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T Graf

Publications and source records attributed to T Graf.

At least 73 records · Page 4Linked to original sources
PubMed1993

Cavitation threshold with respect to dP/dt: evaluation in 29 mm bileaflet, pyrolitic carbon heart valves.

A total of 15 bileaflet mechanical heart valves were studied in a pulse duplicator at the Helmholtz Institute (Aachen, Germany) under conditions approximating first, a physiological pressure curve and subsequently, a sinusoidal pressure curve. In this study Edwards-Duromedics valves of the modified specification were compared with the earlier version of the Edwards-Duromedics valve as well as with St. Jude Medical valves. Each valve was tested at a series of nine (9) conditions. At each condition, without altering the valve installation or the systemic conditions, each valve was filmed by two separate video systems: the Helmholtz Institute strobe light system and a high speed video recording system. All data, as recorded by each system, was then independently analyzed by both of the two contributing groups and subsequently compared. In this manner, it was possible to objectively verify not only the consistency of the data obtained, but to also determine the relative reliability of the methods for cavitation threshold detection.

Carbon↗
PubMed1992

Chicken "erythroid" cells transformed by the Gag-Myb-Ets-encoding E26 leukemia virus are multipotent.

The E26 avian leukemia virus encodes a transcriptional activator-type oncoprotein consisting of Gag, Myb, and Ets domains, and transforms early erythroid cells as well as myeloblasts. Surprisingly, we have found that "early erythroid" transformants obtained in culture are multipotent, since they can be induced to differentiate into myeloblasts and eosinophils after superinfection with retroviruses containing kinase-type or ras oncogenes. In addition, TPA is an efficient inducer that generates predominantly eosinophils at low concentrations and myeloblasts at high concentrations. The determination process involves the complete extinction of erythroid/thrombocytic markers and the subsequent activation of myelomonocytic/eosinophilic properties, including the acquisition of specific growth factor requirements. "Erythroleukemic" cells from virus-infected animals were likewise found to be multipotent, making this a unique system to study the genesis of stem cell leukemias and the molecular basis of lineage commitment during hematopoiesis.

Animals↗
PubMed1992

DNA binding by c-Ets-1, but not v-Ets, is repressed by an intramolecular mechanism.

The E26 avian retrovirus causes an acute leukemia in chickens and transforms both myeloid and erythroid cells. The virus encodes a 135 kDa fusion protein which contains amino acid sequences derived from the viral Gag protein and the two cellular transcription factors c-Myb and c-Ets-1p68. Previously we have shown that like v-myb, v-ets on its own is also active in transformation, but only within the erythroid lineage. To understand better the mechanisms involved in the oncogenic activation of c-Ets-1p68, we used the polyoma PEA3 element, a known Ets binding site, to compare the sequence-specific DNA binding and transactivating properties of v-Ets and c-Ets-1p68. Using Ets protein synthesized in rabbit reticulocyte lysate in gel retardation assays, we detected little binding of c-Ets-1p68 to an oligonucleotide containing the PEA3 motif whereas v-Ets bound strongly. However, in transient cotransfection assays in chicken embryo fibroblasts both c-Ets-1p68 and v-Ets transactivated transcription from a heterologous promoter linked to PEA3 elements. Interestingly, fragments of c-Ets-1p68 with strong DNA binding activity could be produced by limited proteolysis, indicating that the DNA binding domain is repressed within the full-length molecule. By deletion mapping the DNA binding domain was localized to the most highly conserved region of the Ets-related proteins known as the ETS domain. The C-terminus as well as a region in the middle of the polypeptide chain are involved in repression of DNA binding in c-Ets-1p68. Significantly, v-Ets contains a 16 amino acid substitution at the C-terminus. Our results suggest that intramolecular repression of DNA binding is a regulatory mechanism in c-Ets-1p68 which is lost in v-Ets.

Animals↗
PubMed1992

Prediction of phenotype for acetylation and for debrisoquine hydroxylation by DNA-tests in healthy human volunteers.

The debrisoquine/sparteine-type polymorphism of drug oxidation and the polymorphism for acetylation are two common inherited variations in human drug metabolism. The phenotypes for hydroxylation and acetylation can be predicted be newly developed methods based on mutation-specific amplification of DNA by the polymerase chain reaction (PCR), which also allow for identification of heterozygous carriers of one mutant allele. In the present study, the results of genotyping of 81 healthy European volunteers were compared with the phenotype obtained by the classical biochemical approach using debrisoquine and caffeine as probe drugs. Genotyping correctly predicted all 73 extensive metabolisers (EMs) and 6 out of 8 poor metabolisers (PMs) of debrisoquine. All 48 rapid acetylators and 33 of 35 slow acetylators were predicted. Overall, the DNA analysis result matched the in vivo phenotype in 97.5% of individuals.

Acetylation↗
PubMed1992

Myb: a transcriptional activator linking proliferation and differentiation in hematopoietic cells.

Myb is a transcriptional activator protein with repeated helix-turn-helix DNA-binding motifs distantly related to the homeodomain. In hematopoiesis, c-myb appears to control both cell proliferation and differentiation. The mechanisms by which the leukemogenic potential of c-Myb is activated are complex and involve truncations, point mutations, and fusion or coexpression with other proteins.

Animals↗
PubMed1992

Structure of the chicken myelomonocytic growth factor gene and specific activation of its promoter in avian myelomonocytic cells by protein kinases.

In chicken myeloid cells but not in erythroid cells, kinase-type oncogenes activate expression of the chicken myelomonocytic growth factor (cMGF). The autocrine loop established this way plays a key role in lineage-specific cooperation of nuclear and kinase-type oncogenes in retrovirally induced myeloid leukemia. In this report, we describe the cloning of the cMGF gene, including its promoter. The structure of the cMGF gene is homologous to those of the granulocyte colony-stimulating factor and interleukin-6 genes. Expression from reporter constructs containing the cMGF promoter is specific to myelomonocytic cells. Kinases activate cMGF at the transcriptional level in macrophages and strongly induce reporter expression in myelomonocytic cells.

Amino Acid Sequence↗
PubMed1992

Expression patterns of c-myb and of v-myb induced myeloid-1 (mim-1) gene during the development of the chick embryo.

The v-myb oncogene of the acute avian leukemia virus E26 encodes a transcription factor that directly regulates the promyelocyte-specific mim-1 gene (Ness, S.A., Marknell, A. and Graf, T. Cell, 59, 1115-1125). We have investigated the relationship between the c-myb proto-oncogene and the transcription of the mim-1 gene both in vitro and in vivo. We demonstrate that the c-myb protein can transactivate the transcription of mim-1 in a transient transfection assay. In the chick embryo, we confirm that mim-1 is specifically expressed during granulopoiesis and we show that the expression of c-myb and mim-1 are perfectly correlated in the granulocytic spleen and pancreas. However we suggest that mim-1 is efficiently transcribed in the absence of c-myb in the yolk sac and in the promyelocytes at the onset of the colonization of the bursa of Fabricius. On the other hand c-myb transcripts detected in the early hemopoietic progenitor cells, in lymphoid cells and in proliferative epithelia are never associated with mim-1 transcription. We conclude that the granulocyte-specific mim-1 gene is regulated by c-myb-dependent and c-myb-independent mechanisms depending upon the environment in which granulocytic precursor cells differentiate.

Animals↗
PubMed1992

Cavitation of mechanical heart valves under physiologic conditions.

Cavitation has recently entered the discussion of factors leading to blood and material damage after implantation of mechanical heart valves. Since direct evidence for this phenomenon cannot yet be demonstrated in vivo, an in vitro method had to be developed to permit the investigation whether cavitation actually occurs under (simulated) physiologic conditions and to clarify its clinical relevance. Previous studies have shown that different types of commercially available mechanical valves exhibit different tendencies to generate cavitation in vitro. The present study presents a test protocol for the assessment of cavitation generated by different replacement valves under simulated physiologic conditions. Comparative investigations were performed in order to transfer the in vitro results in vivo conditions. Although mechanical valves with an overlapping closing body/valve ring configuration tend to create cavitation earlier, cavitation could not be demonstrated for any investigated mechanical valve type under simulated resting conditions. The danger of continuous cavitation damage is therefore low. Certain valve types exhibit cavitation only at higher heart rates (more than 120-140 beats/min). Some valve types do not show cavitation even during heavy exercise. Based on mathematical models and experimental investigations, it is very likely that if cavitation does occur, blood and material damage may be expected.

Blood↗
PubMed1992

The nuclear oncogenes v-erbA and v-ets cooperate in the induction of avian erythroleukemia.

The nuclear oncogenes v-erbA and v-ets are known to cooperate with other viral oncogenes in the induction of avian erythroleukemia. Thus, in the case of avian erythroblastosis virus (AEV), v-erbA enhances the effect of the tyrosine kinase-encoding v-erbB oncogene by blocking the terminal differentiation of erythroid cells. In the case of E26 virus a fusion of the product from v-ets to that of the nuclear oncogene v-myb is a prerequisite for leukemogenicity. Here we show that an artificial virus carrying both v-erbA and v-ets induces a rapid, acute erythroleukemia phenotypically similar to that induced by AEV. In contrast, virus constructs containing either v-erbA or v-ets alone are non-leukemogenic, although they are capable of transforming erythroid cells in vitro. Analysis of in vitro-transformed cells showed that v-erbA induces a block of differentiation without abrogating dependence on anemic serum, while v-ets predominantly causes anemic serum independence. As expected, cells transformed by both oncogenes exhibit an increased proliferative potential, are blocked in differentiation and are anemic serum independent. These data demonstrate that two separately expressed nuclear oncoproteins can complement each other in vitro and in vivo. They also show that the v-Ets protein on its own can contribute to leukemogenesis.

Animals↗
PubMed1992

Cell surface proteins of chicken hematopoietic progenitors, thrombocytes and eosinophils detected by novel monoclonal antibodies.

E26 is an acute avian leukemia virus that contains two nuclear oncogenes, v-myb and v-ets, and that is capable of transforming early cells of the erythroid and myeloid lineages. In another study, we have found that TPA (phorbol 12,13-dibutyrate) treatment of E26-transformants displaying an 'early erythroid' phenotype results in the production of cells with either myeloid or eosinophil characteristics. To analyze this induction in greater detail we have produced a panel of four monoclonal antibodies against E26-transformants before and after TPA-induced differentiation. Two antibodies, MEP21 and MEP26, reacted with proteins of 150 and 47-60 kDa, respectively, which are expressed on the surface of E26 progenitor cells but whose expression is extinguished following TPA-induced differentiation. A third antibody, EOS47, recognizes a 100 kDa molecule that is expressed on the surface of TPA-induced peroxidase positive cells (an enzyme that in avian species is restricted to cells of the eosinophilic lineage). MEP21, MEP26, and EOS47 do not react with lymphoid, myeloid, or more mature erythroid lineage cell lines. The fourth antibody, MEP17, recognizes a heterodimer of 140 and 150 kDa chains which is expressed at high levels by E26-transformed progenitor cells and at lower levels by TPA-induced cells. Further biochemical characterization of the MEP17 antigen revealed a structure similar to that of the leukocyte adhesion molecule VLA-4; a member of the integrin family of adhesion proteins. All four antibodies react with subpopulations of cells in the bone marrow and spleens of 1-day-old chickens. Although the MEP21 and MEP26 antibodies do not appear to react with mature cells of most hematopoietic lineages they are expressed at high levels by mature thrombocytes. In addition, MEP17 is expressed at high levels by the majority of bursal B-cells, thrombocytes, and more weakly by thymocytes. The reagents described should be useful as markers for the study of development, migration, and differentiation of normal avian hematopoietic progenitor cells and eosinophilic precursors, and for the study of retrovirus-induced neoplasia.

Animals↗
PubMed1992

Identification of genes differentially expressed in two types of v-myb-transformed avian myelomonocytic cells.

In an earlier study we found that different forms of the v-myb oncogene transform myeloid cells which resemble either monoblasts [when v-myb of avian myeloblastosis virus (AMV) was used] or promyelocytes [when a point mutant in v-myb of AMV was used; Introna, M., Golay, J., Frampton J., Nakano, T., Ness, S.A. & Graf, T. (1990). Cell, 63, 1287-1297]. In the present study we have searched for genes expressed in AMV mutant-transformed promyelocytes that are not expressed in AMV-transformed monoblasts using a differential screening approach. Eight different genes were identified among more than 500 differentially expressed clones. The most abundant of these was the previously identified myb-regulated mim-1 gene. The others were found to encode a small calcium-binding (MRP-like) protein; the p20K protein; goose-type lysozyme; a ribonuclease A/angiogenin-related protein; and three non-identified proteins. Although these genes appear to be rather lineage restricted, their expression varied in different subtypes of transformed myelomonocytic cells, and only two of them (goose lysozyme and ribonuclease) showed a similar expression pattern in normal promyelocytes and macrophages, suggesting an aberrant gene regulation in the transformed cells. Co-transfection experiments of a reporter construct containing the promoter of the ribonuclease A-related gene indicated that this promoter is regulated by the v-Myb oncoprotein without the involvement of Myb-specific binding sequences.

Amino Acid Sequence↗
PubMed1991

Goose-type lysozyme gene of the chicken: sequence, genomic organization and expression reveals major differences to chicken-type lysozyme gene.

This report describes the cloning, sequencing and expression pattern of the chicken goose-type lysozyme gene. The cDNA sequence was found to have no homology to that of the chicken-type lysozyme gene and exhibits a completely different exon-intron organization. In addition, goose-type lysozyme displays an overlapping but different tissue expression pattern in chickens than chicken-type lysozyme.

Amino Acid Sequence↗
PubMed1991

Fusion of the nuclear oncoproteins v-Myb and v-Ets is required for the leukemogenicity of E26 virus.

The highly leukemogenic avian retrovirus E26 expresses the two transcriptional activator-type oncogenes v-myb and v-ets as a nuclear fusion protein. Previous studies have shown that both oncogenes cooperate in the transformation of erythroid cells in vitro and that the phenotypes of transformed cells differ, depending on whether the oncogenes are coexpressed as separate proteins or as a fusion protein. Here we show that virus constructs encoding either v-Myb or v-Ets as their only oncoprotein are nonleukemogenic and that constructs coexpressing nonfused v-Myb and v-Ets proteins appear to be weakly leukemogenic. Surprisingly, leukemic animals injected with the latter contain highly leukemogenic variant viruses that exhibit internal deletions in their genome, resulting in the synthesis of novel Myb-Ets fusion proteins. These results show that v-Myb and v-Ets must be fused to cause leukemia and establish a new mechanism of oncogene activation and cooperation.

Alpharetrovirus↗
PubMed1991

Activation of cMGF expression is a critical step in avian myeloid leukemogenesis.

A non-leukemogenic version of the v-myb oncogene causes in vitro transformation of avian myeloblasts, which are dependent on chicken myelomonocytic growth factor (cMGF). We have shown that this version of v-myb, when combined with the erythroleukemia-inducing v-erbB oncogene, is capable of causing a mixed myeloid and erythroid leukemia. Myeloid leukemic cells transformed by this construct produce cMGF. To test whether autocrine growth stimulation via cMGF is the essential contribution of the tyrosine kinase oncogene v-erbB in avian myeloid leukemogenesis we constructed another retrovirus containing both the non-leukemogenic v-myb and the cMGF cDNA. This virus induced myeloid leukemia at high efficiency. In a third construct we combined v-myb with the human EGF-receptor gene. Myeloid cells transformed by this construct could be stimulated to grow by the addition of cMGF or EGF. Growth stimulation with EGF was blocked by a cMGF antiserum indicating that activation of a normal tyrosine kinase-type receptor induces cMGF expression but does not bypass the cMGF requirement. We conclude that cMGF plays a key role in the growth regulation of normal and transformed avian myeloid cells.

Animals↗
PubMed1991

Proposed structure for the DNA-binding domain of the Myb oncoprotein based on model building and mutational analysis.

Myb-related proteins from plants to humans are characterized by a DNA-binding domain which contains two to three imperfect repeats of approximately 50 amino acids each. Based on the evolutionary conservation of specific residues, secondary structural predictions suggest an arrangement of alpha helices homologous to that seen in the homeodomains, members of the helix-turn-helix family of DNA-binding proteins. We have used molecular modelling in conjunction with site-directed mutagenesis to test the feasibility of this structure. We propose that each Myb repeat consists of three alpha helices packed over a hydrophobic core which is built around the three highly conserved tryptophan residues. The C-terminal helix forms part of the helix-turn-helix motif and can be positioned into the major groove of B-form DNA, allowing prediction of residues critical for specificity of interaction. Modelling also allowed positioning of adjacent repeats around the major groove over an 8 bp binding site.

Amino Acid Sequence↗