Norimichi Watanabe

cDNA cloning and structure of mouse putative Ah receptor

Mouse cDNA clones for a putative Ah receptor have been isolated from a cDNA library of mRNA from Hepa-1 cells by an oligonucleotide probe produced by PCR with a pair of primers which was synthesized according to the reported N-terminal sequence of 26 amino acids. The cDNA clones encode a polypeptide of 805 amino acids with a helix-loop-helix motif and with some similarity to a certain region designated PAS of Drosophila Per and Sim, and human Arnt protein. Cotransfection of an expression vector of the Ah receptor with a reporter plasmid pMC6.3k consisting of CYP1A1 promoter and CAT structural gene into CV-1 cells enhanced the CAT expression in response to added 3-methylcholanthrene.

Inhibition by hemin of in vitro translocation of chicken liver δ-aminolevulinate synthase into mitochondria

The precursor form of chicken liver delta-aminolevulinate synthase was synthesized in a reticulocyte lysate cell-free translation system and then incubated with the homologous liver mitochondria. The precursor enzyme was incorporated into the mitochondria with an attendant processing to the mature enzyme. In this in vitro experimental system, both the transport and processing of the enzyme were significantly inhibited by the addition of hemin as low as about 3 microM. This provides further support to the view, which had been derived from the studies in vivo, that inhibition by hemin of the translocation of delta-aminolevulinate synthase into mitochondria could be one of the regulatory mechanisms for heme biosynthesis in the liver cells.

δ-Aminolevulinate synthase isozymes in the liver and erythroid cells of chicken

Antibodies raised against the purified chicken liver delta-aminolevulinate synthase showed a partial cross-reactivity with the chicken erythroid delta-aminolevulinate synthase. delta-Aminolevulinate synthase synthesized in vitro using polysomes from erythroid cells showed a subunit molecular weight of 55,000, whereas the enzyme synthesized in vitro using liver polysomes had a subunit molecular weight of 73,000. delta-Aminolevulinate synthase isolated from mitochondria of erythroid cells showed a molecular weight of 53,000, while the enzyme in liver mitochondria had a value of 65,000. These observations imply that the erythroid delta-aminolevulinate synthase differs from the hepatic enzyme.

Altered surface expression of effector cell molecules on neutrophils in myelodysplastic syndromes

The surface expression of effector cell molecules on neutrophils was examined in 18 patients with myelodysplastic syndromes (MDS) and 20 healthy control subjects. The MDS patients were further classified as low clinical risk (L‐MDS, n = 7) and high clinical risk (H‐MDS, n = 11). The expression of Fc receptors for IgG (FcR), complement receptors (CR) and cellular adhesion molecules on neutrophils was determined by flow cytometry and monoclonal antibodies. The effect of granulocyte colony‐stimulating factor (G‐CSF) and tumour necrosis factor‐alpha (TNF) on L‐selectin shedding and CR up‐regulation on neutrophils was also examined. The percentage of FcRI‐positive neutrophils and CD11b/CR3 expression on neutrophils were significantly increased in the H‐MDS patients when compared to the controls. In contrast, the expression of FcRII, FcRIII, L‐selectin, LFA‐1 and CD18 on neutrophils was significantly reduced in the H‐MDS patients compared with the controls. The L‐MDS neutrophils exhibited lower expressions of CR1, L‐selectin, LFA‐1 and CD18 than those of the controls. Neutrophils from some H‐MDS patients showed impaired L‐selectin shedding and CR up‐regulation after stimulation with G‐CSF or TNF, although these were not significantly different when assessed in the whole H‐MDS group. These findings suggest that an altered surface expression of effector cell molecules and an impaired modulation of cellular adhesion molecules on neutrophils may contribute to the increased susceptibility to bacterial infections in MDS patients.

Two chronic myelogenous leultaemia cell lines which represent different stages of erythroid differentiation

Summary We established two cell lines, YN‐1 and Y‐1K. from the peripheral blood of two chronic myelogenous leukaemia patients in blastic crisis. Characterization of the YN‐1 and Y‐1K cells revealed that these cells expressed erythroid lineage markers. However, there was a marked difference in the level of γ‐globin mRNA and haenioglobin in YN‐1 and Y‐1K cells. YN‐1 contained approximately 1–5% benzidine‐positive staining cells, whereas no benzidine‐positive cells were observed in Y‐1K cells. Haemoglobin production in YN‐1 cells was markedly increased with various chemical inducers of erythroid differentiation, but was not in Y‐1K cells. In contrast, Y‐1K cells expressed CD34 stem cell antigen and CD41 megakaryocyte‐specific antigen. These observations suggested that, although both cell lines were committed to the erythroid lineage. each cell line represented a distinct differentiation stage in the erythroid differentiation programme. Y‐1K seemed to correspond to an early stage of cells in erythroid lineage, whereas YN‐1 represented a more advanced stage in human erythropoiesis.

T cell-mediated inhibition of erythropoiesis in aplastic anaemia: the possible role of IFN-γ and TNF-α

Summary. The inhibitory activity of T cells on autologous erythroid colony‐forming units (CFU‐E) (T cell inhibitory activity) in patients with aplastic anaemia (AA) was investigated. In 11 (32·4%) out of 34 AA cases, T cell inhibition on autologous CFU‐E growth was greater than that in normal individuals. In order to evaluate the mechanism of this inhibitory activity, T cell surface markers, interferon (IFN) production in peripheral blood mononuclear cell (PBMNC) liquid culture, and cytokine levels such as IFN and tumour necrosis factor‐α (TNF‐α) in CFU‐E clot cocultured with T cells, were measured in a portion of the patients. In five patients investigated for IFN production in PBMNC liquid culture, all produced statistically more IFN activity than normal individuals under phytohaemagglutinin (PHA‐P) stimulation (P<0·01) with no relation to T cell inhibitory activity. In only one patient whose T cells displayed increased CD8 and HLA‐DR antigen (CD8+HLA‐DR+) and inhibitory activity, a significant amount of IFN‐γ was observed in CFU‐E clot cocultured with T cells, and the addition of anti‐IFN‐γ antibody to the coculture resulted in recovered CFU‐E colony growth. These results suggest that IFN‐γ production by T cells may explain, at least in part, the pathogenesis of haematopoietic defects in AA. In other patients however, T cell inhibitory activity neither correlated to the T cell subpopulations (CD4+/CD8+, CD8+HLA‐DR+), IFN production in PBMNC liquid culture, nor to IFN and TNF‐α levels in CFU‐E clot culture. The roles played by cytokines other than IFN and TNF‐α on haematopoietic precursor cells require further evaluation in a larger sample of patients with AA.

An immunochemical study of δ-aminolevulinate synthase and δ-aminolevulinate dehydratase in liver and erythroid cells of rat

Abstract The relationship between erythroid δ-aminolevulinate (ALA) synthase and hepatic ALA synthase in rat was analyzed immunochemically, using antibodies directed against rat liver ALA synthase and against chicken liver ALA synthase. Rat erythroid ALA synthase showed no cross-reactivity with anti-liver ALA synthase antibodies, but hepatic ALA synthases from rat, mouse, and chicken share substantial cross-reactivity with one another. These results clearly distinguish the isozyme relationship between erythroid ALA synthase and hepatic ALA synthase in rat and suggest that there may be at least two different ALA synthase genes in rat. ALA dehydratase in rat liver, on the other hand, could not be immunochemically distinguished from ALA dehydratase in rat erythroid cells when antibody against rat erythroid ALA dehydratase was used. The finding that erythroid ALA synthase is an entity different from hepatic ALA synthase may provide a clue to understanding the different features in hepatic and erythropoietic porphyrias.

Tetrasomy 21 as a sole chromosome abnormality in acute myeloid leukemia: fluorescence in situ hybridization and spectral karyotyping analyses

We report a case of acute myeloid leukemia with tetrasomy 21 as the sole chromosome abnormality in a constitutionally normal patient. Tetrasomy 21 was observed at presentation, disappeared in remission, but reappeared in recurrence of the disease. Fluorescence in situ hybridization analysis using a probe specific for the AML1 gene showed four distinct signals in 82.4% and three signals in 10.8% of interphase nuclei, although conventional G-banding revealed tetrasomy 21 alone in mosaicism with normal karyotype. Spectral karyotyping further confirmed the presence of extra copies of chromosome 21. Tetrasomy 21 as the only anomaly is relatively rare in patients with hematologic disorders other than Down syndrome, and to our knowledge has been reported previously in only seven cases. In a review of the literature, tetrasomy 21 as the only anomaly may be associated with myeloid disorders, although simultaneous numeric abnormalities other than chromosome 21 have been reported in acute lymphoblastic leukemia with hyperdiploid karyotype.

Development of diffuse large B-cell lymphoma in a patient with Waldenström’s macroglobulinemia/ lymphoplasmacytic lymphoma: clonal identity between two B-cell neoplasms

Waldenströms macroglobulinemia (WM)/ lymphoplasmacytic lymphoma (LPL) is an indolent mature B-cell neoplasm. In rare cases of WM/LPL, diffuse large B-cell lymphoma (DLBCL) develops as a result of histologic transformation. In this report, we present a case of DLBCL developing in a patient with WM/LPL. Combination chemotherapy for DLBCL was effective and complete remission was eventually achieved. We attempted to determine the clonal relatedness between WM/LPL and DLBCL in the patient by analyzing complementarity-determining region 3 (CDR3) in the immunoglobulin heavy chain gene. A common CDR3 sequence was found in tumor cells of DLBCL and those of WM/LPL, indicating that tumor cells of DLBCL are clonally identical to those of WM/LPL. Therefore, in the present case, DLBCL is developed from WM/LPL cells by clonal evolution.

Complement receptor type 1 (CR1) deficiency on neutrophils in myelodysplastic syndrome

Summary. We present a patient with myelodysplastic syndromes (MDS) whose neutrophils exhibited defective expression of complement receptor type 1 (CR1). A 73‐year‐old man was admitted with an evolution of MDS from RA into RAEBT according to the FAB classification of MDS. The neutrophil alkaline phosphatase (NAP) score was zero. The surface expression of membrance effector melecules on neutrophils was determined by indirect immunofluorescence using flow cytometry and monoclonal antibodies. The expression of CR1 on neutrophils as identified by staining with CD35 was defective in the patient, and the expression of other complement receptors (CR3 and CR4), Fc receptors and adhesion molecules was normal. CR1 deficiency and defective NAP score on neutrophils in the patient might account for impairment of common storage pool, presumably novel intracellular secretory vesicles.