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P Jeppesen

Publications and source records attributed to P Jeppesen.

15 recordsLinked to original sources
PubMed1992

Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA.

Methylation of mammalian DNA can lead to repression of transcription and alteration of chromatin structure. Recent evidence suggests that both effects are the result of an interaction between the methylated sites and methyl-CpG-binding proteins (MeCPs). MeCP1 has previously been detected in crude nuclear extracts. Here we report the identification, purification, and cDNA cloning of a novel MeCP called MeCP2. Unlike MeCP1, the new protein is able to bind to DNA that contains a single methyl-CpG pair. By staining with an antibody, we show that the distribution of MeCP2 along the chromosomes parallels that of methyl-CpG. In mouse, for example, MeCP2 is concentrated in pericentromeric heterochromatin, which contains a large fraction (about 40%) of all genomic 5-methylcytosine.

Amino Acid Sequence↗
PubMed1992

Antibodies to defined histone epitopes reveal variations in chromatin conformation and underacetylation of centric heterochromatin in human metaphase chromosomes.

Unfixed metaphase chromosome preparations from human lymphocyte cultures were immunofluorescently labelled using antibodies to defined histone epitopes. Both mouse monoclonal antibody HBC-7, raised against the N-terminal region of H2B, and rabbit serum R5/12, which recognizes H4 acetylated at Lys-12, gave non-uniform labelling patterns, whereas control antibodies against total histone fractions H4 and H1 produced homogeneous fluorescence. HBC-7 bound approximately uniformly to the bulk of the chromosomes, but the major heterochromatic domains of chromosomes 1, 9, 15, 16 and the Y showed significantly brighter fluorescence. Serum R5/12 indicated an overall reduction in acetylation of H4 in metaphase chromosomes compared with interphase nuclei, although some specific chromosomal locations had considerably elevated acetylation levels. Acetylation levels in the major heterochromatic domains appeared extremely low. To investigate further the differences noted in heterochromatin labelling, metaphases from cultures grown in the presence of various agents known to induce undercondensation of the major heterochromatic domains were similarly immunolabelled. Decondensed heterochromatin no longer exhibited higher than normal immunofluorescence levels with HBC-7. The higher resolution afforded by "stretching" the centromeric heterochromatin of chromosomes 1, 9 and 16 confirmed the low level of H4 acetylation in these domains. We consider the implications of these observations in relation to chromatin conformation and activity.

Acetylation↗
PubMed1992

The organisation of repetitive DNA sequences on human chromosomes with respect to the kinetochore analysed using a combination of oligonucleotide primers and CREST anticentromere serum.

The spatial relationship between the families of repetitive DNAs present at the centromeres of human chromosomes and the position of the kinetochore was examined by combining immunocytochemistry with the PRINS oligonucleotide primer extension technique. Heterochromatic domains were decondensed with 5'-azacytidine to facilitate this study. Using this approach our results clearly show that the alphoid DNA sequences are closely associated with the kinetochore of human chromosomes. Simple-sequence satellite DNAs occupy separate, non-overlapping domains within the centromere. These two major families are separated by a third, relatively low-copy repetitive DNA family, SAU-3A. Pulse-field gel electrophoresis was employed to analyse the centromeric domain of human chromosome no. 9 in more detail and the results although preliminary support the conclusions drawn from the immunocytochemistry/PRINS approach.

Antibodies, Antinuclear↗
PubMed1992

Transcriptional repression by methylation of CpG.

Methylated DNA in mammals is associated with transcriptional repression and nuclease resistant chromatin. In this review we discuss how these effects may be mediated by proteins that bind to methylated DNA.

Animals↗
PubMed1987

Diagnosis and treatment of neuroblastoma using metaiodobenzylguanidine.

Neuroblastoma is a lethal and not uncommon tumor in childhood. Early detection and display of the spread of the tumor is highly desirable for proper treatment. Nine children suspected of having neuroblastomas were examined by I-131 metaiodobenzylguanidine (I-131 MIBG) imaging. In two recent studies I-123 metaiodobenzylguanidine (I-123 MIBG) was used. A primary adrenal neuroblastoma was correctly identified in three cases. In two patients additional tumor sites were found. In one patient, who was in complete remission, no pathologic accumulation of I-131 MIBG was found. I-131 MIBG images were also normal in four patients with other types of neoplastic diseases. A boy with multiple metastases was treated with 100 mCi of I-131 MIBG. He developed transient gastrointestinal illness and there was no regression of the tumor deposits. In one girl with a large adrenal neuroblastoma high uptake of I-131 MIBG was observed. She received two therapy doses of I-131 MIBG (35 mCi and 75 mCi) with curative intention giving a total absorbed dose in the tumor of approximately 76 Gy. In spite of high retention of radioactivity in the tumor, regression did not occur, but her general condition was improved. In the present study, images of superior quality were obtained with I-123 MIBG imaging. It is concluded that imaging using I-131 MIBG or I-123 MIBG should be used in both the initial evaluation and the follow-up of children with neuroblastoma.

3-Iodobenzylguanidine↗
PubMed1986

Characterization of a polypeptide associated with coated vesicles and the cytoskeleton which is recognized by a CREST serum.

Serum from an individual with the CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dismotility, sclerodactyly, telangiectasia) reacts not only with kinetochores, but also with a cytoplasmic, phosphorylatable polypeptide, which is shown by immunofluorescence in whole cells and immunoelectronmicroscopy in sections to be associated with actin stress fibres in cultured mammalian cells. The antigen shows some variation in molecular weight between species, estimated by immunoblotting to range from 68 to 76 kD between mouse, Chinese hamster, sheep and human cells. Much of the polypeptide copurifies with coated vesicles, of which approx. 5% bound antibody from the serum, as detected by immunogold electronmicroscopy.

Actins↗
PubMed1986

Non-kinetochore directed autoantibodies in scleroderma/CREST. Identification of an activity recognizing a metaphase chromosome core non-histone protein.

Three scleroderma/CREST sera possessing anti-kinetochore autoantibody were tested by indirect immunofluorescence for reaction with isolated Chinese hamster metaphase chromosomes and histone-dissociated chromosome "scaffolds". All three sera revealed kinetochore staining on both whole chromosomes and histone-dissociated chromosomes, and confirmed previous reports that the kinetochore is part of the chromosome scaffold. Unexpectedly, one serum also recognized a non-centromere-located chromosomal antigen which is retained at the axial non-histone protein core following histone dissociation. The antigen is exclusively nuclear in origin, does not appear to be DNA or residual histone, and seems to correspond to a major component of the chromosome core. The existence of this antigen has important implications in terms of chromosome structure. Another CREST serum was shown to possess autoantibody against a cytoplasmic structural component, in addition to anti-kinetochore activity. The presence of anti-kinetochore and other autoantibody activities in CREST patient sera is discussed in relation to disease processes.

Animals↗
PubMed1986

Diagnosis and treatment of neuroblastoma using 131I-meta-iodobenzylguanidine.

Neuroblastoma is a lethal and not uncommon tumor in childhood. Early detection and display of the spread of the tumor is highly desirable for proper treatment. Nine children suspected of having neuroblastoma were examined by 131I-meta-iodobenzylguanidine (131I-MIBG) scintigraphy. In two recent studies 123I-meta-iodobenzylguanidine (123I-MIBG) was used. A primary adrenal neuroblastoma was correctly identified in 3 cases. In 2 patients additional tumor sites were found. In one patient, who was in complete remission, no pathological accumulation of 131I-MIBG was found. 131I-MIBG scintigraphies were also normal in 4 patients with other types of tumor. In one girl with a large adrenal neuroblastoma high uptake of 131I-MIBG was observed. She received two therapy doses of 131I-MIBG (1300-2800 MBq) with curative intention giving a total absorbed dose in the tumor of approximately 90 Gy. In spite of high retention of radioactivity in the tumor, regression did not appear, but her general condition was improved. In the present study images of superior quality were obtained by 123I-MIBG scintigraphy. It is concluded that scintigraphy with 131I- or 123I-MIBG should be used for primary visualization and control of neuroblastomas.

3-Iodobenzylguanidine↗
PubMed1985

Effects of sulphydryl reagents on the structure of dehistonized metaphase chromosomes.

Dehistonized metaphase chromosomes lose their apparent axial organization (the 'scaffold') and sediment more slowly following exposure to beta-mercaptoethanol (BME). We have subsequently treated BME chromosomes with reagents that oxidize protein sulphydryls to disulphides, and found that if calcium is also present during the oxidation an apparently similar axial structure is restored following dehistonization, as seen by microscopic examination. In general, however, we do not find that oxidation restores the higher sedimentation rate of dehistonized control chromosomes. Analysis of residual core protein in dehistonized chromosomes by sodium dodecyl sulphate/polyacrylamide gel electrophoresis fails to detect any differences in polypeptide composition related to the state of oxidation or to the presence or absence of visible axial organization. Combining our results with those of other workers, we conclude that the axial structure evident in dehistonized metaphase chromosomes is maintained, at least partially, by inter-protein cross-linking, although in vivo this may not be via simple disulphide bridges. Additional factors, which we have not yet characterized, but which possibly include heavy metal ions, appear to be involved in the axial organization existing in vivo.

Animals↗
PubMed1983

Dot-blotting--a novel screening assay for antibodies in hybridoma cultures.

A novel solid phase radioimmunoassay using nitrocellulose filters, is described. The method is of particular value in screening hybridoma cultures for monoclonal antibodies against chromosomal protein. Also it permits detection of antibodies directed against epitopes stable to SDS denaturation.

Animals↗
PubMed1983

Chinese hamster metaphase chromosomes isolated under physiological conditions. A partial characterization of associated non-histone proteins and protein cores.

In a previous report [2] we have described a non-histone protein core which could be isolated from Chinese hamster metaphase chromosomes. This core structure maintained the overall morphology of the metaphase chromosome even after removal of all of the histones, together with many of the non-histone proteins and the bulk of the DNA. As part of our work on the characterization of these core structures, we have developed a novel procedure for the isolation of metaphase chromosomes which avoids the use of high pH buffers and hexylene glycol, as well as eliminating the numerous centrifugation and resuspension steps previously employed. Chromosome cores prepared by 2 M NaCl extraction and DNase I digestion from metaphase chromosomes isolated under these more gentle, quasi-physiological conditions, are shown to contain a relatively simple subset of non-histone proteins. One-dimensional SDS-polyacrylamide gel electrophoresis shows two major groups of polypeptides having molecular weights 48 000-52 000 and 65 000-72 000 D respectively, with similarities in mobilities to the nuclear pore complex-lamina polypeptides and tubulins. However, more detailed analysis by two-dimensional gel electrophoresis and peptide mapping has failed to detect these proteins. A 52 000 D polypeptide component of the core is tentatively identified as the intermediate filament protein vimentin. The in vivo significance of chromosome cores is discussed.

Animals↗