Search PubMed⌕ Search

Biomedical subjects

C M Price

Publications and source records attributed to C M Price.

At least 19 recordsLinked to original sources
PubMed1996

Frequent somatic deletion of the 13q12.3 locus encompassing BRCA2 in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has consistent 13q chromosomal abnormalities detected by conventional cytogenetics. Using interphase cytogenetics we show deletion of a 1-megabase 13q12.3 locus, encompassing the BRCA2 gene, in 80% of 35 CLL cases studied. Homozygous deletion of BRCA2, located within the minimal deletion consensus, was detected in a significant population of cells in 60% of the cases. Deletion of the previously described 13q14 locus (analyzed with RB1 and D13S25 probes) was seen in 63% of the cases. Homozygous deletion of RB1 was seen in one case. Seven of the cases (32%) with D13S25 deletion had a population of cells with homozygous deletion. Deletions at the 13q12 and 13q14 loci result from distinct events because they were not contiguous. These data provide evidence for the existence of a new tumor suppressor locus in B-cell CLL located at 13q12.3. BRCA2, located within the minimal deletion consensus, is a candidate for the gene whose somatic inactivation could play a role in the initiation and or progression of B-cell CLL.

Alleles↗
PubMed1996

Myocardial ischaemia associated with Ehlers-Danlos syndrome.

A 38-yr-old man with an unusual type of Ehlers-Danlos syndrome presented for elective abdominal aortic aneurysm repair. During surgery he developed acute myocardial ischaemia, resulting in abandonment of the procedure. He was shown subsequently to have severe triple vessel coronary artery disease. Silent ischaemia associated with severe coronary artery disease, although rare, may be associated with the syndrome and is difficult to recognize as other cardiac abnormalities are frequently present.

Acute Disease↗
PubMed1996

Correlation of trisomy 12 with proliferating cells by combined immunocytochemistry and fluorescence in situ hybridization in chronic lymphocytic leukemia.

Conventional G-banding and fluorescence in situ hybridization (FISH) were performed on peripheral blood samples of 340 consecutive untreated cases of chronic lymphocytic leukemia (CLL) for the detection of trisomy 12 and other chromosome abnormalities. These findings were correlated with the proliferative activity of CLL lymphocytes assessed by the monoclonal antibody Ki-67. Cytogenetic analysis displayed a normal karyotype in 131 (38.5%) cases, trisomy 12 in 68 (20%), 31 by G-banding and an additional 37 cases by FISH, other clonal abnormalities in 47 (14%), and no metaphases in 94 (27.5%). The percentage of Ki-67-positive cells was significantly higher in cases with trisomy 12 (4.1 +/- 4.48) than in cases with a normal karyotype (1.5 +/- 2.0), those with other clonal abnormalities (1.35 +/- 1.37) and cases with no metaphases (1.14 +/- 1.6) (P< 0.0001). Cases with trisomy 12 were associated with more advanced clinical stage, atypical morphology and a higher percentage of Ki-67+ve cells than cases lacking trisomy 12 (P< 0.0001). Although there was no direct correlation between the percentage of trisomic and proliferating cells, the combination of immunocytochemistry and FISH showed that most Ki-67-positive cells were trisomic for chromosome 12. Our results suggest that the association of trisomy 12 with a higher proliferative activity supports the view that this abnormality is a secondary event associated with disease progression in CLL.

Adult↗
PubMed1995

Demonstration of spontaneously dividing male fetal cells in maternal blood by negative magnetic cell sorting and fish.

We investigated a case of massive feto-maternal bleeding by using negative magnetic cell sorting (MACS) and fluorescent in situ hybridization (FISH). A 37-year-old pregnant woman had an uncomplicated amniocentesis for advanced maternal age at 16 weeks' gestation. The fetal karyotype was 46,XY. At 19 weeks' gestation, she had a minor car accident and slight vaginal bleeding. A subsequent Kleihauer-Betke test showed a 140 ml feto-maternal haemorrhage. Serial sonographic examinations indicated a normal fetus and placenta. We performed FISH analysis on maternal peripheral blood at 25 weeks. Anti-CD45 and MACS were used to deplete maternal leucocytes, enriching the proportion of fetal nucleated erythrocytes present. The isolated cells were analysed by using dual-colour FISH with X and Y specific probes. Approximately 65,800 nucleated cells were obtained after MACS depletion. A total of 234 cells were analysed by FISH. The results revealed that 70 of the nucleated cells (30 per cent) were male with one X and one Y signal. Among these cells, six male metaphases were observed in spontaneously dividing cells.

Adult↗
PubMed1995

Persistence of multilineage host haemopoiesis following allogeneic bone marrow transplantation.

The survival of host cells following high-dose cytotoxic therapy and allogeneic marrow transplantation has been established previously, but the identity of these cells has not been elucidated in detail. Four patients who received sex-mismatched marrow have been studied for up to 12 months post-transplant using a simultaneous immunophenotyping/fluorescence in situ hybridization technique. The results demonstrate residual host T cells (CD3+), B cells (CD22+) and myeloid cells (CD11c+ and CD13+), and additionally cells of progenitor cell phenotype (CD34+). The long-term persistence of host haemopoiesis may have major relevance to the post-transplant complications of marrow rejection, graft-versus-host disease, and malignant relapse.

B-Lymphocytes↗
PubMed1994

DNA bound by the Oxytricha telomere protein is accessible to telomerase and other DNA polymerases.

Macronuclear telomeres in Oxytricha exist as DNA-protein complexes in which the termini of the G-rich strands are bound by a 97-kDa telomere protein. During telomeric DNA replication, the replication machinery must have access to the G-rich strand. However, given the stability of telomere protein binding, it has been unclear how this is accomplished. In this study we investigated the ability of several different DNA polymerases to access telomeric DNA in Oxytricha telomere protein-DNA complexes. Although DNA bound by the telomere protein is not degraded by micrococcal nuclease or labeled by terminal deoxynucleotidyltransferase, this DNA serves as an efficient primer for the addition of telomeric repeats by telomerase, a specialized RNA-dependent DNA polymerase (ribonucleoprotein reverse transcriptase), EC 2.7.7.49. Moreover, in the presence of a suitable complementary C-rich DNA template, AMV reverse transcriptase and the E. coli Klenow fragment will also elongate DNA bound by the telomere protein. These findings indicate that the 3' terminus and the Watson-Crick base pairing positions are exposed in the protein complex. We propose that the telomere protein can serve a dual role at the telomere by protecting the DNA phosphate backbone from degradation while simultaneously exposing the DNA bases for replication.

Animals↗
PubMed1994

Trisomy 12 in B-cell chronic lymphocytic leukaemia: assessment of lineage restriction by simultaneous analysis of immunophenotype and genotype in interphase cells by fluorescence in situ hybridization.

We have studied the lineage restriction of trisomy 12 in six patients with B-cell chronic lymphocytic leukaemia (CLL) by simultaneous analysis of immunophenotype and fluorescence in situ hybridization (FISH) signals in single interphase cells. Fresh uncultured cells from each patient were immunophenotyped by the alkaline phosphatase anti-alkaline phosphatase method (APAAP) using monoclonal or polyclonal antibodies and hybridized with a chromosome 12 specific alpha-satellite DNA probe. In all cases trisomy 12 was restricted to the clonal B-cells, kappa positive or lambda positive, whereas T-cells (CD3 positive) and non clonal B-cells had only two chromosome 12 signals. Within the clonal B-cell population a large proportion of cells were disomic for chromosome 12, whilst trisomic cells ranged from 21% to 37%. The absence of trisomy 12 in T-cells and the mosaicism demonstrated in the clonal B-cells suggests that this abnormality is a secondary event during the leukaemic transformation of CLL and develops in an already established neoplastic B-cell population.

Aged↗
PubMed1994

Sequential acquisition of trisomy 8 and N-ras mutation in acute myeloid leukaemia demonstrated by analysis of isolated leukaemic colonies.

Specific chromosomal aberrations and point mutations of the N-ras proto-oncogene are characteristic genetic alterations in acute leukaemias. However, the relationships between these two different genetic changes are unclear. Here we have determined the order of genetic events in a patient with acute myeloid leukaemia characterized by trisomy 8 and a point mutation of N-ras at codon 12 (N12-cys) and codon 61 (N61-his). 30 colonies obtained by in vitro clonogenic assay of leukaemic cells from a patient with AML were individually analysed for the presence of trisomy 8 and each of two different N-ras mutations by fluorescence in situ hybridization (FISH) and the polymerase chain reaction (PCR). Trisomy 8 was detected in 25/26 evaluable colonies. 19/26 colonies contained the N12-cys mutation. The N61-his mutation was not detected in any of the colonies obtained. All the colonies with the N12 cys mutation were also trisomic from chromosome 8, whereas 6/25 colonies with trisomy 8 had no N-ras mutation. These data suggest that trisomy 8 was acquired before N12 cys mutation in the pathogenesis of this leukaemia and that two genetic events can co-operate within a single subclone.

Adult↗
PubMed1994

Telomeric DNA sequence and structure following de novo telomere synthesis in Euplotes crassus.

To learn more about the mechanism of de novo telomere synthesis, we have characterized the sequence and structure of newly synthesized telomeres from Euplotes crassus. E. crassus is a particularly useful organism for studying telomere synthesis because millions of telomeres are made in each cell at a well-defined time during the sexual stage of the life cycle. These newly synthesized telomeres are approximately 50 bp longer than mature macronuclear telomeres. We have investigated the structure of the newly synthesized telomeres and have found that they are much more heterogeneous in length than mature telomeres. Most of the heterogeneity is present on the G-rich strand, indicating that the length of this strand is rather loosely controlled. In contrast, the length of the C-rich strand is much less variable, suggesting that synthesis of this strand is the more precisely regulated step in telomere addition. The G-rich strand exhibits variability both in the total number of G4T4 repeats and in the identity of the terminal nucleotide. In most cases, the G-rich strnd extends beyond the C-rich strand to leave a 3' overhang. While the size of this overhang is variable, the median length is 10 nucleotides. This research provides the first detailed picture of a newly synthesized telomere and has allowed us to formulate a model to describe the various steps involved in de novo telomere synthesis.

Animals↗
PubMed1993

Telomere processing in Euplotes.

In Euplotes crassus millions of telomeres are synthesized during the sexual phase of the life cycle. Since these newly synthesized telomeres are longer than normal macronuclear telomeres, they must be trimmed to the mature size. We have examined the timing and mechanism of this trimming step. We have shown that a sudden decrease in telomere length takes place at a specific time during macronuclear development. The decrease in telomere length is not caused by incomplete replication of the most terminal DNA sequences; rather it is the result of an active processing event that occurs independently of DNA replication. The developmentally regulated telomere shortening that takes place in Euplotes is reminiscent of the sudden reductions in telomere length which have been observed in other eukaryotes.

Animals↗
PubMed1993

Fluorescence in situ hybridization.

Spectacular advances in the use of fluorescence in situ hybridization (FISH) for the visualisation of specific DNA sequences in metaphase chromosomes and interphase cells have been made over the last few years making the technique a useful tool in clinical research. One of the biggest impacts has been in the field of detection and diagnosis of human malignancies. Chromosomal translocations, deletions, amplification of specific genes and changes in chromosome number can all be detected in the non-dividing interphase nucleus using probes ranging from whole chromosome 'paints' to individual gene specific probes. Gene mapping has also benefited from advances in FISH technology. Target sequences ranging from one to several hundred kilobases can be visualised on metaphase chromosomes and spatial resolution in interphase cells permits the ordering of two probes over a distance as small as 1000 base pairs. The potential uses of FISH continue to increase with each new technical innovation.

Chromosome Aberrations↗
PubMed1993

The in vitro effects of interferon-gamma, interferon-alpha, and tumour-necrosis factor-alpha on erythroid burst-forming unit growth in patients with non-leukaemic myeloproliferative disorders.

We have studied the effects of interferons gamma (IFN-gamma) and alpha (IFN-alpha), and tumour-necrosis factor-alpha (TNF) on circulating 14-day erythroid progenitor cell (BFU-E) growth in vitro from patients with non-leukaemic myeloproliferative disorders (MPD) compared with normal controls. IFN-gamma (1000 U/ml) inhibited BFU-E growth in all controls studied (mean growth +/- SE = 61% +/- 6%, n = 10). In 7 of 11 MPD studied there was no inhibition, and in some cases clear enhancement of BFU-E growth by IFN-gamma. When cultured in the presence of recombinant erythropoietin (rEpo) 1 U/ml, both IFN-alpha and TNF (at 100 and 1000 U/ml) produced a similar degree of inhibition of BFU-E growth in MPD and controls. The inhibition by 100 U/ml IFN-alpha was abrogated, partially in controls but completely in MPD, by increasing the dose of rEpo to 5 U/ml. Similarly, the increase in rEpo dose enhanced BFU-E growth in cultures with 100 U/ml TNF, but had little effect on cultures containing 1000 U/ml of either IFN-alpha or TNF. The aberrant in vitro response to IFN-gamma demonstrated in some of these patients may be of relevance to the pathophysiology of MPD. These results fail to demonstrate a differential in vitro effect for IFN-alpha on MPD BFU-E growth compared with controls and suggest that the in vitro suppression of haemopoiesis by IFN-alpha when used in MPD treatment is non-specific.

Cells, Cultured↗
PubMed1993

Prenatal diagnosis from maternal blood: simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative magnetic cell sorting.

Fetal nucleated cells in the maternal circulation constitute a potential source of cells for the non-invasive prenatal diagnosis of fetal genetic abnormalities. We have investigated the use of the Magnetic Activated Cell Sorter (MACS) for enriching fetal nucleated erythrocytes. Mouse monoclonal antibodies specific for CD45 and CD32 were used to deplete leucocytes from maternal blood using MACS sorting, thus enriching for fetal nucleated erythrocytes which do not express either of these antigens. However, significant maternal contamination was present even after MACS enrichment preventing the accurate analysis of fetal cells by interphase fluorescence in situ hybridisation (FISH). To overcome this problem, we used simultaneous immunophenotyping of cells with the mouse antifetal haemoglobin antibody, UCH gamma, combined with FISH analysis using chromosome X and Y specific DNA probes. This approach enables selective FISH analysis of fetal cells within an excess of maternal cells. Furthermore, we have confirmed the potential of the method for clinical practice by a pilot prospective study of fetal sex in women referred for amniocentesis between 13 and 17 weeks of gestation.

Cell Separation↗
PubMed1992

DNA recognition and binding by the Euplotes telomere protein.

The 51-kDa telomere protein from Euplotes crassus binds to the extreme terminus of macronuclear telomeres, generating a very salt-stable telomeric DNA-protein complex. The protein recognizes both the sequence and the structure of the telomeric DNA. To explore how the telomere protein recognizes and binds telomeric DNA, we have examined the DNA-binding specificity of the purified protein using oligonucleotides that mimic natural and mutant versions of Euplotes telomeres. The protein binds very specifically to the 3' terminus of single-stranded oligonucleotides with the sequence (T4G4) > or = 3 T4G2; even slight modifications to this sequence reduce binding dramatically. The protein does not bind oligonucleotides corresponding to the complementary C4A4 strand of the telomere or to double-stranded C4A4.T4G4-containing sequences. Digestion of the telomere protein with trypsin generates an N-terminal protease-resistant fragment of approximately 35 kDa. This 35-kDa peptide appears to comprise the DNA-binding domain of the telomere protein as it retains most of the DNA-binding characteristics of the native 51-kDa protein. For example, the 35-kDa peptide remains bound to telomeric DNA in 2 M KCl. Additionally, the peptide binds well to single-stranded oligonucleotides that have the same sequence as the T4G4 strand of native telomeres but binds very poorly to mutant telomeric DNA sequences and double-stranded telomeric DNA. Removal of the C-terminal 15 kDa from the telomere protein does diminish the ability of the protein to bind only to the terminus of a telomeric DNA molecule.

Amino Acid Sequence↗
PubMed1992

Simultaneous genotypic and immunophenotypic analysis of interphase cells using dual-color fluorescence: a demonstration of lineage involvement in polycythemia vera.

Fluorescent in situ hybridization has become a useful technique by which chromosomal abnormalities may be shown in interphase cells. We present a dual-fluorescence method whereby a chromosomal and immunophenotypic marker can be visualized simultaneously in the same interphase cell. Two patients with the myeloproliferative disorder polycythemia vera and trisomy for chromosome 8 have been studied using this technique and selective involvement of the myeloid and erythrocyte lineages has been shown by the detection of the trisomy in immunophenotyped cells. Simultaneous analysis of genotype and immunophenotype in individual cells from patients with myeloproliferative disorders or leukemia may help identify the developmental and lineage status of cells in which molecular alterations have resulted in clonal advantage.

Aged↗