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C G Begley

Publications and source records attributed to C G Begley.

At least 91 records · Page 5Linked to original sources
PubMed1996

Molecular basis of the soluble and membrane-bound forms of the murine leukemia inhibitory factor receptor alpha-chain. Expression in normal, gestating, and leukemia inhibitory factor nullizygous mice.

The murine leukemia inhibitory factor receptor alpha-chain (mLIFR) exists in a membrane-bound and a soluble form. The two major classes of mRNA transcript correspond to either the soluble or membrane-bound form of the mLIFR. In this study we have identified a complex and heterogeneous pattern of expression of mRNA transcripts for this receptor in normal mouse tissues and cell lines. In order to understand the molecular basis of these transcripts, genomic clones encompassing the region of divergence from the soluble to the membrane-bound form of the receptor were isolated. cDNAs encoding the membrane-bound form of the mLIFR were generated by an alternative splicing event where an exon that is specific to the soluble mLIFR was skipped. The membrane-bound form of the mLIFR was heterogeneously polyadenylated with at least five different sites of polyadenylation. The mRNA transcript encoding the soluble form of the mLIFR contained a region highly homologous to a murine B2 repetitive element, thus providing a possible explanation for the genesis of this transcript. The different forms of the mLIFR were analyzed in a wide range of mouse tissues in pseudopregnant mice and in mice at various stages of pregnancy. Only liver, placenta, and uterus showed an increase in the levels of mLIFR mRNA expression during pregnancy, indicating an important role for the LIFR in this process. However, somewhat surprisingly, there was no detectable difference in mLIFR mRNA levels or levels of soluble protein in leukemia inhibitory factor nullizygous mice when compared with normal mice.

Animals↗
PubMed1996

The human IL-11 receptor requires gp130 for signalling: demonstration by molecular cloning of the receptor.

We describe the molecular cloning of a cDNA for the alpha chain of the human IL-11 receptor (IL-11R alpha) and demonstrate the requirement of either the human or mouse gp130 molecule for signalling. cDNA clones encoding IL-11R alpha were isolated from a bone marrow cDNA library using a fragment from the murine IL-11R alpha as a probe. The human receptor was predicted to consist of 422 amino acids and was found to share 84% identity with the murine protein. In the extra-cellular region it exhibited a single hemopoietin domain with conserved cysteine residues and WSTWS motif. The transmembrane region was followed by a short cytoplasmic tail which did not contain a tyrosine kinase domain. Interaction of the human IL-11R alpha with murine gp130 was demonstrated: expression of the human IL-11R alpha in murine M1 cells which constitutively express murine gp130 (and murine LIF receptor), resulted in the generation of specific high-affinity binding sites for IL-11 (Kd = 250 pM). In addition, expression of the human IL-11R alpha in these cells permitted the induction of macrophage differentiation in response to IL-11. These results suggested that the human IL-11R alpha chain was able to form a functional receptor complex in association with murine gp130. The requirement of gp130 for signalling was confirmed by expression of the human IL-11R alpha in Ba/F3 cells. BaF3 cells that expressed the human IL-11R alpha alone showed binding of radiolabelled IL-11 but no proliferative response. Introduction of human gp130 into these cells resulted in high-affinity IL-11 binding sites and IL-11 dependent cellular proliferation. Thus these results demonstrated the absolute requirement of gp130 for signalling.

Amino Acid Sequence↗
PubMed1996

The helix-loop-helix gene SCL: implicated in T-cell acute lymphoblastic leukaemia and in normal haematopoietic development.

The SCL gene encodes a member of the helix-loop-helix family of transcription factors and was first identified through its involvement in a chromosomal translocation in a human leukaemic cell line. SCL is now recognized to be aberrantly expressed in most human T-cell leukaemias. In around 25% of cases SCL overexpression is associated with identifiable chromosomal abnormalities but in other cases the mechanism of ectopic expression has not yet been identified. SCL is normally expressed in haematopoietic progenitor cells, erythroid cells, mast cells and megakaryocytes. Gene delivery experiments using haematopoietic cell lines and the recent creation of mice with a null mutation of the SCL gene have demonstrated that SCL plays a crucial role in haematopoietic commitment and differentiation.

Animals↗
PubMed1996

Characteristics of corneal staining in hydrogel contact lens wearers.

Corneal fluorescein staining is widely used in clinical practice and research, but little information exists on the distribution of staining in a large group of asymptomatic contact lens wearers. This cross-sectional study took place at 3 centers, and investigated the pattern of corneal fluorescein staining in both eyes of 98 hydrogel contact lens wearers. We also investigated the strategies used by three experienced clinicians to grade corneal staining. Overall corneal staining was graded using a scale from 0 to 4 in one-half steps, and five corneal zones, superior, inferior, nasal, temporal, and central, were also graded. The average overall staining grade for both eyes of our subjects was 0.50, with an average of 0.57 for the right eye and 0.44 for the left. This difference between the eyes was statistically significant (p = 0.011). In addition, a comparison of the zones within each eye showed a significant difference (p = 0.0001) among the zones. Corneal staining between the two eyes was also positively and significantly correlated (r = 0.58; p = 0.0001). Grading strategies among clinical investigators were significantly different (p = 0.0001), indicating a potential source of bias in multi-centered studies. The difference in corneal staining between the eyes may represent a source of systematic bias, and could be due to grading the right eye before the left. The correlation in corneal staining between the eyes indicates that the two eyes of a subject cannot serve as independent data points. One-third of the subjects who participated in this study had notable corneal staining. This finding underlines the importance of regularly checking corneal staining in clinical practice.

Contact Lenses↗
PubMed1996

Murine flt3 ligand protects M1 leukemic cells from LIF-induced differentiation and suppression of self-renewal.

Self-renewing cell divisions are an important characteristic exhibited by both normal hematopoietic stem cells and leukemic cell populations. We have examined the action of flt3/flk2 ligand (FL) on physiologic suppression of self-renewal during growth factor-induced differentiation of M1 leukemic cells. Unstimulated M1 cells expressed high levels of flt3 receptor mRNA and protein, with approximately 20,000 molecules present at the cell surface. Consistent with data obtained from normal macrophage populations, expression of both mRNA and protein for flt3 receptor was suppressed as cells were induced to differentiate into mature macrophages in response to leukemia inhibitory factor (LIF). Although FL alone had no detectable action on unstimulated M1 cells, an effect was revealed during LIF-induced differentiation. FL overcame LIF-induced suppression in clonal cultures of M1 cells, prevented morphologic changes associated with macrophage differentiation and interfered with the LIF-induced responsiveness of M1 cells to macrophage colony-stimulating factor (M-CSF). This action of FL was evident on both parental M1 cells and M1 cells whose differentiation program was perturbed by enforced expression of the transcription factor SCL. The action of FL was most striking in clone transfer experiments in which FL rescued M1 cells from LIF-induced suppression of self-renewal. The ability of FL to maintain self-renewal characteristics satisfies one of the criteria predicted for a stem-cell-active molecule and contrasts with the action of FL in stimulating proliferation and differentiation of normal hematopoietic cells.

Animals↗
PubMed1996

The effectiveness of soft contact lens disinfection systems against Acanthamoeba on the lens surface.

BACKGROUND: This investigation compared the efficacy of three widely used contact lens disinfection systems against an ocular isolate of Acanthamoeba polyphaga. METHODS: Twenty-seven worn Ciba NewVues lenses were quartered, heat sterilized and inoculated with Acanthamoeba. Lens quarters were then randomly assigned to three experimental groups, with Group A lenses exposed to cleaner and saline rinse only, Group B to disinfection only, and Group C to both cleaner and disinfection. One quarter of each lens served as a control and the other three quarters were experimental. Quantification of viable Acanthamoeba remaining on the lens was performed after each step of the disinfection process. RESULTS: Group A lenses showed no significant difference between the treatments, or the treatments and the control. Group B lenses demonstrated a significant difference (p = 0.0001) between the treatments and the control. In Group C (cleaning and disinfection), the control lens quarters were significantly different (p = 0.037) from the experimental group, but there was no significant difference between the treatments. CONCLUSIONS: All three disinfection regimens were very effective in reducing the number of viable Acanthamoeba on the contact lens surface. In the absence of proper cleaning (Group B), AOSept was the most effective of the three. These results also show the importance of thoroughly rubbing the contact lens surface to decrease the number of Acanthamoeba.

Acanthamoeba↗
PubMed1995

Hematopoietic and lung abnormalities in mice with a null mutation of the common beta subunit of the receptors for granulocyte-macrophage colony-stimulating factor and interleukins 3 and 5.

Gene targeting was used to create mice with a null mutation of the gene encoding the common beta subunit (beta C) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3; multi-CSF), and interleukin 5 (IL-5) receptor complexes (beta C-/- mice). High-affinity binding of GM-CSF was abolished in beta C-/- bone marrow cells, while cells from heterozygous animals (beta C+/- mice) showed an intermediate number of high-affinity receptors. Binding of IL-3 was unaffected, confirming that the IL-3-specific beta chain remained intact. Eosinophil numbers in peripheral blood and bone marrow of beta C-/- animals were reduced, while other hematological parameters were normal. In clonal cultures of beta C-/- bone marrow cells, even high concentrations of GM-CSF and IL-5 failed to stimulate colony formation, but the cells exhibited normal quantitative responsiveness to stimulation by IL-3 and other growth factors. beta C-/- mice exhibited normal development and survived to young adult life, although they developed pulmonary peribronchovascular lymphoid infiltrates and areas resembling alveolar proteinosis. There was no detectable difference in the systemic clearance and distribution of GM-CSF between beta C-/- and wild-type littermates. The data establish that beta C is normally limiting for high-affinity binding of GM-CSF and demonstrate that systemic clearance of GM-CSF is not mediated via such high-affinity receptor complexes.

Animals↗
PubMed1995

Lineage-restricted regulation of the murine SCL/TAL-1 promoter.

The SCL/TAL-1 gene encodes a basic helix-loop-helix transcription factor that is expressed in multipotent hematopoietic progenitors before lineage commitment. Its expression is maintained during differentiation along erythroid, mast, and megakaryocytic lineages, but is repressed after commitment to nonexpressing lineages. To begin to address the molecular mechanisms underlying this complex pattern of expression, we have studied the regulation of the murine SCL promoter in erythroid and T-cell lines. Analysis of the methylation and chromatin structure of the SCL promoter region showed that SCL mRNA expression correlated with DNase hypersensitive sites and methylation status of the promoter. Transient reporter assays showed that promoter 1a was active in erythroid cells but not in T cells. Sequences between -187 and +26 were sufficient for lineage-restricted activity of promoter 1a. A joint promoter construct containing both promoter 1a and promoter 1b also exhibited lineage-restricted activity. Conserved GATA (-37), MAZ (+242), and ETS (+264) motifs were all shown to contribute to SCL promoter activity in erythroid cells, but several other motifs were not required for full promoter activity. The pattern of complexes binding to the +242 MAZ and +264 ETS sites were the same in erythroid and T cells. However, GATA-1 bound the -37 GATA site in erythroid cells, whereas in T cells GATA-3 was only able to bind weakly, if at all. Moreover, GATA-1 but not GATA-2 or GATA-3 was able to transactivate SCL promoter 1a in a T-cell environment. These results suggest that inactivity of SCL promoter 1a in T cells reflected the absence of GATA-1 rather than the presence of trans-dominant negative regulators.

Amino Acid Sequence↗
PubMed1995

Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene.

The scl gene encodes a basic-helix-loop-helix transcription factor which was identified through its involvement in chromosomal translocations in T-cell leukemia. To elucidate its physiological role, scl was targeted in embryonic stem cells. Mice heterozygous for the scl null mutation were intercrossed and their offspring were genotyped. Homozygous mutant (scl-/-) pups were not detected in newborn litters, and analysis at earlier time points demonstrated that scl-/- embryos were dying around embryonic day 9.5. The scl-/- embryos were pale, edematous, and markedly growth retarded after embryonic day 8.75. Histological studies showed complete absence of recognizable hematopoiesis in the yolk sac of these embryos. Early organogenesis appeared to be otherwise normal. Culture of yolk sac cells of wild-type, heterozygous, and homozygous littermates confirmed the absence of hematopoietic cells in scl-/- yolk sacs. Reverse transcription PCR was used to examine the transcripts of several genes implicated in early hematopoiesis. Transcripts of GATA-1 and PU.1 transcription factors were absent from RNA from scl-/- yolk sacs and embryos. These results implicate scl as a crucial regulator of early hematopoiesis.

Animals↗
PubMed1995

Human gene for the large subunit of ribonucleotide reductase (RRM1): functional analysis of the promoter.

Ribonucleotide reductase comprises two nonidentical protein subunits R1 and R2, both of which are required for enzyme activity and show cell cycle-dependent regulation. The TATA-less promoter of the human RRM1 gene (encodes R1 protein) was examined with reference to regulatory domains upstream of the transcription start site. A region from nt -195 to +3 was found to give maximal expression of a reporter gene when transfected into the human cell line K562. Overall, this 198-bp region shows 58% identity with the equivalent region of the murine promoter; however, it contains two 22-bp domains that were 81 and 91% identical between species. Electrophoretic mobility shift assays were performed using a fragment of the domain closest to the transcription start site. These experiments revealed that several factors were able to bind this region in a sequence-specific manner. One of these factors was shown to be Sp1 by specific competition and supershift using antibody to Sp1. The data presented suggest that Sp1 is involved in the transcription of RRM1.

Animals↗
PubMed1995

Differential regulation of macrophage differentiation in response to leukemia inhibitory factor/oncostatin-M/interleukin-6: the effect of enforced expression of the SCL transcription factor.

The physiologic program of macrophage differentiation normally proceeds in a coordinated manner in response to several different growth factors. Although the utilization of common receptor subunits may explain in part overlapping biologic functions, mechanisms by which unique actions are mediated remain obscure. We examined growth factor-induced macrophage differentiation in M1 leukemia cells that simultaneously display receptors for interleukin-6 (IL-6), leukemia inhibitory factor (LIF) and Oncostatin-M (OSM). Differentiation induced by all three factors was associated with decreased expression of transcription factors myb and SCL, increased expression of macrophage markers, and suppression of proliferation. Cell lines were established in which SCL expression was enforced. In the absence of growth factors, cells were indistinguishable from parental cells. However, LIF (or OSM)-induced macrophage differentiation was perturbed; there was failure to undergo morphologic differentiation, disturbed expression of lysozyme and Mac1 alpha, and failure to suppress proliferation. Surprisingly the perturbation of macrophage differentiation did not apply to induced expression of macrophage colony-stimulating factor (M-CSF) or granulocyte colony stimulating factor (G-CSF) receptors. This dissociation of elements normally coordinated in a macrophage differentiation program applied at a clonal level. There was no disturbance of IL-6-induced macrophage differentiation. These data directly implicate SCL in components of the macrophage differentiation program (suggesting that LIF receptor/gp130 heterodimers utilize an SCL-inhibitable pathway while gp130 homodimers do not) and demonstrate differential-regulation of components of the mature macrophage phenotype.

Animals↗
PubMed1995

scl, a gene frequently activated in human T cell leukaemia, does not induce lymphomas in transgenic mice.

The scl gene is implicated in human T cell acute lymphoblastic leukaemia (T-ALL) through its involvement in the t(1;14)(p32;q11) chromosomal translocation and, more frequently, as a result of a tumour-specific interstitial deletion on chromosome 1. The consequence of both these chromosomal alterations is overexpression of scl in the leukaemic cells. Despite the strong inference of a role in human T-ALL, scl has not yet been demonstrated to be causally involved in neoplastic transformation. We attempted to do this by generating transgenic mice in which scl expression was directed to the T cell lineage using the CD2 enhancer and the strong SR alpha viral promoter (CD2-scl mice). Three transgenic lines, all of which expressed the scl transgene at a high level, were bred and analysed. No alterations in T cell development were seen in the mice. Unexpectedly CD2-scl mice did not develop tumours, nor did the transgene enhance tumourigenesis by Moloney murine leukaemia virus. These findings throw into question the mechanism by which aberrant scl expression contributes to T cell leukaemogenesis.

Animals↗
PubMed1995

Broad inter-individual variations in circulating progenitor cell numbers induced by granulocyte colony-stimulating factor therapy.

The elevated white cell counts (WCC) and myeloid progenitor cell levels in the blood induced by granulocyte colony-stimulating factor (G-CSF) treatment were studied in three settings: cancer patients previously treated with chemo/radiotherapy (n = 13), untreated cancer patients (n = 5) and normal volunteers (n = 9). The inter-individual variations in progenitor cell mobilization responses to G-CSF and the impact of previous chemo/radiotherapy were investigated. The absolute levels of circulating progenitor cells, but not total white cells, were reduced significantly in the pretreated cancer patients (median 961, range 289-3355 per ml blood) as compared to untreated cancer patients (median 9891, range 2219-16625 per ml blood). In each setting, wide ranges of circulating progenitor cell numbers were observed, and the variation in progenitor cell numbers was considerably greater than observed for the WCC. However, progenitor cell numbers in normal volunteers (942-25296 per ml blood) demonstrated as much variance as observed in pretreated cancer patients. This broad physiological variation in progenitor cell levels induced by G-CSF needs to be considered when designing strategies for allogeneic peripheral blood stem cell transplantation.

Cohort Studies↗
PubMed1995

Reconstitution of mice with bone marrow cells expressing the SCL gene is insufficient to cause leukemia.

Rearrangement or translocation of the SCL gene is the most common genetic abnormality observed in human T-cell acute lymphocytic leukemia and results in the aberrant expression of SCL. To examine the oncogenic potential of this gene, an SCL-retrovirus was used to infect mouse bone marrow cells, which were then used to reconstitute C57/BL6 mice. Expression of SCL did not perturb the composition nor number of day 12 or day 13 colony forming unit-spleen. In total, 141 mice reconstituted with SCL-infected bone marrow and 103 control-mice were monitored for up to 2 years with no difference in survival, hematocrit, white cell count, or differential white cell count. As expected, from day 200 onwards, mice died due to radiation-induced thymomas; SCL provirus was not detected in these tumors. Thus, despite SCL being strongly implicated in the development of human leukemia, its enforced expression in mice using a retrovirus and bone marrow reconstitution was insufficient to generate leukemia.

Animals↗
PubMed1995

The cytokine receptor repertoire specifies autocrine growth factor production in factor-dependent cells.

Mechanisms of helper virus-induced growth factor-independence were examined in FDC-P1 cells and FDC-P1 cells expressing the erythropoietin receptor (FDER cells). Retroviral mutagenesis of FDC-P1 cells led to factor-independent (FI) colonies from which cell lines could readily be established; whereas control cells exhibited at least 20 to 40-fold lower rates of factor-independence. From 44 independent experiments using either FDC-P1 or FDER cells, 205 autonomous cell lines were obtained. Sixteen colonies displayed a novel ("satellite-inducing") appearance in agar and produced up to 4.1 x 10(5) U/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (some with altered GM-CSF transcript sizes) and/or interleukin-3 (IL-3). Retroviral mutagenesis of FDER cells increased the repertoire of autocrine growth factors now responsible for stimulating autocrine proliferation: 3% of FI cell lines produced erythropoietin (Epo) (0.5 U/mL). Unexpectedly, in every autonomous FDC-P1 cell line, reverse transcriptase-PCR demonstrated expression of a growth factor normally required for proliferation. Thus, a profound selection for cells able to produce growth factors as the mechanism for achieving autonomous proliferation was documented. The ectopic expression of a receptor lacking a cognate ligand ("orphan") followed by retroviral mutagenesis and selection for autocrine mutants may offer an effective method for identifying new ligands.

Animals↗
PubMed1995

Emergence of myeloid cells from cultures of J2E erythroid cells is linked with karyotypic abnormalities.

J2E cells are an erythroid cell line immortalized at the proerythroblast stage of differentiation by the J2 retrovirus which contains the raf and myc oncogenes. In response to erythropoietin, these cells terminally differentiate into mature, hemoglobin-producing erythroid cells. We have shown previously that B cells overexpressing raf and myc acquired the phenotype of macrophages, and here we demonstrate that, under adverse growth conditions, myeloid cells can also emerge from J2E cultures. Morphologically, ultrastructurally, and by cytochemical analyses, these cells resembled monocytic precursor cells at different stages of differentiation. They no longer responded to erythropoietin, failed to express an erythroid-specific surface antigen, and ceased producing transcripts for globin genes, GATA-1 and SCL. Most of the converted cells displayed surface antigens typically found on myeloid cells and in vivo produced histiocytomas with severe cachexia, instead of erythroleukemias. All of the myeloid convertants had karyotypic abnormalities, and we speculate that these mutations may have triggered the transition from erythroid to myeloid phenotype. Overexpression of raf and myc oncogenes may have generated genetic instability, which then influenced the commitment of cells to specific lineages.

Animals↗
PubMed1995

The flt3/flk-2 ligand: receptor distribution and action on murine haemopoietic cell survival and proliferation.

In this study the distribution and quantitation of the flt3/flk-2 receptor was examined on bone marrow cells and defined haemopoietic subpopulations. Undifferentiated cells expressed the greatest numbers of flt3/flk-2 receptors: 19% of primitive lin-kit+sca-1+ bone marrow cells and 16% of fetal liver lin-aa4.1+ cells exhibited over 15 000 receptors per cell as determined by binding of the radiolabeled cognate ligand (flt3/flk-2 ligand, FL). Moderate binding was demonstrated on early B lymphocyte subsets (4400 receptors per cell) and very low levels were detected on monocytes. Binding was not detected on promyelocytes, myelocytes, promonocytes, metamyelocytes, polymorphonuclear cells, eosinophils or nucleated erythroid cells. FL enhanced the survival of primitive lin kit+sca-1+ cells with an efficacy s with an efficacy equivalent to stem cell factor (SCF). FL stimulated predominantly blast and granulocyte-macrophage colony formation in cultures of bone marrow cells by both direct and indirect mechanisms. Marked synergistic effects of FL with combinations of colony stimulating factors (CSFs) or interleukin-6 occurred in the proliferation of primitive lin-kit+sca-1+ cells, but not lin-kit+sca-1- progenitor cells. Surprisingly, recloning experiments revealed that FL plus IL-3 increased the generation of progenitor cells by lin-kit a-1- cells compared with SCF plus IL-3. Thus FL functions as a factor with both direct and indirect stimulatory activities directed to the expansion, maintenance of clonogenic potential, and possibly limited self-renewal, of early haemopoietic cells.

Animals↗