Biomedical subjects

M Peacocke

Publications and source records attributed to M Peacocke.

At least 55 records · Page 3Linked to original sources

PubMed1994

A beta 2RARE-LacZ transgene identifies retinoic acid-mediated transcriptional activation in distinct cutaneous sites.

Retinoic acid and its derivatives (retinoids) exert profound influences on epithelial growth differentiation in a variety of tissues, including the skin. How retinoic acid mediates these effects is not fully understood. The recent cloning of a series of nuclear receptors for retinoic acid (RARs) has demonstrated that these proteins can function as ligand-inducible transcriptional enhancing factors. Moreover, all receptors are members of the steroid/thyroid hormone multigene family. In vitro studies have demonstrated the expression of RAR alpha, RAR beta, and RAR gamma in various cell types found in the skin. While multiple isoforms exist for each of the three RARs, it is unclear where each of these receptors functions in vivo to mediate the tissue-specific effects of retinoic acid. As a first step in determining sites of retinoic acid-mediated transcriptional activation in the skin and its appendages, we developed a transgenic model in which the retinoic acid response element (RARE) of the RAR beta 2 isoform is linked to a beta-galactosidase reporter gene. Our observations consistently demonstrate that retinoic acid transcriptionally activates the beta 2RARE in distinct areas of the skin. Of interest, certain of these areas are known to contain stem cells. These data clearly demonstrate that this type of transgenic "reporter" model can be used to further define retinoic acid-regulated signal transduction pathways in the skin, as well as other complex tissues. Furthermore, these observations raise the possibility that transcriptional activation of RAR beta 2 may regulate the growth and differentiation programs of selected populations of stem cells in the skin and its appendages.

Animals↗

PubMed1994

Down-regulation of retinoic acid receptor beta in mammary carcinoma cell lines and its up-regulation in senescing normal mammary epithelial cells.

Retinoids are important cellular, dietary factors that regulate differentiation and cellular growth. They serve as ligands for specific nuclear receptors, the retinoic acid receptors (RARs). Ligand-activated receptors regulate gene transcription through target retinoic acid-responsive elements (RAREs) found in promoter regions. We have investigated the expression of retinoic acid receptor genes (alpha, beta, gamma) and retinoid X receptor beta in normal, senescing, and tumorigenic human mammary epithelial cells. We find that most tumor cells show a loss of RAR-beta expression, but that RAR-alpha and -gamma as well as retinoid X receptor beta are variably expressed in both normal and tumor cells. RAR-beta gene expression is induced both by retinoic acid and by fenretinide in normal cells, but tumor cells fail to respond to either. In contrast, RAR-beta expression increases with serial passage in senescing cells. Paradoxically, both normal and tumor cells can trans-activate an exogenous beta-RARE, as demonstrated by reporter gene assays. Oligonucleotide mobility shift assays with the beta-RARE show a single discrete complex in normal cells, whereas tumor cells exhibit a heterogeneous set of larger complexes, which indicates that tumor cells utilize a different array of factors within the beta-RARE. Reporter gene assays with extended promoter regions indicate the presence of negative regulatory elements and/or factor binding sites that reside between -1500 and the RARE located at -59, and that the promoter is down-regulated in MCF-7 tumor cells. Our findings reveal a dichotomy: RAR-beta transcription is down-regulated in tumor cells compared with normal human mammary epithelial cells, and up-regulated in senescence.

Base Sequence↗

PubMed1993

Linkage of Wiskott-Aldrich syndrome with three marker loci, DXS426, SYP and TFE3, which map to the Xp11.3-p11.22 region.

Linkage analysis was performed in 19 families segregating for the Wiskott-Aldrich syndrome (WAS) and in 1 family with X-linked thrombocytopenia using nine polymorphic DNA markers spanning the interval DXS7-DXS14. The results confirm close linkage of WAS to the DXS7, TIMP, OATL1, DXS255, DXS146, and DXS14 loci and reveal three additional marker loci, DXS426, SYP, and TFE3, to be closely linked to WAS. The linkage data are also consistent with the localization of X-linked thrombocytopenia to the same chromosomal region as WAS and support localization of the WAS gene between the TIMP and DXS146 loci. However, the data were insufficient for positioning these disease genes with respect to the four marker loci that map within this latter interval. Analysis of recombination events between the marker loci place the TFE3 gene distal to DXS255 and favor the marker loci order Xpter-DXS7-(DXS426, TIMP)-(OATL1, SYP, TFE3)-DXS255-DXS146-DXS14.

Base Sequence↗

PubMed1993

Cultured human melanocytes express the intermediate filament vimentin.

Human melanocytes are neural crest-derived cells that synthesize the pigment melanin. These cells migrate from a central location to the dermal-epidermal junction early in gestation and situate themselves between keratinocytes of the basal layer of the epidermis, extending thin dendritic processes upwards into the epidermis. In vitro, neonatal melanocytes can assume a variety of morphologies, depending on the culture conditions. Using standard immunofluorescent, immunoblotting, and Northern blotting techniques, we investigated the expression of intermediate filament proteins and demonstrate here that cultured human melanocytes express vimentin gene and protein under a variety of culture conditions. Vimentin is a 57-kD intermediate filament protein synthesized primarily by cells of mesenchymal origin. It is transcribed as a single-messenger RNA species of 2.0 kb and the human gene is located on chromosome 10. As a member of the intermediate filament family of proteins, we suggest that vimentin is an important component of the cytoskeleton of neonatal, human melanocytes.

Blotting, Northern↗

PubMed1993

Clonal origin of dermatofibrosarcoma protuberans.

Dermatofibrosarcoma protuberans (DFSP) is a malignant tumor originating in the dermis. Although it is known to be locally aggressive, it only rarely metastasizes and will recur unless completely excised. The exact cell responsible for the development of a DFSP has been a matter of controversy for several decades; however, most histochemical and electron microscopic studies support a fibroblastic origin, with the tumor cells staining uniformly for vimentin and containing active endoplasmic reticulum synthesizing collagen. Cytogenetic analysis of some of these tumors has demonstrated at least two specific chromosomal abnormalities in DFSP and suggested that this tumor may be polyclonal in origin. To further address the clonal origin of this locally invasive, mesenchymal tumor, we analyzed DNA from two female patients by restriction fragment length polymorphisms and methylation analysis. Our data strongly support the concept that DFSP is monoclonal in origin and that this tumor mass reflects the clonal expansion of a single cell.

Adult↗

PubMed1993

The Wiskott-Aldrich syndrome.

Skin diseases manifesting classic sex-linked recessive patterns of inheritance provide straightforward problems in the mapping of disease loci. In contrast to autosomal disorders, in which the abnormal gene might be found on any chromosome, sex-linked diseases are found only on the human X chromosome. Thus, with a reasonable number of polymorphic DNA probes and families with living affected and unaffected males, disease loci can be easily mapped to the relevant subregions of the X chromosome. The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by eczema, immunodeficiency, and thrombocytopenia. Boys with WAS suffer from skin diseases, bleeding problems, recurrent infections, and lymphoid malignancies. In contrast, females who carry the WAS gene are entirely normal, as the abnormal X chromosome is selectively inactivated in cells of hematopoietic origin. Using restriction fragment length polymorphisms (RFLPs) and appropriate families, the WAS locus has been mapped to the proximal portion of the short arm of the X chromosome (Xp11). Prenatal diagnosis is now possible using specific RFLP markers. Moreover, combining RFLP studies with methylation analysis has allowed identification of all female carriers. Although the abnormal gene and protein that are responsible for WAS are currently unknown, studies using yeast artificial chromosomes containing portions of human Xp11 should ultimately allow for the cloning and characterization of the WAS gene. As this gene is expressed primarily in cells of bone marrow origin, WAS is an excellent candidate disease for gene therapy.

Chromosome Mapping↗

PubMed1993

Selective impairment of CD43-mediated T cell activation in the Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is associated with defective glycosylation and altered membrane expression of the leukocyte sialoglycoprotein CD43. To investigate whether such modifications of CD43 are relevant to T cell dysfunction in WAS, we have analyzed peripheral blood mononuclear cells from WAS patients for proliferative responses to both CD43-interacting and other T cell mitogenic stimuli. While patient lymphocytes proliferated in response to phytohaemagglutinin, concanavalin A, interleukin-2 and neuraminidase/galactose oxidase, no responses were elicited upon attempted triggering of the CD43 signalling pathway using periodate or anti-CD43 antibody. Cells from four of five patients with clinical profiles resembling, but not identical, to that of classic WAS also failed to respond to periodate or anti-CD43 antibody stimulation. These results indicate that the aberrant expression of CD43 on WAS lymphocytes is associated with selective impairment of CD43-induced T cell proliferation and that detection of this defect may be useful in the diagnosis of WAS and its variant forms.

Adolescent↗

PubMed1992

The Wiskott-Aldrich syndrome: refinement of the localization on Xp and identification of another closely linked marker locus, OATL1.

The Wiskott-Aldrich syndrome (WAS) has previously been mapped to the proximal short arm of the X chromosome between the DXS14 and DXS7 loci. In this study, further segregation analysis has been performed using a newly identified WAS family as well as an additional marker probe, HOATL1. The results indicate close linkage between the WAS and OATL1 loci (Z = 6.08 at theta = 0.00) and localize the TIMP, OATL1, DXS255, and WAS loci distal to DXS146 and the OATL1 and WAS loci proximal to TIMP. These linkage data narrow the boundaries within which the WAS locus maps to the chromosomal region bracketed by TIMP and DXS146 and support the loci order Xpter-DXS7-TIMP-(OATL1, WAS, DXS255)-DXS146.

Alleles↗

PubMed1992

Wiskott-Aldrich syndrome: new molecular and biochemical insights.

The Wiskott-Aldrich syndrome is an uncommon X-linked recessive disease characterized by eczema, thrombocytopenia, and immunodeficiency. The clinical features begin early in life and include recurrent infections, bleeding, and severe eczema. Unless the condition is treated by bone marrow transplantation, the prognosis of Wiskott-Aldrich syndrome is grave, and premature death caused by sepsis, hemorrhage, or lymphoreticular malignancy is common. Although the biochemical defect responsible for the syndrome is not known, recent investigations with restriction fragment length polymorphisms have mapped the Wiskott-Aldrich syndrome locus to the proximal portion of the short arm of the human X chromosome (Xp11). The isolation of these DNA markers makes feasible both carrier detection and prenatal diagnosis of Wiskott-Aldrich syndrome and provides an important adjunct to the management of Wiskott-Aldrich syndrome for patients and their families. These genetic data, in conjunction with the recent identification of a specific O-glycosylation defect in lymphocytes from patients with Wiskott-Aldrich syndrome, present an opportunity for the eventual isolation of the Wiskott-Aldrich syndrome gene and identification of the underlying cellular defect. We review the clinical and laboratory features of this syndrome and summarize the new molecular and biochemical approaches that can be used in diagnosis, genetic counseling, and treatment.

Genetic Linkage↗

PubMed1992

Induction of c-fos but not c-myc in S-91 cells by melanization signals.

Synthesis of the pigment melanin is a complex differentiated function performed by pigment cells in response to a variety of stimuli. The possible roles of the proto-oncogenes c-fos and c-myc in the control of pigmentation were studied using subconfluent, actively proliferating Cloudman S-91 murine melanoma cells stimulated to synthesize melanin by melanocyte stimulating hormone (MSH) or forskolin. Stimulation caused a significant increase in melanin synthesis when compared to control cells, but had no effect on cell growth. Northern analysis of total cellular RNA demonstrated rapid, transient induction of c-fos mRNA as early as 30 min after stimulation with MSH or forskolin. In contrast, there was no effect on the high constitutive expression of c-myc in these actively proliferating cells. These data strongly suggest that the induction of c-fos mRNA is an early genetic event in stimulation of melanin synthesis and thus this proto-oncogene may play a major role in the regulation of this differentiated function, as reported for other forms of cellular differentiation. In contrast, c-myc expression is unaffected and instead correlated with cellular proliferative capacity. These results are consistent with the hypothesis that the down-regulation of c-myc frequently observed during cell differentiation is not a necessary event, but rather reflects an associated decrease in cell growth rate. The S-91 melanoma system appears to provide a convenient model for study of the regulation for a single, well defined differentiated function that is independent of growth rate.

Animals↗

PubMed1992

Fibroblasts protect the Lyme disease spirochete, Borrelia burgdorferi, from ceftriaxone in vitro.

The Lyme disease spirochete, Borrelia burgdorferi, can be recovered long after initial infection, even from antibiotic-treated patients, indicating that it resists eradication by host defense mechanisms and antibiotics. Since B. burgdorferi first infects skin, the possible protective effect of skin fibroblasts from an antibiotic commonly used to treat Lyme disease, ceftriaxone, was examined. Human foreskin fibroblasts protected B. burgdorferi from the lethal action of a 2-day exposure to ceftriaxone at 1 microgram/mL, 10-20 x MBC. In the absence of fibroblasts, organisms did not survive. Spirochetes were not protected from ceftriaxone by glutaraldehyde-fixed fibroblasts or fibroblast lysate, suggesting that a living cell was required. The ability of the organism to survive in the presence of fibroblasts was not related to its infectivity. Fibroblasts protected B. burgdorferi for at least 14 days of exposure to ceftriaxone. Mouse keratinocytes, HEp-2 cells, and Vero cells but not Caco-2 cells showed the same protective effect. Thus, several eukaryotic cell types provide the Lyme disease spirochete with a protective environment contributing to its long-term survival.

Adenocarcinoma↗

PubMed1991

Cellular senescence: a reflection of normal growth control, differentiation, or aging?

Normal cells, with few exceptions, cannot proliferate indefinitely. Cell populations--in vivo and in culture--generally undergo only a limited number of doublings before proliferation invariably and irreversibly ceases. This process has been termed the finite lifespan phenotype or cellular senescence. There is long-standing, albeit indirect, evidence that cellular senescence plays an important role in complex biological processes as diverse as normal growth control, differentiation, development, aging, and tumorigenesis. In recent years, it has been possible to develop a molecular framework for understanding some of the fundamental features of cellular senescence. This framework derives primarily from the physiology, genetics, and molecular biology of cells undergoing senescence in culture. Our understanding of senescence, and the mechanisms that control it, is still in its infancy. Nonetheless, recent data raise some intriguing possibilities regarding potential molecular bases for the links between senescence in culture and normal and abnormal growth control, differentiation, and aging.

Adult↗

PubMed1990

Dissociation of proto-oncogene induction from growth response in normal human fibroblasts.

Proto-oncogenes are cellular homologues of viral oncogenes that are known to be associated with regulation of growth and differentiation. The c-myc and c-fos proto-oncogenes have been extensively studied by using established cell lines but to a lesser extent by using normal cells. Using physiologic growth modulators, we have shown that mitotic stimulation of normal human dermal fibroblasts is associated with induction of c-myc and c-fos, but that growth inhibition of these cells is not necessarily accompanied by their down-regulation. When treated with both serum and interferon-alpha during quiescence, fibroblasts were delayed in their progress into the S-phase of the cell cycle as compared to cells treated with serum alone and displayed substantial growth inhibition as measured by cell number at the end of 1 week. However, this growth inhibition was not preceded by down-regulation or delay in induction of c-myc and c-fos mRNA. The above studies suggest that in normal fibroblasts growth inhibition is not necessarily dependent on down-regulation of transcription of either c-myc or c-fos and that interferon may act to inhibit cell growth either through a post-transcriptional effect on cellular proto-oncogenes necessary for cell proliferation or through induction of other, as yet unrecognized gene(s) associated with growth arrest.

Cell Cycle↗

PubMed1990

Linkage relationships of the Wiskott-Aldrich syndrome to 10 loci in the pericentromeric region of the human X chromosome.

Twelve families with Wiskott-Aldrich syndrome (WAS) were studied by linkage analysis using 10 polymorphic marker loci from the X-chromosome pericentromeric region. The results confirm close linkage of WAS to the DXS14, DXS7, TIMP, and DXZ1 loci and are consistent with previous data suggesting that WAS maps to the proximal Xp and is flanked by the DXS14 and DXS7 loci. The strongest linkage (Z = 10.19 at theta = 0.00) was found to be between WAS and the hypervariable DXS255 locus, a marker locus already mapped between DXS7 and DXS14 and which was informative for all meioses included in this analysis. Linkage of the WAS to two pericentromeric Xq loci, DXS1 and PGK1, was also established. On the basis of these results, accurate predictive testing should now be feasible in the majority of WAS families.

Chromosome Mapping↗

PubMed1989

Linkage studies of the Wiskott-Aldrich syndrome: polymorphisms at TIMP and the X chromosome centromere are informative markers for genetic prediction.

Eleven families segregating for the X-linked recessive immune deficiency disorder, Wiskott-Aldrich syndrome (WAS), were studied by linkage analysis with an alpha satellite DNA probe, pBamX-7, which detects polymorphisms at the X chromosome centromere, locus DXZ1, as well as three other polymorphic markers defining loci on the proximal short arm of the X chromosome. Linkage has been established between WAS and DXZ1 (zeta (theta) = 7.08 at theta = 0.03) and WAS and the TIMP gene locus (zeta (theta) = 5.09 at theta = 0.0). We have also confirmed close linkage between DXZ1 and two marker loci, DXS14 and DXS7, previously shown to be linked to the WAS locus. The probe pBamX-7 detected allelic variation in all females tested, reflecting the high frequency of polymorphism at the centromere. One WAS carrier revealed a recombination between WAS and both marker loci DXZ1 and DXS14, indicating that WAS does not map between these loci. In conjunction with previous data from genetic mapping studies of WAS, these results confirm the pericentromeric Xp localization of WAS and demonstrate the usefulness of alpha satellite DNA probes as tools for genetic prediction in WAS as well as other pericentric X-linked diseases.

Centromere↗

PubMed1989

Interferon and the epidermis: implications for cellular senescence.

Human keratinocytes are epithelial cells that cover the external surface of the body and function primarily to protect against physical, chemical and biological injury. Since 1975, serial cultivation of disaggregated human keratinocytes in vitro has been possible and these cultures have been utilized to assess in vitro physiological age. As has been demonstrated in human dermal fibroblasts harvested from donors of different ages, human epidermal keratinocytes from older donors manifest a decreased proliferative response to serum and to selected mitogens when compared to younger donors. Recent work suggests an increased sensitivity to negative growth modulators as well. The epidermis constantly renews itself. In normal skin, the majority of the cells in the germinative, basal cell layer are blocked in Go and do not cycle unless stimulated. Interferons (IFN) are a family of glycoproteins well known for their antiviral activity and their ability to inhibit growth and alter the behavior of various normal and transformed cell types, both in vitro and in vivo. As such, we examined intact skin for the presence of negative modulators of growth and demonstrated the presence, by immunofluorescence and Western blotting, of a protein of approximate molecular weight 40 kilodaltons with polyclonal antiserum directed against recombinant IFN-alpha. Extracts from healthy, non-virally infected keratinocyte cultures contained IFN activity as determined by viral plaque inhibition assays. Further, we have demonstrated that IFN inhibits the growth of the human keratinocyte in a non-cytotoxic, reversible manner and that the keratinocytes harvested from older adult donors are significantly more sensitive to the growth inhibitory effects of IFN than are the keratinocytes of young donors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1989

X-chromosome inactivation in the Wiskott-Aldrich syndrome: a marker for detection of the carrier state and identification of cell lineages expressing the gene defect.

Recently developed techniques for the direct analysis of DNA have made possible the determination of patterns of cellular X-chromosome inactivation. These techniques provide a potential method for carrier detection for several X-linked human disorders in which obligate carriers show nonrandom X inactivation. By using restriction fragment length polymorphic (RFLP) gene-specific probes in conjunction with methylation-sensitive enzymes, we have characterized the patterns of X-chromosome inactivation in cell subsets from females belonging to 10 kindreds segregating for the X-linked immune deficiency disorder Wiskott-Aldrich syndrome (WAS). We show that selective inactivation of the X chromosome distinguishes obligate WAS carriers from noncarrier females and constitutes a valuable marker of the WAS carrier state. Selective inactivation phenomena were observed in the monocytes and T and B lymphocytes of obligate carriers, implying that the WAS gene defect is expressed in each of these cellular lineages. In conjunction with the use of linked DNA markers, RFLP-methylation analysis should render carrier detection feasible for the majority of females from WAS families. The results of such analyses also provide an initial step toward identifying the cellular level and molecular basis for WAS.

DNA Probes↗

PubMed1989

Altered expression of leucocyte sialoglycoprotein in Wiskott-Aldrich syndrome is associated with a specific defect in O-glycosylation.

The Wiskott-Aldrich syndrome (WAS) is an X-linked immune deficiency disorder characterized clinically by both lymphocyte and platelet dysfunction. Studies of WAS T lymphocytes have revealed deficient or defective cell surface expression of the highly O-glycosylated leucocyte sialoglycoprotein CD43. To further elucidate the basis for, and functional relevance of, CD43 modifications on WAS lymphocytes, we have studied lymphocytes from two WAS patients with regard to membrane glycoprotein profile and mitogen-induced proliferative responses. CD43 was found to be either absent or altered in size on peripheral blood lymphocytes and lectin-stimulated T cells from both patients. Compared with control cells, the WAS lymphocytes displayed reduced, but measurable proliferative responses to lectins and neuraminidase/galactose oxidase, and virtually no response to periodate, a mitogenic agent which targets sialic acid residues on membrane glycoproteins such as CD43. Analysis of activities of three glycosyltransferases involved in O-glycosylation revealed marked reduction in the level of activity of UDP-N-acetylglucosamine: Gal beta 1-3GalNAc-R beta-1,6-N-acetylglucosamine (beta-1,6-GlcNAc) transferase in one WAS patient and no detectable activity of this enzyme in a second. beta-1,6-GlcNAc transferase activity has recently been shown to increase during T cell activation coincident with changes in the O-linked glycans on CD43. A selective reduction of this glycosyltransferase in WAS lymphocytes suggests that O-linked oligosaccharides may be important to the structure of membrane glycoproteins involved in lymphocyte activation.

Cell Division↗