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Biomedical subjects

N M Gough

Publications and source records attributed to N M Gough.

At least 19 recordsLinked to original sources
PubMed1992

Synergistic suppression: anomalous inhibition of the proliferation of factor-dependent hemopoietic cells by combination of two colony-stimulating factors.

Cells of the continuous murine hemopoietic cell line FDC-P1 expressing macrophage-colony-stimulating factor (M-CSF) receptors following retroviral insertion of murine c-fms cDNA proliferated clonally when stimulated by granulocyte/macrophage (GM)-CSF, multipotential CSF, or M-CSF. However, M-CSF combined with either GM-CSF or multi-CSF, even at low CSF concentrations, strongly inhibited colony formation, with loss of clonogenicity in affected cells accompanied by increased macrophage differentiation. Stimulation by these CSF combinations did not induce short-term changes in CSF receptor expression or internalization. FDC-P1 cells expressing another inserted tyrosine kinase receptor, basic fibroblast growth factor receptor, did not exhibit suppression when GM-CSF was combined with fibroblast growth factor. This phenomenon of synergistic suppression may have relevance for the future clinical use of combinations of CSFs, because a potentially similar suppression is also observable with some normal macrophage progenitor cells.

Animals↗
PubMed1992

Cross-species comparison of the sequence of the leukaemia inhibitory factor gene and its protein.

Leukaemia inhibitory factor (LIF) is a pleiotropic growth factor active in diverse cell systems in both the adult and the embryo. The LIF gene from a number of mammalian species is highly conserved. The ovine and porcine LIF genes were cloned, sequenced and compared to the previously published murine and human LIF gene sequences. While the coding regions of the LIF gene are highly conserved, the non-coding regions are largely non-conserved. In a region of approximately 340 bp, the 5' end of the translational initiation codon is highly conserved (84%). This region includes four conserved TATA boxes, two transcriptional start-sites identified in the murine gene and the minimal region required to function as the LIF promoter. A sequence in the murine gene adjacent to this highly conserved region which appears to contain a negative control element is, however, poorly conserved between the four species compared, except for a sequence of 16 conserved nucleotides. Within the largely non-conserved first intron, there is a block of approximately 150 nucleotides which is highly conserved between all four species (approximately 72%). However, a sequence in intron 1 of the murine LIF gene which corresponds to an alternative exon of a putative variant LIF transcript is very poorly conserved between species, with only relics of this exon evident in the other three species. A comparison of the five LIF protein sequences available (murine, rat, human, ovine and porcine) revealed that the protein displays a high degree of similarity, ranging from 74% between mouse and sheep to 92% between rat and mouse. Several large blocks of absolutely conserved amino acid sequence were identified. The ovine LIF gene was modified to allow production of recombinant ovine LIF in yeast cells, which was shown to be biologically active on murine cells.

Amino Acid Sequence↗
PubMed1992

Molecular characterization of NSCL, a gene encoding a helix-loop-helix protein expressed in the developing nervous system.

We report here the molecular cloning and chromosomal localization of an additional member of the helix-loop-helix (HLH) family of transcription factors, NSCL. The NSCL gene was identified based on its hybridization to the previously described hemopoietic HLH gene, SCL. Murine NSCL cDNA clones were obtained from a day 11.5 mouse embryo cDNA library. The coding region is 399 base pairs and encodes a predicted protein of 14.8 kDa. The nucleotide sequence shows 71% identity and the amino acid sequence shows 61% identity to murine SCL in the HLH domain. The NSCL protein-coding region terminates six amino acids beyond the second amphipathic helix of the HLH domain. Expression of NSCL was detected in RNA from mouse embryos between 9.5 and 14.5 days postcoitus, with maximum levels of expression at 10.5-12 days. Examination of 12- and 13-day mouse embryos by in situ hybridization revealed expression of NSCL in the developing nervous system. The NSCL gene was mapped to murine chromosome 1. The very restricted pattern of NSCL expression suggests an important role for this HLH protein in neurological development.

Amino Acid Sequence↗
PubMed1992

Arrangement and localization of the human GM-CSF receptor alpha chain gene CSF2RA within the X-Y pseudoautosomal region.

The gene encoding one subunit of the receptor for the hemopoietic growth factor, GM-CSF, has been previously localized to the short arm of the human sex chromosomes. By pulsed-field gel electrophoresis, the precise localization of this gene, CSF2RA, within the pseudoautosomal region has been determined. The gene is located 1180 to 1300 kb from the telomere, in close proximity to the CpG island B5. The CSF2RA gene spans at least 45 kb, and a representation of most of the gene on three overlapping cosmid clones has been obtained. The exon(s) encoding the first 35 bp of cDNA sequence lies outside these cosmids. The CSF2RA gene is characterized by abundant hypervariable sequences, and a number of informative restriction fragment length polymorphisms have been defined.

Amino Acid Sequence↗
PubMed1992

Localization of the human GM-CSF receptor beta chain gene (CSF2RB) to chromosome 22q12.2-->q13.1.

The gene for the beta-chain of the human GM-CSF receptor (CSF2RB) has been mapped to chromosome 22 by PCR analysis of a series of human x rodent somatic cell hybrids. In situ hybridization to normal human chromosomes and two translocations involving chromosome 22 and the chromosome expressing the rare fragile site FRA22A place the gene in the region 22q12.2-->q13.1, proximal to the fragile site.

Chromosomes, Human, Pair 22↗
PubMed1992

Inhibition of differentiation in a murine F9 embryonal carcinoma cell subline by leukemia inhibitory factor (LIF).

Leukemia inhibitory factor (LIF) is a cytokine previously shown to maintain pluripotent embryonic stem cells in their undifferentiated state. We have examined the effects of LIF in nullipotent embryonal carcinoma cell lines, and have found that LIF blocks differentiation induced by retinoic acid and at low temperature in OTF9 cells. LIF did not block differentiation in a parent F9 cell line. For OTF9 cells, LIF acts early in differentiation, inhibiting the appearance of parietal endoderm-type product cells. However, it acts subsequent to retinoic acid, and at least one early retinoic acid-induced event is unaltered in the presence of LIF. This finding provides both a means of dissecting the cascade of events leading to EC cell differentiation, and a well-characterised target cell type for studying the mechanism of action of LIF.

Animals↗
PubMed1992

Presence of single-stranded DNA in PCR products of slow electrophoretic mobility.

PCR products were characterized by electrophoresis, blotting and hybridization. In addition to the bands of expected size, bands of slower electrophoretic mobility were often detected. The slower bands completely disappeared when the PCR products were subjected to slow cooling, treated with S1 nuclease or run on an alkaline gel, whereas the bands of expected size were unaffected. The slower bands are therefore likely to contain single-stranded DNA.

Base Sequence↗
PubMed1992

Molecular biology of the leukaemia inhibitory factor gene.

Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine that has been identified and characterized in several laboratories by virtue of a number of different biological activities. LIF is encoded by a unique gene located at 11A1 in the mouse and at 22q12 in man. However, loci related to sequences in the 3' untranslated region of the mRNA have been detected and located elsewhere in the genome. The LIF gene from four mammalian species has been cloned and sequenced; the sequences are highly conserved within the coding regions and largely non-conserved within the non-coding regions. However, a number of non-coding segments displaying high interspecies similarity are evident; these are candidate control regions. Intriguingly, an exon corresponding to the 5' end of a variant LIF transcript in the mouse that encodes a potentially matrix-associated form of LIF is not conserved in the human, ovine and porcine genes. The promoter region of the LIF gene contains four well-conserved TATA elements, and two start sites of transcription have been identified. Three regions within the 5' flanking region have been identified as important for the function of the LIF promoter, including a candidate repressor sequence. The LIF gene is transcribed at only very low levels in normal tissues, but its expression can be increased by various stimuli.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and chromosomal localization of the murine homolog of the human helix-loop-helix gene SCL.

The human SCL gene is a member of the family of genes that encode the helix-loop-helix (HLH) class of DNA-binding proteins. A murine SCL cDNA was isolated from a normal macrophage cDNA library by using HLH-specific oligonucleotides as hybridization probes. The coding region is 987 base pairs and encodes a predicted protein of 34 kDa. The nucleotide sequence of the coding region shows 88% identity to the human SCL gene, and the amino acid sequence is 94% identical. The HLH motif and upstream hydrophilic region are entirely conserved in the murine and human proteins. The identity between the mouse and human sequences was less marked in the 5' and 3' untranslated regions. Two murine SCL transcripts that differ in the 3' noncoding region have been detected in fetal liver and various cell lines. Variation was also observed in the 5' untranslated region. Interestingly, immediately downstream of the protein-termination codon, both the human SCL sequence and the murine homolog share an E-box element--the suggested target site for DNA binding of HLH proteins. The murine SCL homolog was mapped to the central part of chromosome 4.

Alleles↗
PubMed1991

Leukemia inhibitory factor: a biological perspective.

The notion that a single hormone may exert a broad range of effects has become well established. As such, leukemia inhibitory factor (LIF) is a prime example. LIF was initially described, purified, and genetically cloned on the basis of its ability to induce the differentiation and suppress the clonogenicity of the monocytic leukemia cell line, M1. Subsequently, it has become apparent that in vitro LIF inhibits the differentiation of pluripotential ES cells, stimulates the synthesis of hepatic acute-phase proteins, induces a switch in neurotransmitter phenotype from adrenergic to cholinergic, suppresses adipocyte lipoprotein lipase activity, and results in an increase in bone resorption. Moreover, elevation of LIF levels in vivo has a number of patho-physiological consequences, many of which parallel those effects observed in vitro. The challenge that lies ahead is to determine whether other sites of LIF action exist and to define more clearly the physiological role LIF plays in vivo. A major mechanism of cell-cell communication is by the production and secretion of polypeptide hormones by one cell type, which act either systemically or locally, via interaction with specific receptors on the surface of responsive cells. Recently, it has become apparent that hormones initially described and named, on the basis of a specific action, in many cases exert a spectrum of effects on a broad range of cell types. Moreover, the effects exerted are often mimicked closely by other hormones. Hormones that act in a pleiotropic manner are, for example, transforming growth factor-beta (TGF-beta), the various fibroblast growth factors (FGFs), interleukin-6 (IL-6), and leukemia inhibitory factor (LIF). This review will focus on the various biological effects ascribed to LIF.

Animals↗
PubMed1991

Characterization of cytokine production by the metrial gland and granulated metrial gland cells.

The metrial gland and its population of bone marrow-derived, large, granulated, lymphocyte-like cells, termed granulated metrial gland (GMG) cells, are consistent but poorly understood, decidua-associated features of pregnancy in the mouse and other species. Decidua, a complex maternal tissue, is thought to be a source of cytokines important for placental development. Thus, it is important to determine if lymphokine or cytokine production is among the activities of the metrial gland and GMG cells. Media conditioned by culture of either metrial gland explants or migrating GMG cells were evaluated for various cytokine activities. At least four activities were present: CSF-1, IL-1, a factor promoting proliferation of DA-1 cells that was not GM-CSF, IL-3 or erythropoietin and an activity cytotoxic to the CSF-1-dependent macrophage cell line 5/10.14. CSF-1 and IL-1 appeared to be products of the GMG cells. Cytokines not present at detectable levels included IL-2, IL-4, TNF-alpha and TGF-beta. Qualitatively, the cytokine profiles remained constant throughout days 8-16 of gestation. mRNA from migratory GMG cells was isolated and assayed for eleven cytokine mRNAs by polymerase chain reaction-based amplification of cDNA synthesized from mRNA. GMG cell RNA contained transcripts for LIF and CSF-1 but did not contain transcripts for GM-CSF, G-CSF, IL-2, IL-3, IL-4, IL-6, IL-7, IFN-gamma or TNF-alpha. TGF-beta transcripts were detected in occasional samples at very low levels. Since GMG cells are highly mobile cells that migrate throughout the placenta and into trophoblast-lined maternal blood spaces, their function in pregnancy may involve the delivery of very localized differentiation or growth regulatory signals to the developing fetal trophoblast and placenta.

Animals↗
PubMed1991

Heterogeneity in lymphokine profiles of CD4+ and CD8+ T cells and clones activated in vivo and in vitro.

Analysis of lymphokine mRNA expression and protein secretion by about 100 short-term alloreactive T-cell clones revealed marked heterogeneity in the combinations of lymphokines synthesized. This finding argues against a simple model in which T cells express either an unrestricted (Th0) or a restricted (Th1 or Th2) lymphokine profile. Lymphokine titers appeared to be normally distributed, with the percentage of positive clones for any one product determined by the threshold of detection. Accordingly, the observation that CD4+ clones on average produced higher titers of most lymphokines than CD8+ clones indicated that apparent differences between the lymphokine profiles of these two subsets were quantitative rather than qualitative. Patterns of lymphokine gene expression detected in whole tissues or by analysis of single cells and clones were markedly influenced by in vivo priming. Relative levels of expression of IL-4, IFN-gamma and GM-CSF in lymphoid tissues differed in mice undergoing a GvHR or following contact sensitization with OX or immunization with KLH in adjuvant. Consistent with the finding that IL-4 was the major lymphokine mRNA detected in lymph nodes of KLH-primed mice, most short-term KLH-specific clones derived from such mice also expressed IL-4. A similar approach to the detection of lymphokine-secreting T-cell precursors activated late in L. major infection showed that most clones from the L. major-resistant strain, C57BL/6, secreted IFN-gamma without IL-4 whereas most clones from the susceptible strain, BALB/c, secreted IL-4 without IFN-gamma. Differences were also noted in anti-CD3-induced IL-3 production at the single-cell level between CD8+ cells activated in the GvHR or against a tumor allograft. Con A-induced, filler cell-dependent cloning of CD4+ T cells from unprimed mice gave rise both to IFN-gamma-producing and to IL-4-producing clones. A requirement for an undefined, filler cell-dependent signal for development of IL-4-secreting clones was suggested by the finding that clones of normal CD4+ and CD8+ T cells activated in an anti-CD3-induced, filler cell-free system exclusively produced IFN-gamma and IL-3 without detectable IL-4 or IL-6. With a view to developing a single-cell approach to the analysis of lymphokine profiles of in vivo-activated T cells, sensitive assays for IL-3 and other lymphokines were used to measure secreting cells activated in the GvHR or against a tumor allograft.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Localization of the human GM-CSF receptor gene to the X-Y pseudoautosomal region.

Mammalian sex chromosomes share a small terminal region of homologous DNA sequences, which pair and recombine during male meiosis. Alleles in this region can be exchanged between X and Y chromosomes and are therefore inherited as if autosomal. Genes from this so-called pseudoautosomal region (PAR) are present in two doses in both males and females, and escape inactivation of the X chromosome in females. Indirect evidence suggests that there must be several pseudoautosomal genes, and several candidates have been proposed. Until now, the only gene that has been unequivocally located in the PAR is MIC2, which encodes a cell-surface antigen of unknown function. We now report the localization of a gene of known function to this region--the gene for the receptor of the haemopoietic regulator, granulocyte-macrophage colony stimulating factor. The chromosomal localization of this gene may be important in understanding the generation of M2 acute myeloid leukaemia.

Chromosome Mapping↗
PubMed1990

Structural organization of the genes for murine and human leukemia inhibitory factor. Evolutionary conservation of coding and non-coding regions.

Leukemia inhibitory factor, LIF, is a glycoprotein with multiple activities in both the adult and the embryo. LIF appears to be encoded by a unique gene in both mouse and man, although the 3'-untranslated region of the mouse LIF gene gives a complex hybridization pattern on Southern blots. The complete nucleotide sequences of both the murine and human LIF genes and their flanking regions (8.7 and 7.6 kilobase pairs, respectively) were determined and compared. Both genes comprise three exons, two introns and an unusually long 3'-untranslated region (3.2 kilobase pairs), specificying a mRNA of approximately 4.1 kilobases. Two start sites of LIF-transcription were determined, by S1-nuclease protection and by a novel approach involving the polymerase chain reaction. S1-nuclease protection revealed a start site 60-64 base pairs upstream of the translational start codon and immediately downstream of a TATA box (TATATAAAT). The PCR approach identified a second transcriptional start site 160 base pairs 5' of the start codon and adjacent to a "TATA-like" element (CATAATTT). A comparison of the murine and human LIF gene sequences revealed a high degree of conservation in the coding regions and in segments of the untranslated and flanking regions. Seven segments displaying greater than 75% homology were identified, with the 5' and 3' ends of the transcription unit revealing the highest degree of homology. These conserved regions represents potential cis-acting control elements.

Amino Acid Sequence↗