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D J Williamson

Publications and source records attributed to D J Williamson.

At least 73 records · Page 4Linked to original sources
PubMed1992

Interaction of basal positive and negative transcription elements controls repression of the proximal rat prolactin promoter in nonpituitary cells.

The proximal rat prolactin (rPRL) promoter contains three cell-specific elements, designated footprints I, III, and IV, which restrict rPRL gene expression to anterior pituitary lactotroph cells. Footprint II (-130 to -120) binds a factor, which we have termed F2F, present in pituitary and nonpituitary cell types. Here we demonstrate that a key role of the footprint II site is to inhibit rPRL promoter activity in nonpituitary cells, specifically, by interfering with the basal activating function of a vicinal element. Gene transfer analysis revealed 20-fold activation of the rPRL promoter in nonpituitary cell types when footprint II was either deleted or specifically mutated. Similar activation of the intact rPRL promoter was obtained by in vivo F2F titration studies. In GH4 rat pituitary cells, the footprint II inhibitory activity was masked by the redundant, positively acting cell-specific elements and was inhibitory only if the two upstream sites, footprints III and IV, were deleted. Deletion of the -112 to -80 region in the footprint II site-specific mutant background resulted in complete loss of rPRL promoter activity in both pituitary and nonpituitary cell types, mapping a basal activating element that is operative irrespective of cell type to this region. While the basal activating element imparted an activating function in a heterologous promoter assay, the footprint II sequence did not display any inherent repressor function and actually induced several minimal heterologous promoters. However, the inhibitory activity of the footprint II site was detected only if it was in context with the basal activating element. These data underscore the importance of ubiquitous activating and inhibitory factors in establishing cell-specific gene expression and further emphasize the complexity of the molecular mechanisms which restrict gene expression to specific cell types. We provide a novel paradigm to study rPRL promoter function and hormone responsiveness independently of lactotroph cell-specific requirements.

Animals↗
PubMed1991

Change in charge of an unvaried heme contact residue does not cause a major change of conformation in cytochrome c.

The structure of the Ala38 variant of yeast iso-1-cytochrome c, in which the previously unchanged Arg38 has been replaced, has been characterised by NMR. The NMR data indicate that the structure of the Ala38 variant is very similar to that of the wild type protein. In particular, the heme environment and interactions of the heme macrocycle are shown to be preserved. Analysis of the chemical shift perturbations to the resonances of Ile35 is shown to be consistent with the change in charge at position 38. The only significant area of conformational change detected was at residues 39 and 58, close to the site of modification. Therefore the redox potential change accompanying the modification [1988, Biochemistry 28, 3188-3197] appears to be a direct consequence of the altered side-chain of residue 38 and not a result of secondary conformational changes induced by the modification.

Alanine↗
PubMed1990

Activation of the murine thyrotropin beta-subunit promoter by GH4 rat pituitary cell-free extracts.

Expression of the TSH beta subunit gene is restricted to the thyrotroph cells of the anterior pituitary. Previously we identified several AT-rich DNA elements within the murine (m) TSH beta 5'-flanking region, denoted as D1 (-253 to -227), P4 (-142 to -131), P3 (-126 to -112), P2 (-106 to -98), and P1 (-76 to -68) which bind thyrotroph-specific factor(s). These sites are related to, but distinct from GHF-1 and LSF-1 binding sites, which restrict GH and PRL gene expression to pituitary somatotrophs and lactotrophs, respectively. To determine whether different pituitary cell types contain related factors capable of activating the mTSH beta promoter, cell-free transcription studies were performed using extracts from GH4 rat pituitary somatomammotroph cells. AI-through the endogenous mTSH beta gene is not expressed in GH4 cells, in vitro transcription of the mTSH beta promoter, normalized to the Rous sarcoma virus internal control, revealed faithful transcription initiation from the authentic mTSH beta CAP sites in GH4 but not in HeLa cell extracts. Cell-free transcription analysis of mTSH beta 5'-deletion mutants revealed consistent promoter activity with deletion to position -46 but complete loss of activity when deleted to position -9. To better define the specific factors in pituitary somatomammotrophs which interact with and activate the mTSH beta promoter, DNase I protection and gel-shift studies were performed using extracts from GC rat pituitary somatomammotroph cells and DNA affinity-purified lactotroph-specific transcription factor, LSF-1, required for rat PRL promoter activity, and purified from GC cells. These cells contain a factor(s) which binds to thyrotroph-specific elements of the mTSH beta promoter. These studies also show that LSF-1 binds the D1 and proximal thyrotroph-specific elements of the mTSH beta promoter and is capable of reconstituting the trans-activation of the mTSH beta promoter in HeLa nonpituitary cell extracts in vitro. Conversely, nuclear factors present in TtT-97 murine thyrotrophs bind the proximal lactotroph-specific elements on the rPRL promoter. This in vitro transcription assay provides a means to biochemically dissect the trans-activation of the mTSH beta promoter and to determine the functional overlap of distinct pituitary cell-specific factors in regulating GH, PRL, and TSH beta gene expression.

Animals↗
PubMed1989

Detection of colony-stimulating factor messenger RNA in single T cells by in situ hybridization.

In situ hybridization has been used to study the accumulation of colony-stimulating factor (CSF) mRNA in single cells of a T lymphocyte clone (E9.D4) following antibody-mediated (F23.1) activation via the Ti-T3 complex in filler-independent bulk cultures. The specificity of hybridization for cellular RNA was demonstrated by pretreating the cells with the Ca2+-dependent enzyme micrococcal nuclease by using a novel protocol developed for use with riboprobes. Maximal levels of granulocyte-macrophage (GM) and multipotential-CSF (interleukin 3) mRNA were detected after 8-10 h, with GM-CSF mRNA being detected earlier and at a lower concentration of stimulus. The rise in intracellular mRNA was accompanied by an increase in the corresponding CSF bioactivity in the supernatant. In situ hybridization was of comparable sensitivity to Northern blot analysis and revealed significant heterogeneity in the accumulation of CSF mRNA within individual cells of the clone following stimulation with F23.1. This could account for the corresponding heterogeneity in CSF production by single cells. Under optimal conditions at least 25% of cells contained both transcripts. The method has been used to examine CSF production by normal spleen cells and should be useful in the further analysis of lymphokine gene regulation in single T cells.

Animals↗
PubMed1988

1H-n.m.r. and c.d. studies of haem orientational disorder in sperm-whale myoglobin and human haemoglobin.

1H-n.m.r. and c.d. studies on sperm-whale myoglobin show that the c.d. signal in the Soret region is inversely and linearly related to the proportion of minor isomer present. An alternative method, 'pH jump', is described for inducing orientational disorder in sperm-whale myoglobin without recourse to reconstitution. 1H-n.m.r. studies on human haemoglobin A indicate little heterogeneity in freshly isolated haemoglobin A, but the effect is enhanced in freeze-dried Sigma haemoglobin A.

Animals↗
PubMed1988

Specificity of riboprobes for intracellular RNA in hybridization histochemistry.

The specificity of the probe for intracellular RNA during hybridization histochemistry is usually confirmed by loss of the signal following ribonuclease pre-treatment of the target. Interpretation of such controls is complicated when single-stranded (asymmetric) RNA probes (riboprobes) are used for in situ hybridization, since the probes themselves may be degraded by residual ribonuclease. A protocol using the Ca2+-dependent enzyme, micrococcal nuclease, is presented to circumvent these difficulties.

Animals↗
PubMed1988

The detection and initial characterization of colony-stimulating factors in synovial fluid.

In this study which included 16 patients with inflammatory or non-inflammatory arthropathies, human granulocyte-macrophage colony-stimulating activity was detected in synovial fluid. This was attributable to the presence of colony-stimulating factor(s) (CSF), as a direct action on human bone marrow progenitor cells was demonstrated using clone transfer experiments. Samples of synovial fluid also stimulated the growth of murine macrophage colonies and induced differentiation in the murine myelomonocytic leukemia cell line, WEHI-3B(D+), which are characteristic properties of human macrophage-CSF or granulocyte-CSF respectively. These findings and the results of preliminary fractionation procedures suggested that the colony-stimulating activity in synovial fluid was not explicable by the presence of any one of the well-characterized human CSF acting in isolation. This provides a new insight into the pathogenesis of inflammatory arthropathies and supports the hypothesis that CSF have important roles in vivo in addition to the regulation of haemopoiesis.

Adult↗
PubMed1987

Transgenic mice expressing a hemopoietic growth factor gene (GM-CSF) develop accumulations of macrophages, blindness, and a fatal syndrome of tissue damage.

Transgenic mice carrying the murine granulocyte-macrophage colony stimulating factor (GM-CSF) gene expressed from a retroviral promoter exhibit elevated levels of GM-CSF in the serum, urine, peritoneal cavity, and eye. The eyes of transgenic mice are opaque, contain accumulations of macrophages, and develop retinal damage. Similarly, lesions containing macrophages develop in striated muscle. The mice also display an accumulation of large, often multinucleate, activated macrophages in the peritoneal and pleural cavities. The transgene is transcribed in peritoneal cells, as well as in eyes and infiltrated striated muscle. A high proportion of transgenic mice die with muscle wasting when aged 2-4 months, possibly because of macrophage activation resulting from the high levels of GM-CSF.

Animals↗
PubMed1987

Evidence for local synthesis of antibodies to denatured collagen in the synovium in rheumatoid arthritis.

Synovial fluid samples from 36 patients with rheumatoid arthritis (RA) and 31 patients with other articular diseases (OAD) were examined for the presence of antibodies to denatured or native human type II collagen. Levels of IgG antibodies to denatured or native human type II collagen, rheumatoid factor, immunoglobulins, and total proteins were assessed in paired samples of serum and synovial fluid from 21 patients with RA and from 14 patients with OAD. Solid-phase radioimmunoassay showed that levels of antibodies to denatured collagen in synovial fluid were significantly higher in RA patients than in OAD patients (median 3,270, range 44-16,816 versus median 919, range 119-5,814; P less than 0.001). These antibody levels were higher in synovial fluid than in the serum of RA patients, but not in patients with OAD. Paired serum and synovial fluid samples showed no correlation between the level of antibodies to denatured collagen and levels of either IgG, IgA, IgM, or rheumatoid factor. Synovial fluid antibodies to native collagen were higher in RA patients. Antibodies to collagen may be synthesized preferentially in synovial tissues and, hence, participate in the perpetuation of RA.

Adult↗
PubMed1987

Enhancement of neutrophil-mediated phagocytosis by human granulocyte-macrophage colony-stimulating factor demonstrated using a novel mathematical model.

A mathematical model is presented which may be applied to describe and analyse data from microscopic phagocytosis assays. The method has been used to investigate the phagocytosis of opsonized yeast by peripheral blood neutrophils treated with purified recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) in vitro. Under limiting conditions of serum opsonization, rH GM-CSF decreased the proportion of non-phagocytic cells and increased the mean number of ingested yeast per cell. Stimulation of phagocytosis was dose-dependent and occurred with concentrations of rH GM-CSF in the range 10-320 units/ml. The effect was dependent on a heat-labile component in serum and was not attributable to endotoxin contamination of the preparation.

Colony-Stimulating Factors↗
PubMed1987

Purification and properties of bacterially synthesized human granulocyte-macrophage colony stimulating factor.

Human granulocyte-macrophage colony stimulating factor (GM-CSF) has been synthesized in high yield using a temperature inducible plasmid in Escherichia coli. The human GM-CSF is readily isolated from the bacterial proteins because of its differential solubility and chromatographic properties. The bacterially synthesized form of the human GM-CSF contains an extra methionine residue at position 1, but otherwise it is identical to the polypeptide predicted from the cDNA sequence. The specific activity of 2.9 X 10(7) units/mg of protein for purified bacterially synthesized human GM-CSF indicates that despite the lack of glycosylation, the molecule is substantially in its native conformation. This molecule stimulated the same number and type of both seven- and 14-day human bone marrow colonies as the CSF alpha preparation from human placental conditioned medium. Human GM-CSF had no activity on murine bone marrow or murine leukemic cells. There was no detectable, direct stimulation of adult human erythroid burst forming units (BFU-E) by the bacterially synthesized human GM-CSF. Although impure preparations containing native human GM-CSF (eg, human placental conditioned medium) stimulated the formation of mixed colonies, even in the presence of erythropoietin, the bacterially synthesized human GM-CSF failed to stimulate the formation of mixed colonies from adult human bone marrow cells. The bacterially synthesized human GM-CSF increased N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced superoxide production and lysozyme secretion. Antibody-dependent cytotoxicity and phagocytosis by human neutrophils was stimulated by the bacterially synthesized human GM-CSF and eosinophils were also activated in the antibody-dependent cytotoxicity assay.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1987

Hemopoietic responses in mice injected with purified recombinant murine GM-CSF.

Normal adult BALB/c, C57BL, and C3H/HeJ mice were injected intraperitoneally three times daily for six days with 6-200 ng purified, bacterially synthesized, murine recombinant GM-CSF. Mice injected with 200 ng rGM-CSF developed a twofold increase in blood neutrophils. In the peritoneal cavity, a dose-related rise was observed in macrophages (up to 15-fold), neutrophils (10- to 100-fold) and eosinophils (10- to 100-fold). Peritoneal macrophages exhibited 15-fold increased mitotic activity (to 7.6/10(3) cells) and increased phagocytic activity for antibody-coated erythrocytes. Increased numbers of infiltrating neutrophils and monocytes were observed in the liver and lung. Dose-related rises were observed in spleen weight (up to 50%) and the spleen content of monocytes (twofold) and nonerythroid progenitor cells (up to fourfold). A dose-related fall occurred in total marrow cellularity (40%) and total nonerythroid progenitor cells (37%-66%), but levels of neutrophils and monocytes remained constant. The data indicate that the injection of rGM-CSF to normal mice increases overall numbers of granulocytes and macrophages and the phagocytic activity of macrophages and provides direct evidence for the conclusion that GM-CSF is likely to function in vivo as a regulator of these cell populations.

Animals↗
PubMed1986

Murine eosinophil differentiation factor. An eosinophil-specific colony-stimulating factor with activity for human cells.

A purified murine lymphokine, eosinophil differentiation factor (EDF), was found to be a selective stimulus for the clonal proliferation and differentiation of murine eosinophil progenitor cells, establishing it as the murine eosinophil colony-stimulating factor (Eo-CSF). EDF was also active on human eosinophil progenitors and mature blood eosinophils, but had no effect on neutrophil or macrophage precursor cells, nor on blood neutrophils. In culture of human bone marrow cells, EDF stimulated equal numbers and equal sizes of eosinophil colonies to develop when compared with human placental conditioned medium, a source of human CSFs, suggesting that all responsive progenitor cells were stimulated. Clone transfer experiments and the linear relationship between number of bone marrow cells plated and colonies produced confirmed that the action of EDF was directly on eosinophil progenitor cells. EDF increased the capacity of human blood eosinophils, but not neutrophils, to kill antibody-coated tumor cells and to phagocytose serum-opsonized yeast cells. This functional activation was associated with the enhanced expression of functional antigens (GFA-1, GFA-2, and the receptor for C3bi) on eosinophils. The possession by EDF (Eo-CSF) of all the properties expected of a human eosinophil CSF raises the possibility that a human analog of this molecule exists, and is involved in the regulation of production and function of human eosinophils in vivo.

Animals↗
PubMed1986

A comparison of eosinophil-activating factor (EAF) with other monokines and lymphokines.

Monocytes from moderately eosinophilic individuals secrete material that enhances the cytotoxic activity of eosinophils against antibody-coated schistosomula of Schistosoma mansoni. This material is not a single substance, but can be fractionated into several active components of different size and different charge. Gel filtration of mononuclear cell supernatants separated the eosinophil-activating activity into a major component of molecular mass of 40 kDa and a minor component of molecular mass of less than 10 kDa. The major component exhibited further heterogeneity on fractionation by high performance liquid chromatography. The bulk of the eosinophil-activating activity could be separated from both colony-stimulating factor (CSF) alpha activity and from tumor necrosis factor (TNF) activity. However, human recombinant CSF alpha (GM-CSF), human recombinant TNF and rabbit tumor necrosis serum all had eosinophil-activating activity when tested against schistosomula. Eosinophils were not activated by interleukin 1, interleukin 2, interferon-alpha, lipopolysaccharide or phorbol myristate acetate.

Colony-Stimulating Factors↗
PubMed1986

Recombinant human granulocyte-macrophage colony-stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression, and survival.

A purified recombinant human granulocyte-macrophage colony stimulating factor (rH GM-CSF) was a powerful stimulator of mature human eosinophils and neutrophils. The purified rH GM-CSF enhanced the cytotoxic activity of neutrophils and eosinophils against antibody-coated targets, stimulated phagocytosis of serum-opsonized yeast by both cell types in a dose-dependent manner, and stimulated neutrophil-mediated iodination in the presence of zymosan. In addition, rH GM-CSF enhanced N-formylmethionylleucylphenylalanine(FMLP)-stimulated degranulation of Cytochalasin B pretreated neutrophils and FMLP-stimulated superoxide production. In contrast, rH GM-CSF did not promote adherence of granulocytes to endothelial cells or plastic surfaces. rH GM-CSF selectively enhanced the surface expression of granulocyte functional antigens 1 and 2, and the Mo1 antigen. rH GM-CSF induced morphological changes and enhanced the survival of both neutrophils and eosinophils by 6 and 9 h, respectively. These experiments show that granulocyte-macrophage colony stimulating factor can selectively stimulate mature granulocyte function.

Antibody-Dependent Cell Cytotoxicity↗