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A Neubauer

Publications and source records attributed to A Neubauer.

At least 163 records · Page 9Linked to original sources
PubMed1992

Analysis of gene amplification in archival tissue by differential polymerase chain reaction.

Oncogene amplification is found in many human tumors, and its detection may have important prognostic value. However, analysis of gene amplification may be hampered by inadequate tissue or poor DNA quality. We have previously described a polymerase chain reaction (PCR)-based procedure called differential PCR that can detect variations in gene dosage using miniscule amounts of tumor DNA [Frye, R.A., Benz, C.C. & Liu, E. (1989). Oncogene, 4, 1153-1157]. We now report the optimization of this technique for the analysis of oncogene amplification in paraffin-embedded archival tissues. We find that differential PCR is able to detect amplification of the HER2 (c-erbB-2) and the epidermal growth factor receptor (EGFR) genes and can be used to arrive at a semiquantitative estimate of gene dosage. Furthermore, our approach can determine gene amplification in samples in which the DNA is significantly degraded. Using differential PCR on paraffin-embedded tissues from cases previously investigated by standard DNA extraction and dot-blot procedures, good correlation between the two methods was found. Approaches are described to overcome technical problems posed by factors that affect the differential PCR, including the method of DNA extraction and extreme fragmentation of the DNA (less than 200 base pairs). Furthermore, the resulting analytical algorithm reported herein has proved effective in detecting oncogene amplification in archival breast cancer specimens from standard pathology laboratories. Thus, differential PCR will be particularly helpful in the analysis of tumor specimens that are archived, small in size or rare in occurrence.

Algorithms↗
PubMed1991

[Long-term results of coronary angioplasty in relation to vessel size].

Repeat angiography was performed after 3-4 months in 927 of 1386 consecutive patients (67%) who had a successful percutaneous coronary angioplasty (PTCA) between 1986 and 1988. The degree of coronary artery stenosis was determined angiographically before PTCA, immediately after and 3-4 months later. Patients were assigned to one of four groups according to balloon diameter at dilatation: Group 1: 1.5-2.0 mm; group 2: 2,5 mm; group 3: 3.0 mm; group 4: 3.4-4.2 mm. Vessel wall proliferation occurred in all four groups after PTCA. In patients with angiographically demonstrated recurrence (first recurrence: 308 patients, second recurrence: 43 patients) another balloon dilatation was undertaken and a repeat angiography 3-4 months later. Long-term success rate (less than 50% stenosis) differed significantly according to the post-PTCA vessel diameter: 48% in group 1, 63% in group 2, 66% in group 3 and 80% in group 4 (analysis of variance: P less than 0.001). PTCA thus produces better long-term results in large than in small vessels.

Adult↗
PubMed1991

Loss of heterozygosity on the short arm of chromosome 17 is associated with high proliferative capacity and DNA aneuploidy in primary human breast cancer.

Loss of heterozygosity (LOH) on the short arm of chromosome 17 (17p) was found in 27 of 52 (52%) previously untreated primary breast cancers. There was a significant correlation between this 17p allelic loss and two parameters associated with aggressive tumor behavior: high cellular proliferative fraction and DNA aneuploidy. These correlations with high cellular proliferative fraction and DNA aneuploidy were not found in tumors with LOH at nine other chromosome locations. The p53 gene, a putative tumor suppressor gene located at 17p13, was examined for aberrations to determine whether it is the target for the 17p LOH in breast cancer. Unlike other types of human cancer, there were no homozygous deletions or rearrangements of the p53 gene, and only 2 of 13 (15%) were mutated in the conserved region where mutational "hot spots" have been previously located. Therefore, we hypothesize that, in breast cancer, either loss or inactivation of gene(s) on chromosome 17p other than the p53 gene or a different mechanism of p53 gene inactivation may be responsible for the observed high labeling index and DNA aneuploidy associated with LOH at 17p.

Base Sequence↗
PubMed1991

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system. Purification and protein sequencing of the Staphylococcus carnosus histidine-containing protein, and cloning and DNA sequencing of the ptsH gene.

The histidine-containing protein (HPr) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Staphylococcus carnosus and purified to homogeneity. The protein sequence was determined by Edman degradation of peptides obtained by proteolytic digestion with proteases V8, trypsin and chemical cleavage with BrCN. Furthermore, immunological screening of a chromosomal S. carnosus DNA gene library in pUC19 vector enabled us to isolate S. carnosus HPr-expressing colonies. The nucleotide sequence of this ptsH gene and its flanking regions was determined by the dideoxy-chain-termination technique. Upstream, the 264-bp open reading frame of the ptsH gene is flanked by a putative S. carnosus promoter structure and a putative ptsI gene downstream suggesting that ptsH gene is the first gene in the PTS operon of S. carnosus. Comparison of the amino acid sequence of S. carnosus HPr with the HPr sequence of Staphylococcus aureus (derived from peptide sequencing) showed a high degree of similarity.

Amino Acid Sequence↗
PubMed1991

Mutations of the ras proto-oncogenes in childhood monosomy 7.

ras gene mutations are the most frequent molecular changes found in the preleukemic syndromes of adults and may play a role in initiating these diseases and in their progression to acute leukemia. However, little is known about the incidence or importance of these genetic mutations in childhood myeloproliferative states (MPS). The bone marrow (BM) monosomy 7 syndrome accounts for a large percentage of childhood MPS. Although the duration of the MPS is quite variable, children with monosomy 7 eventually develop acute myeloid leukemia (AML). We investigated 20 children (13 with MPS, 7 with AML) with BM monosomy 7 or 7q- for the presence of ras gene mutations using the polymerase chain reaction and hybridization with mutation-specific oligonucleotides. Mutations of N-ras and K-ras were detected in three children. Two patients carrying a ras mutation were in the myeloproliferative phase, and one had acute leukemia. All three patients with ras mutations either died of their disease or relapsed after BM transplantation as compared with 8 of 17 without ras mutations. However, this difference is not statistically significant (P = .14, not significant). We conclude that ras mutations are observed in childhood monosomy 7, though less frequently than in adult MDS, and may play a limited role in the progression of this disease to acute leukemia. More patients are needed to address the prognostic role of ras mutations in this rare disease.

Bone Marrow↗
PubMed1991

axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase.

Using a sensitive transfection-tumorigenicity assay, we have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antiphosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. The juxtaposition of fibronectin type III and immunoglobulinlike repeats in the extracellular domain, as well as distinct amino acid sequences in the kinase domain, indicate that the axl protein represents a novel subclass of receptor tyrosine kinases.

Amino Acid Sequence↗
PubMed1990

[Long-term success of coronary angioplasty after repeated treatment].

To ascertain the long-term results after second and third angioplasties for coronary stenosis the coronary angiograms from 1504 consecutive patients with coronary heart disease (1211 men, 293 women, mean age 57 [27-82] years) were retrospectively surveyed. A good initial response (at least 20% reduction in stenosis) was achieved in 295 out of 306 second angioplasties (95.5%), and in all 36 third angioplasties. Viewed overall, these results are significantly better (P less than 0.005) than those achieved at the initial angioplasty (1386 out of 1504 patients; 92.2%). The reduction in the severity of the stenosis achieved at the second angioplasty (from 86 to 24%) and at the third angioplasty (from 86 to 26%) was the same as at the first angioplasty (from 88 to 28%). Serious complications after the first angioplasty were infrequent (death in 0.2%, emergency bypass in 0.4%, myocardial infarction in 0.5%), and no complications were noted after second and third angioplasties. A good long-term outcome (at least 20% reduction in stenosis at 3-4 months) was slightly more frequent after the second and third angioplasties (103 out of 170 [60.6%] and 14 out of 17 patients, respectively) than after the first intervention (532 out of 926 patients; 57.5%). In keeping with these results, the degree of stenosis found at follow-up angiography was significantly lower (first intervention 54.8%, second intervention 50.3%, third intervention 36.9%). There were only 57 patients (3.8%) who ultimately required operative treatment. These figures indicate that the probability of a good long-term outcome from coronary angioplasty increases each time the stretching operation is repeated. Only a very small proportion of patients will require bypass surgery.

Adult↗
PubMed1990

Polymerase chain reaction based assay to detect allelic loss in human DNA: loss of beta-interferon gene in chronic myelogenous leukemia.

We have developed a polymerase chain reaction (PCR) based technique to detect allelic loss. In this differential PCR a target gene and a reference gene are coamplified in the same reaction vessel. The ratio of the intensity of the two resultant bands is an indication of relative gene dosage. This procedure is sensitive in that gene copy ratios of 2:1 and 3:2 (reference: target gene) can readily be detected. Using this differential PCR, we have examined 64 cases of chronic myelogenous leukemia (CML) for the loss of the beta 1-interferon gene, a relatively common event in certain human leukemias and lymphomas. Only one patient who was Philadelphia chromosome positive and who was in blast crisis exhibited allelic loss of the beta-interferon gene. Thus despite deletions at the beta-interferon locus in the CML cell line, K562, this perturbation is rarely seen in primary CML samples.

Alleles↗
PubMed1990

[CEA as a tumor marker on the cell surface and in the exudate. Comparison in thoracoscopy examined pleural effusions].

A prospective comparison was conducted in 43 patients to assess comparatively the ranking of cytology and CEA determined in the pleural effusions and by immunocytology on the cell surface (the latter two compared with each other) and then juxtaposed with the information obtained by histology via thoracoscopy. It was found that the yield achieved by cytology was comparable with that obtained by immunocytology. The maximum sensitivity was established with the adenocarcinoma.

Biomarkers, Tumor↗
PubMed1989

Binding of mitogenic plant lectins to human lymphocytes. Flow cytometric analysis.

Several plant lectins, such as phytohaemagglutinin (PHA), pokeweed mitogen (PWM), concanavalin A (ConA), Maclura pumifera (MPA) and Pisum sativum agglutinin (PSA), are potent mitogens for human lymphocytes. The pattern of activation induced, however, is not uniform for all mitogenic lectins. The different biological effects following lectin activation of human lymphocytes might be due at least in part to a differential binding of the various lectins to lymphocyte subsets. We have therefore studied the binding of five mitogenic plant lectins, namely PHA, PWM, ConA, MPA and PSA to three major human lymphocyte subsets as defined by anti-CD4, anti-CD8 and anti-CD16 monoclonal antibodies. Dual colour, flow cytometric analysis employing PE-conjugated monoclonal antibodies and FITC-conjugated lectins revealed that all subsets uniformly show high binding of PHA, whereas two different populations, one high binding and the other low binding, can be detected with PWM, ConA, MPA and PSA.

Antigens, Differentiation↗
PubMed1989

Lymphocytosis of large granular lymphocytes associated with anemia and neutropenia: proof of monoclonality of the LGL-population, but benign clinical course.

A 37-year-old man with severe transfusion-requiring anemia and neutropenia associated with lymphocytosis of large granular lymphocytes (LGLs) has been followed over a period of 3.5 years. Detailed phenotyping of the LGL has been performed, revealing a phenotype of CD3+, CD8+, CD16+, HLA-DR+, and Leu-7+. Southern-blot analysis of the T-cell receptor beta-chain locus detected a gene rearrangement, thus providing proof of monoclonality of the peripheral blood LGLs.

Adult↗
PubMed1989

[Effect of the method on the results of determining T-cell subpopulations].

Two methods of determining lymphocyte subpopulations were compared on samples obtained from 18 healthy blood donors and 16 HIV-positive patients. In the whole-blood method directly marked monoclonal antibodies were used, whereas Ficoll-separated mononuclear cells were assayed indirectly. The number of CD4-positive helper cells was significantly lower in both groups in the whole-blood test, but that of CD8-positive suppressor cells significantly increased after Ficoll separation. In HIV-infected patients the values for CD8-positive lymphocytes were even further apart than in the healthy donors. As a result the difference in the ratio CD4/CD8 was higher in HIV-infected patients (0.3 vs 0.65; P less than 0.001) than in the healthy donors (1.35 vs 1.8; P less than 0.001).

Antibodies↗
PubMed1989

Allergic reaction with immune hemolytic anemia resulting from chlorambucil.

In a 73-year-old female with chronic lymphocytic leukemia we observed an acute illness with shivering, high fever, and hemolytic anemia following chlorambucil administration. Reexposure in a controlled clinical situation suggested an allergic drug reaction. In an in vitro assay, we were able to demonstrate chlorambucil as the causative agent of immune hemolysis.

Aged↗
PubMed1989

Immunocytochemical detection of p21ras expression in fresh human leukaemic cells and cell lines.

The expression of p21ras proteins was investigated by immunocytochemistry in permanent cell lines and in fresh human leukaemic cells. While high and low levels of p21ras could be detected in most of the cell lines, no significant p21ras immunoreactivity was noted in cells of ten human acute and chronic leukaemias. Thus, notwithstanding its possible role in the initial transformation process in human leukaemias, p21ras expression appears not to be an irrevocable requirement for the maintenance of the transformed state.

Antibodies, Monoclonal↗
PubMed1989

A flow cytometric assay for the determination of cell proliferation with a monoclonal antibody directed against DNA-methyltransferase.

The enzyme DNA-methyltransferase is responsible for the methylation of a newly synthesized DNA-strand. A monoclonal antibody directed against DNA-methyltransferase was used to determine cell proliferation by means of flow cytometry. The reactivity of DNA-methyltransferase antibody was compared with the known proliferation markers transferrin-receptor and Ki67. All three methods showed comparable reactivity with the erythroblastic cell line K562 (86%, 81%, 76% respectively). In a second set of experiments peripheral blood mononuclear cells were stimulated with phytohaemagglutinin; in three experiments, a mean of 63% of the cells reacted with DNA-methyltransferase antibody after 72 h of culture as compared to a mean of 6% in the case of unstimulated control cells. HL60 cells were incubated with DMSO and harvested on day 5 of culture. The results obtained show that in differentiated cells the fraction positive with DNA-methyltransferase antibody decreased to levels below 10%. It is concluded that the technique described is a fast and easy method for the flow cytometric determination of cellular proliferation.

Antibodies, Monoclonal↗