonal proliferation of B220+ cells.

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PubMed1992

Enrichment of primary lymphocyte-supporting stromal cells and characterization of associated B lymphocyte progenitors.

Studies of Whitlock/Witte long-term bone marrow cultures have revealed the necessity of two cell types for B lymphopoiesis, a stem cell and the stromal cell. While a number of stromal cell lines exist they have been found to be heterogeneous with respect to cell surface marker expression and growth factor production. Separation and analysis of fresh bone marrow stromal cells is, therefore, necessary to understand the regulation of lymphopoiesis in vivo. Here we report the early stages of such studies. We demonstrate that stromal cells, as assessed by morphology and alkaline phosphatase reactivity after short-term culture, are enriched in cellular aggregates that can be separated from bone marrow suspensions. Stromal cells are present in aggregates at a frequency of one per thousand cells, whereas marrow from which the aggregates have been removed contains only one stromal cell per fifty-thousand cells. These aggregates are able to form Whitlock cultures from greatly reduced numbers of initiating cells, indicating that they contain culturable B lineage precursors as well as stromal cells capable of supporting B lymphopoiesis. The aggregates appear to be naturally formed and provide a means to examine native B cell precursor-stromal cell contacts. We find little evidence for sequestering of late-stage B cell precursors within the aggregates. Terminal deoxynucleotidyl transferase-positive cells, on the other hand, are approximately three times more frequent in bone marrow aggregates, suggesting close contact between very early B cell progenitors and stromal cells within the aggregates. The finding that stromal cells are enriched in cellular aggregates is an important first step in the ultimate isolation of these cells from marrow suspensions, which is vital to understanding stromal cell function in vivo.

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