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Biomedical subjects

P M Brickell

Publications and source records attributed to P M Brickell.

At least 55 records · Page 3Linked to original sources
PubMed1995

Epstein-Barr virus replication studies and their application to vector design.

Vectors containing elements of the Epstein-Barr virus genome are used primarily to maintain cloned DNA inserts as plasmids in mammalian cells. However, Epstein-Bar-virus-based vectors have also been valuable tools in the hands of those studying the life cycle of Epstein-Barr virus. In this article, we discuss those characteristics of Epstein-Barr virus and its life cycle that have been used in vector construction and describe methods that are particularly applicable to the use of Epstein-Barr-virus-based vectors.

DNA Replication↗
PubMed1995

Regulation of human growth hormone-binding protein production by human growth hormone in a hepatoma cell line.

The mechanism by which growth hormone-binding protein (GH-BP) is generated in humans remains unclear. To address this question, we analysed human GH-receptor/GH-BP gene expression in a human hepatoma cell line (HuH7). Northern hybridisation showed that HuH7 cells contain a single mRNA species hybridising with a probe for the sequences encoding the extracellular domain of the hGH-receptor/GH-BP. These data were confirmed by solution hybridisation methods. Thereafter, the cells were treated with r-hGH at physiological (12.5, 25, 50 ng/ml) and supra-physiological (150, 500 ng/ml) concentrations over the period of 48 h. At intervals, RNase protection assays were performed to determine GH-receptor/GH-BP mRNA levels, nuclear run-on assays were carried out to determine whether changes in mRNA levels represented changes in transcription rate, and a radio-ligand binding assay was performed to measure levels of GH-BP in the medium. We found that the r-hGH-regulated changes in GH-receptor/GH-BP mRNA levels detected with the probe for sequences encoding the extracellular domain of human GH-receptor/GH-BP were identical to those previously detected using a probe for the sequences encoding the transmembrane/intracellular domain of the human GH-receptor. In addition, we found that r-hGH had a rapid effect on the levels of GH-BP in the culture medium, which differed from its effect on the GH-receptor/GH-BP mRNA levels. Furthermore, lowering of temperature resulted in a decrease of GH-BP released into the medium implying that enzymes may be involved in the releasing mechanism. These data support the idea that GH-receptor and GH-BP are encoded by a single mRNA species in humans. In addition, they suggest that GH-BP levels are not an accurate reflection of GH-receptor/GH-BP mRNA levels, but that GH-BP production is subject to r-hGH-dependent post-transcriptional regulation, perhaps at the level of post-translational cleavage of the full-length GH-receptor protein. The notion that GH-BP measurements might represent GH-receptor status at the functional level must therefore be taken with caution.

Blotting, Northern↗
PubMed1995

Signalling by the p60c-src family of protein-tyrosine kinases.

The c-src gene family has nine known members (blk, c-fgr, fyn, hck, lck, lyn, c-src, c-yes and yrk), each encoding a cytoplasmic protein-tyrosine kinase (PTK) believed to be involved in signal transduction. The c-src PTKs contain three domains (SH1, SH2 and SH3) that are found in many other signalling proteins. The SH1 domain has PTK activity, whilst the SH2 and SH3 domains are involved in mediating protein-protein interactions by binding to phosphotyrosine-containing and proline-rich motifs, respectively. The expression patterns of the c-src PTKs suggest that they function in a broad range of biological situations, in many cases regulating the behaviour of terminally-differentiated, post-mitotic cell types. Targeted disruption of members of the c-src family in transgenic mice has confirmed important roles for p56lck and p59fym(T) in T-lymphocyte maturation and activation, but has also revealed unexpected roles for p60c-src in bone maintenance and for p59fym(B) in learning and memory. There is increasingly detailed information about the biochemical nature of the signalling pathways in which the c-src PTKs operate and about the other signalling proteins with which they interact. The c-src PTKs can associate with activated receptor PTKs, including the receptors for platelet-derived growth factor and epidermal growth factor, by means of SH2-phosphotyrosine binding. The c-src PTKs also associated with transmembrane proteins that lack PTK activity, frequently by means of interactions involving their unique amino-terminal sequences.

Animals↗
PubMed1995

Tumour necrosis factor-alpha (TNF-alpha) synthesis is associated with the skin and peripheral nerve pathology of leprosy reversal reactions.

Leprosy may be complicated by episodes of increased cell-mediated immunity towards Mycobacterium leprae (reversal reactions) which result in severe local immunopathology in skin lesions and peripheral nerves. Using in situ hybridization and MoAb techniques we have demonstrated TNF-alpha mRNA and TNF-alpha protein in macrophages infiltrating leprosy skin and peripheral nerve. Levels of TNF-alpha mRNA are significantly increased in reactional skin and nerve, particularly in borderline tuberculoid patients. TNF-alpha mRNA and TNF-alpha protein levels are higher in reactional nerves then reactional skin. In both reactional skin and nerve TNF-alpha mRNA is more abundant than TNF-alpha protein; this may reflect the rapid turnover of TNF-alpha protein in an immunologically dynamic situation, such as is seen in reversal reaction. Our findings emphasize the importance of documenting both mRNA and protein production when assessing the role of cytokines in pathology. The leprosy reversal reaction may be regarded as a useful model of tissue immunopathology in which TNF-alpha is generated as part of the host response to infection, but also produces local tissue damage.

Humans↗
PubMed1994

Transfection of vitamin D receptor cDNA into the monoblastoid cell line U937. The role of vitamin D3 in homotypic macrophage adhesion.

A 2-kB cDNA for the vitamin D receptor (VDR) was cloned in sense orientation into the plasmid pMEP4 (containing a cadmium-inducible metallothionein II promoter and a hygromycin-resistance selection gene) and transfected into monoblastoid U937 cells. The resultant cell line, DH39, expressed two species of VDR mRNA: 4.6-kb wild-type mRNA (present in native U937 cells or cells transfected with pMEP4 alone) and 2-kb transfected mRNA, which increased with cadmium treatment. Binding studies (using the active vitamin D metabolite, 1,25-dihydroxycholecalciferol (1,25-DHCC)) showed that DH39 cells contained five times more VDR per cell than controls, and ten times more after cadmium treatment. DH39 were sensitive to 1,25-DHCC: adding cadmium with 100 nM 1,25-DHCC for 72 h completely inhibited proliferation and induced concomitant differentiation. Unlike control cells, differentiation of DH39 by 1,25-DHCC led to homotypic cell-cell adhesion and formation of macrophage clusters. FACS analysis showed that 1,25-DHCC increased the number of cells expressing CD11b in both DH39 and controls, and the number of cells expressing CD11c in DH39. There was a quantitative increase in mean fluorescence intensity of expression of CD11a and CD18 in DH39. Northern blotting showed increased CD11a and CD18 mRNA in DH39. Ab inhibition of 1,25-DHCC-induced homotypic adhesion showed that CD11a/18 mediated the cell-cell clustering. CD50 expression was decreased on DH39, but the CD11a/18 ligand implicated was CD54. DH39 provides a model system not only for investigating the VDR role in 1,25-DHCC anti-proliferative effects, but also for regulation of homotypic macrophage adhesion mechanisms that are important in disease pathogenesis.

Blotting, Northern↗
PubMed1994

Src-related protein tyrosine kinases are physically associated with the surface antigen CD36 in human dermal microvascular endothelial cells.

Src-related cytoplasmic PTKs are physically and functionally associated with cell surface receptors and are involved in signal transduction. In this paper we report the identification of src-related proteins p59fyn, pp60c-src and p62yes in human microvascular endothelial cells cultured from normal human skin and their physical association with the thrombospondin receptor CD36. Such an association represents a potential signalling pathway by which thrombospondin may regulate angiogenesis.

Antigens, CD↗
PubMed1994

The chicken retinoid-X-receptor-gamma gene gives rise to two distinct species of mRNA with different patterns of expression.

Retinoids are metabolites of vitamin A that can regulate gene expression in a range of embryonic and adult cell types. They do this by binding to nuclear receptors belonging to the steroid/thyroid hormone receptor superfamily of ligand-activated transcription factors. Vertebrates possess two classes of nuclear retinoid-receptor genes, each with three members. These are the RAR-alpha, RAR-beta and RAR-gamma genes and the RXR-alpha, RXR-beta and RXR-gamma genes. In this paper we show by cDNA cloning and ribonuclease protection that the chicken RXR-gamma gene gives rise to two mRNA species (RXR-gamma 1 and RXR-gamma 2) that differ at their 5' ends. The two mRNAs have different tissue distributions in the 10-day-old chick embryo. RXR-gamma 2 mRNA was present in the eye and dorsal root ganglia but was undetectable in the liver. In contrast, RXR-gamma 1 mRNA was present in liver, was undetectable in dorsal root ganglia and was just detectable in the eye, where it was much less abundant than RXR-gamma 2 mRNA. The predicted protein products of the RXR-gamma 1 and RXR-gamma 2 mRNAs differ at their N-termini, in a region thought to modulate transcriptional transactivation by the receptor. These results show that at least one of the retinoid-X-receptor (RXR) genes gives rise to more than one protein product, a principle previously established for the retinoic acid-receptor (RAR) genes. The existence of multiple RXR protein isoforms would increase the range of heterodimers formed between RXRs and other nuclear receptors, including RARs and the receptors for thyroid hormone, vitamin D and peroxisome proliferators. This could increase the diversity of transcriptional responses mediated by these molecules.

Amino Acid Sequence↗
PubMed1994

Expression patterns of the bone morphogenetic protein genes Bmp-4 and Bmp-2 in the developing chick face suggest a role in outgrowth of the primordia.

Bone morphogenetic proteins BMP-4 and BMP-2 are closely-related members of the transforming growth factor-beta superfamily that have been implicated in signalling in a number of developmental systems. To determine whether they could be involved in the epithelial-mesenchymal interactions that control face development, we mapped the distribution of Bmp-4 and Bmp-2 gene transcripts in the developing chick facial primordia. At stages when primordia were becoming established, Bmp-4 transcripts were present in specific regions of epithelium in all facial primordia, but were undetectable in the mesenchyme. Bmp-4 transcripts appeared subsequently in specific regions of mesenchyme at the distal tips of the primordia. This mesenchymal expression first appeared in the frontonasal mass and then, in turn, in the lateral nasal processes, the maxillary primordia and the mandibular primordia. There was a complex relationship between domains of epithelial and mesenchymal Bmp-4 expression, and at many sites there was an inverse correlation between epithelial and mesenchymal Bmp-4 expression. Bmp-2 transcripts were found in the epithelium and mesenchyme of the maxillary and mandibular primordia at early stages in facial development. Bmp-2 transcripts appeared in the frontonasal mass and lateral nasal processes at later stages, with epithelial expression preceding mesenchymal expression. In general, mesenchymal Bmp-2 expression was associated with overlying epithelial Bmp-2 expression. The domains of Bmp-4 expression overlapped with those of Bmp-2, but detailed examination showed that there was no precise correlation between the expression patterns of the two genes. Indeed, in some places the Bmp-4 and Bmp-2 expression domains were complementary. The expression of the Bmp-4 and Bmp-2 genes in the epithelium and distal mesenchyme of the facial primordia suggests that BMP-4 and BMP-2 may be involved in the epithelial-mesenchymal interactions that control outgrowth of these primordia.

Animals↗
PubMed1994

Bone morphogenetic proteins and a signalling pathway that controls patterning in the developing chick limb.

We show here that bone morphogenetic protein 2 (BMP-2) is involved in patterning the developing chick limb. During early stages of limb development, mesenchymal expression of the Bmp-2 gene is restricted to the posterior part of the bud, in a domain that colocalizes with the polarizing region. The polarizing region is a group of cells at the posterior margin of the limb bud that can respecify the anteroposterior axis of the limb when grafted anteriorly and can activate expression of genes of the HoxD complex. We dissect possible roles of BMP-2 in the polarizing region signalling pathway by manipulating the developing wing bud. Retinoic acid application, which mimics the effects of polarizing region grafts, activates Bmp-2 gene expression in anterior cells. This shows that changes in anteroposterior pattern are correlated with changes in Bmp-2 expression. When polarizing region grafts are placed at the anterior margin of the wing bud, the grafts continue to express the Bmp-2 gene and also activate Bmp-2 expression in the adjacent anterior host mesenchyme. These data suggest that BMP-2 is part of the response pathway to the polarizing signal, rather than being the signal itself. In support of this, BMP-2 protein does not appear to have any detectable polarizing activity when applied to the wing bud. The pattern of Bmp-4 gene expression in the developing wing bud raises the possibility that BMP-2 and BMP-4 could act in concert. There is a close relationship, both temporal and spatial, between the activation of the Bmp-2 and Hoxd-13 genes in response to retinoic acid and polarizing region grafts, suggesting that expression of the two genes might be linked.

Amino Acid Sequence↗
PubMed1993

Effects of the synthetic anti-oxidant, probucol, on the U937 monoblastoid cell line.

Probucol is a clinically important drug that decreases plasma cholesterol in humans and has a marked anti-atherogenic effect in hyperlipidaemic Watanabe rabbits. The action of probucol in this animal model has been partly attributed to its anti-oxidant abilities. Probucol can decrease the oxidative modification of low-density lipoprotein and hence diminish its uptake by macrophages. In this paper, we have examined the effect of probucol on the monoblastic cell line U937 and on U937 cells induced to differentiate towards a macrophage phenotype by 1,25-dihydroxycholecalciferol (DHCC), tumour necrosis factor-alpha (TNF-alpha) or phorbol myristate acetate (PMA). We found that probucol enhanced the proliferation of undifferentiated U937 cells. Probucol also enhanced proliferation in cultures that had been pre-treated with DHCC or TNF-alpha, but had no effect on cultures that had been pre-treated with PMA. In contrast, when U937 cells were treated simultaneously with probucol and DHCC or TNF-alpha, there was a more marked decrease in proliferation than was induced by these agents in the absence of probucol. Probucol had little effect on the phenotype of resultant cells. The surface expression of CD13 (aminopeptidase N), CD4, CD35 (C3b receptor), CD64 (Fc gamma RI), CD71 (transferrin receptor) and HLA Class II was not affected by probucol. Probucol treatment led to a small increase in the surface expression of CD16 (Fc gamma RIII) in TNF-alpha treated cells and to a small decrease in the expression of CD14 (a monocyte marker) in PMA-treated cells. The induction of c-fgr mRNA and TNF-alpha mRNA by DHCC or PMA or TNF-alpha was not significantly altered in the presence of probucol. The affect of probucol on U937 cells does not appear to be due to its anti-oxidant abilities because butylated hydroxytoluene (BHT), an equally powerful anti-oxidant, did not have the same effect on the cell proliferation as probucol and because no changes were detected in the levels of lipid peroxidation in U937 cell culture supernatants.

Antigens, Surface↗
PubMed1993

Retinoic acid induces changes in c-fgr proto-oncogene mRNA levels in Burkitt's lymphoma cells.

The c-fgr proto-oncogene is expressed in Burkitt's lymphoma (BL) cell and cell lines derived from them. When Epstein-Barr virus (EBV)-negative BL cell lines that contain low levels of c-fgr mRNA are infected with EBV, transcription of the c-fgr gene is further induced. In this paper we show that treatment of EBV-negative and EBV-positive BL cell lines with all-trans retinoic acid also stimulates an increase in c-fgr mRNA levels, varying between 2- and 13-fold depending on the cell line. An increase is detectable 12 to 48 h after treatment, depending on the cell line, suggesting that the c-fgr gene is not regulated directly by retinoic acid but responds to other retinoic acid-induced changes in the cell. We also show that treatment of BL cell lines with all-trans retinoic acid either results in a dose-dependent decrease in growth rate, or has no effect on growth, depending on the cell line. It has previously been suggested that the c-fgr gene product might have a role in regulating the growth of BL cells, since treatment of the EBV-positive BL cell line Daudi with alpha-interferon results in a decrease in c-fgr mRNA levels followed by a decrease in growth rate. Our data indicate that there is no general correlation between c-fgr mRNA levels and growth rate in BL cells and so argue against a role for the c-fgr gene product in growth regulation in these cells.

Burkitt Lymphoma↗
PubMed1992

Structural and functional evidence for activation of a chick retinoid X receptor by eicosanoids.

The retinoid X receptors (RXR-alpha, RXR-beta and RXR-gamma) are members of the steroid-thyroid hormone receptor superfamily of ligand-dependent transcription factors. They appear to function as auxiliary proteins that regulate high-affinity DNA binding and enhance transcriptional activity through heterodimer formation with other members of the superfamily. The RXR-alpha, RXR-beta and RXR-gamma proteins bind and are activated by the naturally occurring retinoid, 9-cis-retinoic acid. Structural similarities are apparent between retinoic acid and various eicosanoids, raising the possibility that eicosanoids may also activate retinoid receptors in vivo. We present evidence that lipoxygenase metabolites of arachidonic acid at submicromolar concentrations are capable of activating RXR-gamma activity in transient transfection assays. In addition, molecular modelling predicts conformational similarities between some lipoxygenase products and retinoic acid. Consistent with this, hydroxyeicosatetraenoic acids are known to mimic some actions of retinoids in cell-based assays. These observations raise the possibility that eicosanoids, already known to act both as local hormones and as intracellular second messengers, may also have a direct role in transcriptional activation via nuclear receptors.

Animals↗
PubMed1992

Loss of transcriptional repression contributes to the ectopic expression of the calcitonin/alpha-CGRP gene in a human lung carcinoma cell line.

The calcitonin/alpha-CGRP (CT/CGRP) gene is ectopically expressed in a wide variety of neoplasia. We have investigated the molecular mechanisms responsible for this ectopic expression in the human cell line BEN, which is derived from a poorly differentiated squamous cell lung carcinoma. We show that a trans-acting factor which represses expression of the CT/CGRP gene in HeLa cells is absent or inactive in BEN cells, and have localised the repressor binding site to a 53 bp fragment 1500 bp upstream of the transcription start site.

Base Sequence↗
PubMed1992

Position-dependence of retinoic acid receptor-beta gene expression in the chick limb bud.

Retinoic acid and 3,4-didehydroretinoic acid are metabolites of vitamin A that can induce duplications and other malformations when locally applied to the anterior margin of the chick limb bud. There is evidence that they may be natural signaling substances in the limb bud. Both compounds are thought to act by binding to ligand-dependent transcription factors that belong to the steroid/thyroid hormone nuclear receptor superfamily. In situ hybridization analyses show that in the mesenchyme of the chick wing bud between embryonic stages 20 and 27, retinoic acid receptor-beta (RAR-beta) transcripts are restricted to the proximal region of the bud and are present at highest levels in the region of the limb bud mesenchyme that contributes to the shoulder. We have performed grafting experiments in order to examine whether RAR-beta gene expression in limb bud mesenchyme cells is cell-autonomous or whether it is dependent upon the cell's position within the limb bud. When tissue from the proximal region of the stage 22 wing bud, which contains high levels of RAR-beta transcripts, was grafted to the distal tip of the bud, RAR-beta transcripts were undetectable in the graft 6 hr later. When tissue from the distal tip of the bud was grafted to a proximal site, most of the grafts exhibited a slight increase in the level of RAR-beta transcripts, which was detectable 6 hr after grafting. However, the levels of RAR-beta transcripts in these grafts never approached those found in the proximal core of the bud. These data indicate that RAR-beta gene expression in the chick wing bud is position-dependent in that it is repressed at the distal tip of the bud and partially activated by grafting distal tissue to a proximal site. However, accumulation of RAR-beta transcripts to high levels appears to be a characteristic of mesenchyme that was initially specified to form proximal structures.

Animals↗
PubMed1992

The use of the polymerase chain reaction in prenatal diagnosis of growth hormone gene deletions.

OBJECTIVE: Familial isolated growth hormone deficiency (IGHD) type IA is characterized by a complete absence of human growth hormone (hGH) resulting in most cases from either a 6.7 or 7.7 kb deletion of DNA containing the hGH-1 gene. These patients have a strong initial anabolic response to exogenous recombinant hGH (r-hGH) therapy, frequently associated with the development of immune intolerance to r-hGH which causes an arrest of response to r-hGH replacement. This disorder is inherited as an autosomal recessive trait. PATIENTS AND DESIGN: In two pregnancies at risk, the polymerase chain reaction (PCR) was applied as a method for identifying hGH-1 gene deletions in DNA obtained by chorionic villus sampling (CVS) in the first trimester. RESULTS: Homozygotes for the 6.7kb deletion of DNA containing the hGH-1 gene were easily and conclusively detected by the absence of 1900, 761 and 712bp fragments after SmaI digestion of the polymerase chain reaction products. In contrast, the pattern found in heterozygotes for the hGH-1 gene deletion was difficult to distinguish from the pattern found in normal homozygotes. CONCLUSIONS: We conclude that the polymerase chain reaction method is valuable for diagnosing individuals who are homozygous for hGH-1 gene deletions, while heterozygotes and normal individuals may be difficult to distinguish from each other. We suggest that, in these cases, Southern blotting remains the analysis to perform.

Blotting, Southern↗
PubMed1992

Development of the spatial pattern of retinoic acid receptor-beta transcripts in embryonic chick facial primordia.

Retinoic acid causes a range of embryonic defects, including craniofacial abnormalities, in both birds and mammals and is believed to have a number of roles in normal development. We have previously shown that the distribution of retinoic acid receptor-beta (RAR-beta) transcripts is spatially restricted within the neural-crest-derived upper beak primordia of the chick embryo. We have now used in situ hybridisation to trace the distribution of RAR-beta transcripts during the migration of cranial neural crest cells and during formation of these primordia. RAR-beta transcripts were present in a subset of migrating neural-crest-derived cells in the head of the stage 10 embryo. These cells were situated in pathways followed by cells that migrate from the neural crest overlying the posterior prosencephalic/anterior mesencephalic region of the developing brain. Cells containing RAR-beta transcripts accumulated around the developing eyes and in the regions of the ventral head from which the upper beak primordia later develop. We mapped the distribution of RAR-beta transcripts as the facial primordia were forming, with particular reference to the development of the maxillary primordia. We found that these form in a region of the ventral head that includes the boundary between regions of high and low levels of RAR-beta transcripts. The boundary between these two groups of cells persisted as the maxillary primordia developed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗