PubMed Health⌕ Search

Biomedical subjects

P M Brickell

Publications and source records attributed to P M Brickell.

At least 19 recordsLinked to original sources
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↗
PubMed1992

The p60c-src family of protein-tyrosine kinases: structure, regulation, and function.

In 1911, Peyton Rous reported that a fibrosarcoma could be transmitted between chickens in a cell-free extract of the tumor. The transmissible agent, Rous sarcoma virus (RSV), transforms cells by virtue of the presence within its genome of a viral oncogene, v-src, which is derived from a normal cellular gene that has been picked up, or transduced, by the virus. This cellular proto-oncogene, c-src, encodes a protein, p60c-src, which has the ability to phosphorylate proteins on tyrosine residues. Studies of RSV were thus directly responsible for the discovery of cellular proto-oncogenes and of protein-tyrosine kinases, discoveries which have been fundamental in shaping our ideas about cellular growth control. In spite of this, the normal biological role of p60c-src is still unclear and it remains impossible to provide a full answer to the question of how RSV causes tumors. It is clear, however, that c-src is the prototype of a family of at least 8 closely related genes encoding protein tyrosine kinases, the other family members being blk, c-fgr, fyn, hck, lck, lyn, and c-yes. The purpose of this review is to outline our current knowledge of the structure, expression pattern, and function of each of the members of the c-src gene family and to describe recent data which begins to explain how these proteins interact with other cellular proteins to control cell behavior. The evidence for involvement of these proteins in oncogenesis is also discussed.

Amino Acid Sequence↗
PubMed1992

Regulation of c-fgr messenger RNA levels in U937 cells treated with different modulating agents.

The U937 cell line was used to investigate the induction of messenger RNA (mRNA) for the c-fgr mRNA tyrosine kinase proto-oncogene in cells of the monocyte-macrophage lineage. U937 cells were exposed to tumour necrosis factor-alpha (TNF-alpha), TNF-beta and transforming growth factor-alpha (TGF-alpha), alone and in combination with PMA and 1,25-dihydroxycholecalciferol (1,25-DHCC). TNF-alpha and TNF-beta, but not TGF-alpha, decreased the proliferation of U937 cells in a time- and dose-dependent manner, and both TNF-alpha and TNF-beta enhanced the response of U937 cells to PMA during the first 24 hr of treatment and to 1,25-DHCC over 72 hr. TNF-alpha induced a rapid increase in c-fgr mRNA levels within 4 hr, in contrast to slower induction by PMA and 1,25-DHCC. TNF-alpha and 1,25-DHCC together had an additive effect on c-fgr mRNA levels. In U937 cells exposed to PMA, c-fgr mRNA levels continued to increase over 72 hr. Levels of c-fgr mRNA induced by the various modulating agents did not correlate clearly with the changes in proliferation. Therefore, we suggest that although the c-fgr gene product may have a role in differentiation, the more significant role is likely to be in the fully differentiated macrophage.

Animals↗
PubMed1991

A member of the chicken RXR family of nuclear receptors activates transcription in response to retinoic acid.

The chicken cRXR nuclear receptor is a member of the steroid/thyroid hormone receptor superfamily. In this paper we show that cRXR can transactivate gene expression in response to retinoic acid, but that its sensitivity to retinoic acid is lower than that of the hRAR-beta receptor. We have also compared the ability of cRXR and hRAR-beta to respond to a panel of other retinoids. Unlike hRAR-beta, cRXR failed to respond to the naturally occurring retinoid 3,4-didehydro-retinoic acid or to the synthetic retinoid TTNPB, both of which share the ability of retinoic acid to induce digit duplications when locally applied to chick limb buds.

Animals↗
PubMed1991

Transgenic mice carrying the guinea-pig alpha-lactalbumin gene transcribe milk protein genes in their sebaceous glands during lactation.

We have generated transgenic mice carrying the entire guinea-pig alpha-lactalbumin gene. Lactating transgenic mice expressed high levels of correctly initiated and processed guinea-pig alpha-lactalbumin mRNA in the secretory epithelium of their mammary glands, and secreted guinea-pig alpha-lactalbumin in their milk. Transcripts were detectable after 7 days of pregnancy, indicating that the transgene was under correct hormonal control. Whereas no or negligible transcription was detectable in all other tissues tested, high levels of transcripts were found in the skin of lactating transgenic mice. Guinea-pig alpha-lactalbumin protein was undetectable in the skin, however. In situ hybridization analysis showed that expression was localized to the undifferentiated cells in the basal layer of the sebaceous glands. Further studies revealed high levels of endogenous beta-casein mRNA in normal lactating mouse skin, demonstrating that the transcription of milk protein genes in lactating mouse skin is a normal event, and is not peculiar to the transgene. This surprising finding highlights the developmental relationship of the mammary gland to other specialized structures of the skin, supports a role for epithelial-extracellular matrix interactions in the regulation of milk protein gene expression in vivo, and identifies the skin as a particularly accessible model system in which to study the regulation of milk protein gene expression. In addition, the guinea-pig alpha-lactalbumin gene will be a source of regulatory sequences with which to direct heterologous gene expression to the sebaceous glands of transgenic mice.

Animals↗
PubMed1991

Regulation of human growth hormone receptor gene expression by human growth hormone in a human hepatoma cell line.

We have investigated the effects of recombinant human growth hormone (r-hGH) on the expression of hGH-receptor in a human hepatoma cell line (HuH 7). Levels of hGH-receptor mRNA in HuH 7 cells treated with different doses of r-hGH were measured by means of an RNase protection assay. Treatment with r-hGH at physiological concentrations (12.5, 25 and 50 ng/ml) resulted in an increase in hGH-receptor mRNA levels within 1 h of addition of the hormone. A steady state was reached after 3-4 h and maintained for at least 48 h. In contrast, treatment with supraphysiological r-hGH concentrations (150 and 500 ng/ml) led to a down-regulation of hGH-receptor mRNA levels during the first 3 h after hormone addition followed by an increase in hGH-receptor mRNA levels thereafter. Nuclear run-off assays demonstrated that these changes in hGH-receptor mRNA levels were a result of changes in the rate of transcription of the hGH-receptor gene. Cycloheximide (10 micrograms/ml) did not affect these changes in hGH-receptor gene transcription significantly, indicating that they are mediated by pre-existing factors and do not require new protein synthesis. These data demonstrate that r-hGH specifically regulates the rate of transcription of the hGH-receptor gene in a human hepatoma cell line.

Blotting, Northern↗
PubMed1991

Activation of endogenous retroviral transcription in SV40-transformed mouse cells.

We have previously isolated a number of cDNA clones that correspond to mRNAs present at higher levels in SV40-transformed cells than in the untransformed parental cells (Scott, M.R.D., Westphal, K.-H. and Rigby, P.W.J. (1983) Cell 34, 557-567). We have now determined the nucleotide sequence of the archetypal Set 2 clone, pAG59, and can thus identify it as corresponding to the env gene of the endogenous, ecotropic C-type retrovirus of Balb/c mice, Emv-1. We have shown that in the subset of SV40-transformed cells that express the provirus both of the proteins encoded by env, gp70 and p15E, are synthesised and that the former is displayed on the cell surface. We discuss the significance of these observations for the biology of SV40 transformation.

Animals↗
PubMed1991

Growth characteristics and response to growth hormone therapy in patients with hypochondroplasia: genetic linkage of the insulin-like growth factor I gene at chromosome 12q23 to the disease in a subgroup of these patients.

Hypochondroplasia, a heterogeneous and usually mild form of chondrodystrophy, is a common cause of short stature. It often goes unrecognized in childhood and is diagnosed in adult life when disproportionate short stature becomes obvious. We performed restriction enzyme analysis of the insulin-like growth factor I (IGF-I) gene on the families of 20 white British Caucasian children with short stature attributed to hypochondroplasia by radiological and clinical criteria, who were undergoing human growth hormone (r-hGH) treatment, in 60 children with isolated growth hormone deficiency and in 50 normal individuals. The frequency of the heterozygous pattern (Hind III: 8.2, 5.2, 4.8, 3.2 kb fragments, Pvu: 8.4, 5.1, 4.7, 2.5 kb fragments) in children with hypochondroplasia was significantly higher (chi2: P less than 0.05) than in the control groups. The hypochondroplastic children whose response to r-hGH treatment was characterized by a proportionate increase in both spinal and subischial leg length were all heterozygous for two co-inherited IGF-I gene restriction fragment length polymorphism (RFLP) alleles (Hind III: 5.2, 4.8 kb; Pvu II: 5.1, 4.7 kb). Children whose response was characterized by accentuation of the body disproportion by r-hGH treatment were all homozygous for these alleles (Hind III: 4.8, 4.8 kb; Pvu II: 4.7, 4.7 kb). Their response to r-hGH treatment is significantly different (P less than 0.01). Studies of the families of the heterozygous affected children demonstrated strong linkage (lod score 3.311 at zero recombination) of the IGF-I gene locus at chromosome 12q23 to this subgroup of hypochondroplasia. The 5.2 kb Hind III and 5.1 kb Pvu II alleles are in strong linkage disequilibrium with this trait. These data indicate that IGF-I gene may be a candidate gene for involvement in the aetiology of short stature presenting with hypochondroplastic features and a proportionate response to r-hGH treatment; they also provide support for the concept of genetic heterogeneity in chondrodystrophy.

Achondroplasia↗
PubMed1991

The c-fgr proto-oncogene: expression in Epstein-Barr-virus-infected B lymphocytes and in cells of the myelomonocytic and granulocytic lineages.

The c-fgr proto-oncogene, which is a member of the c-src gene family, encodes the cytoplasmic tyrosine kinase p55c-fgr. Expression of the c-fgr gene is activated in human B lymphocytes following infection with Epstein-Barr virus, and the viral protein EBNA-2 is involved in mediating this effect. The only normal cells in which the c-fgr gene is known to be expressed are peripheral-blood granulocytes and monocytes, and tissue macrophages. In accordance with this, levels of c-fgr mRNA increase when the human promonocytic cell line U937 and the human myelomonocytic cell line HL60 are induced to differentiate. The enzyme activity and the expression pattern of p55c-fgr suggest that it is involved in regulating the responses of terminally differentiated granulocytes and monocytes to external stimuli, perhaps by controlling changes in cytoskeletal structure.

B-Lymphocytes↗
PubMed1991

Constitutionally short stature: analysis of the insulin-like growth factor-I gene and the human growth hormone gene cluster.

It has been suggested that modifications of the IGF-I gene might be responsible for constitutionally short stature (CSS). To assess this possibility, and to examine the possible role of the hGH gene cluster in CSS, we used Southern blotting and restriction fragment length polymorphism (RFLP) linkage analysis to study 57 children with CSS, including 17 children from 10 families with CSS. We studied as control populations 50 children and 25 adults of normal height, including three families, and 60 children with isolated growth hormone deficiency. No gross structural abnormalities of the IGF-I gene or the hGH gene cluster were found in any of the subjects. The allelic frequencies for each of two distinct RFLP associated with the IGF-I gene (EcoRV and HindIII/PvuII) and for each of five distinct RFLP associated with the hGH gene cluster [BglII(A), BglII(B), MspI(A), MspI(B), and HincII] were the same in the CSS, isolated growth hormone deficiency, and normal populations, and the RFLP haplotypes for both the IGF-I gene and the hGH gene cluster also appeared with the same frequency in all three populations. Analysis of the families that contained heterozygotes for the HindIII/PvuII and/or EcoRV IGF-I RFLP excluded a linkage of the IGF-I gene to CSS in these families. Assuming that the inheritance is dominant, the lod score at 1% recombination was -2.2, whereas assuming a recessive inheritance, the lod score at 1% recombination was -1.93.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Southern↗
PubMed1991

A member of the RXR nuclear receptor family is expressed in neural-crest-derived cells of the developing chick peripheral nervous system.

Retinoic acid (RA) affects differentiation and morphogenesis in various developmental systems and is believed to act through nuclear RA receptors that belong to the steroid/thyroid hormone family of ligand-binding transcription factors. Three closely related receptors, RAR-alpha, -beta and -gamma, with distinct expression patterns, have been identified and a fourth receptor, hRXR-alpha, which responds to RA but which has low homology to RAR-alpha, -beta and -gamma, was recently discovered. Here we report the isolation of a cDNA clone encoding a chicken homologue of hRXR-alpha (cRXR) and show that a cRXR transcript of 2.5 kb is expressed in a range of embryonic chick tissues. By in situ hybridization to sections from stage 24 and stage 27 chick embryos, we show that cRXR transcripts are expressed at high levels in the liver and in elements of the developing peripheral nervous system derived from the neural crest, including dorsal root ganglia, cranial ganglia, enteric ganglia and peripheral nerve tracts. At stage 16, in the posterior trunk region, cRXR transcripts are expressed by cells in the neural crest and in neural crest cells migrating into the sclerotome, indicating that neural crest cells express cRXR transcripts before overt differentiation into peripheral nervous tissue. This distribution suggests a novel role for RA in the developing peripheral nervous system, mediated by cRXR. In addition, it identifies cRXR as a marker for a specific population of neural-crest-derived cells.

Amino Acid Sequence↗
PubMed1991

Retinoic acid treatment alters the distribution of retinoic acid receptor-beta transcripts in the embryonic chick face.

Retinoic acid is a metabolite of vitamin A that can act as a signalling molecule in a number of developmental systems. Retinoic acid is also known to be teratogenic in mammals, causing a range of defects including abnormalities in craniofacial development. Exposure of the developing chick face to retinoic acid released slowly from a bead implanted in the wing bud results in a specific facial defect, in which outgrowth of the frontonasal mass is inhibited. This results in clefting of the primary palate and absence of the upper beak. To investigate the role of nuclear retinoic acid receptors in normal and abnormal chick face morphogenesis, we isolated chick retinoic acid receptor-beta (RAR-beta) cDNA clones and probed northern blots of RNA isolated from chick embryos at stages 22, 24 and 25 and from adults. RAR-beta transcripts of 2.8 and 3.5 kb were present in several regions of the embryo, including the facial primordia, and were also present at much lower levels in adult tissues. In situ hybridisation showed that RAR-beta transcripts were present in all of the facial primordia at embryonic stages 20, 24 and 28, but that their distribution was not uniform. Transcripts were abundant in the lateral nasal processes, at the edges and corners of the frontonasal mass and in the anterior part of the maxillary primordia. Lower levels were present elsewhere. Treatment of stage 20 embryos with retinoic acid altered the distribution of RAR-beta transcripts in the maxillary primordia, such that high levels of transcripts were present throughout, rather than being confined to the anterior part. This change was detectable at stage 24, before any alterations in the morphology of the facial primordia were apparent. By stage 28, when the morphology of the facial primordia was clearly abnormal, there were more widespread changes in the distribution of RAR-beta transcripts. These results show that RAR-beta transcripts are particularly concentrated in regions of the primordia that give rise to the upper beak, the development of which is specifically affected by retinoic acid. In addition, they demonstrate that retinoic acid can induce changes in the pattern of expression of RAR-beta transcripts in vivo.

Amino Acid Sequence↗
PubMed1991

Identification of antisense transcripts of the chicken insulin-like growth factor-II gene.

Insulin-like growth factor-II (IGF-II) is a polypeptide mitogen which is believed to play an important role in fetal development. The human and rat IGF-II genes are complex transcription units, which contain multiple promoters and polyadenylation sites and which exhibit alternate splicing of their primary transcripts. In order to study IGF-II gene expression during chick embryonic development, we screened a 10-day chick embryo cDNA library with a human IGF-II cDNA probe. We isolated a clone, designated cigf, that was comprised, in part, of sequences homologous to the second coding exon of the human, mouse and rat IGF-II genes. Comparison of the nucleotide sequence of cigf with that of the corresponding genomic clone indicated that cigf was derived from a spliced antisense transcript of the chicken IGF-II gene, which overlapped the second coding exon. Northern blotting experiments with single-stranded RNA probes synthesized using cigf DNA as a template showed that stage 22 and stage 36 chick embryos contained sense strand IGF-II transcripts of 1.4, 2.2, 4.7 and 7.0 kb and antisense strand transcripts of 0.7, 1.3, 1.8, 2.5, 4.9, 6.0 and 8.0 kb. The pattern of sense strand IGF-II transcripts was similar to that previously found in rat fetal tissues. Whilst there are precedents for the transcription of both strands of a single gene, this is the first evidence for antisense transcription of an IGF gene. The functions of the antisense transcripts remain to be determined. These findings demonstrate a further level of complexity in the IGF-II transcription unit and indicate that studies of IGF-II transcript distribution performed with double-stranded probes should be interpreted with caution. They also suggest explanations for the recent finding that IGF-II peptides are present at much lower levels in embryos than expected from the high levels of IGF-II transcripts present.

Animals↗