Phylogeographic origin of Hokkaido house mice (Mus musculus) as indicated by genetic markers with maternal, paternal and biparental inheritance.

We examined intraspecies genetic variation in house mice (Mus musculus molossinus) from the northern third of the Japanese Islands, in order to obtain evidence of the history of mouse colonization that might have shaped the current genetic diversity. We extended the previous sampling of mitochondrial cytochrome b sequence and added information from the Y-linked Sry gene and ribosomal RNA gene surveys. We distinguish mitochondrial haplotypes characteristic of the North Asian musculus subspecies group (involving M. m. musculus and M. m. molossinus) as 'MUS', and that of the Southeast Asian castaneus subspecies group as 'CAS' (although the mice resemble MUS morphologically). There was a clear geographic partition of MUS and CAS types into southern and northern Hokkaido, respectively. Conversely, on Tohoku, the MUS and CAS types were interspersed without clear geographic subdivision. In contrast to the mtDNA data, all Hokkaido and Tohoku mice examined were found to possess a unique type for the Y-linked Sry gene, specific to Korea and Japan. Restriction site analysis of nuclear rDNA probe showed a consistent distribution of MUS and CAS types, as major and minor components, respectively, in the Hokkaido and Tohoku mice. These data support the previous notion that the Hokkaido and Tohoku mice experienced genetic hybridization between primary residents of CAS origin and MUS newcomers arriving via a southern route. The invasion of the MUS type could correspond with the evidence for arrival of prehistoric peoples. There are, however, alternative interpretations, including genetic admixture between MUS arriving by a southern route and CAS from a northern route.

Cloning and characterization of a chicken platelet-derived growth factor B-chain cDNA.

Avian thrombocytes are nucleated blood cells homologous in function to mammalian platelets. In the present study, we obtained a cDNA from chicken thrombocyte polyadenylated RNA [Poly(A)+RNA], which coded for the chicken PDGF-B chain. The sequence was 1083-bp long and had an open reading frame (ORF) of 753-bp. At the amino acid level, the predicted mature protein showed 69% homology with the processed coding region of human PDGF-B. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that PDGF-B mRNA was expressed at high levels in thrombocytes and in the lung. The expression of PDGF-B chain mRNA in thrombocytes reached its maximum level 12h following type 1 collagen treatment. These results suggest that chicken PDGF-B chain may play an important role in the vascular system and in healing wounded tissue.

Circumvention of acquired resistance to doxorubicin in K562 human leukemia cells by oxatomide.

We studied the effect of oxatomide, an antiallergic drug, on the resistance of K562 cells to doxorubicin. Oxatomide synergistically potentiated the cytotoxicity of doxorubicin in doxorubicin-resistant K562 cells (K562/DXR) at a concentration of 1-10 microM, but had hardly any synergistic effect on the parental cell line (K562) at the same concentration. Oxatomide inhibit P-glycoprotein pump-efflux activity and the binding of [3H]-azidopine to the cell-surface protein P-glycoprotein, in a dose-related manner. These results indicate that oxatomide reverses the multidrug-resistance phenotype through direct interaction with P-glycoprotein.

Translocation (14;19)(q32;q13) detected by spectral karyotyping and lack of BCL3 rearrangement in CD5-positive B-cell lymphoma associated with hemophagocytic syndrome.

It has been shown that some cases of B-cell non-Hodgkin lymphoma associated with a hemophagocytic syndrome (B-LAHS) have chromosomal abnormalities at 14q32 or 19q13. We report here a 64-year-old woman with B-LAHS and a complex karyotype including add(14)(q32). We applied spectral karyotyping and revealed that the add(14)(q32) was derived from a der(14)t(14;19)(q32;q13). However, rearrangement of the BCL3 gene at 19q13 could not be detected by Southern blot analysis. Our results indicate that the translocation involving 19q13 may be one of the recurrent aberrations in B-LAHS and that the molecular mechanism of t(14;19)(q32;q13) in B-LAHS appear to be different from that observed in chronic lymphocytic leukemia.

Expression of steroidogenic factor-1 in frog embryo and developing gonad.

Steroidogenic factor-1 (SF-1), originally identified as an orphan nuclear receptor that regulates expression of genes encoding cytochrome P-450 steroid hydroxylases, is an essential transcriptional factor for adrenal and gonadal development in mammals. Since sex steroid hormones have been shown to play important roles in the sex determination of frogs, it is of interest to know how SF-1 gene expression is regulated during the sexual development of this organism. A previous study isolated the cDNA of the frog Rana rugosa SF-1 (rrSF-1) and found sexual differences in its gene expression in adult frogs; positive in testis and negative in ovary (Kawano et al., 1998). This study examined rrSF-1 gene expression in frog embryos and developing and mature gonads by immunohistochemical staining using anti-rrSF-1 protein antibody for protein localization and by in situ hybridization analysis for mRNA transcription. The results obtained in this study indicated that cells expressing SF-1 that originate in the mesoderm and endoderm probably migrate into the developing gonad via the dorsal mesentery, genital ridge, and mesorchium or mesovarium. Thus, SF-1 may play an important role in gonadal development in amphibians.

Apoptosis induced by doxorubicin and cinchonine in P388 multidrug-resistant cells.

Acquired drug resistance is a major factor in the failure of doxorubicin-based cancer chemotherapy. We determined the ability of cinchonine to reverse doxorubicin drug resistance in a doxorubicin-resistant leukaemia cell line (mouse P388/DOX). A non-cytotoxic concentration of cinchonine (10 microM) increased the sensitivity to doxorubicin of multidrug-resistant P388/DOX cells and significantly enhanced the doxorubicin-induced apoptosis and DNA fragmentation in resistant cells, but had no effect in parent cells. Time-course studies demonstrated that DNA fragmentation was present 24 h after incubation with doxorubicin and cinchonine, indicating that DNA degradation was a preceding event. In cultured cells, cinchonine increased the intracellular accumulation of doxorubicin in the resistant cells in a dose-dependent manner. Using flow cytometry to measure the inhibition of the P-glycoprotein (P-gp) dependent efflux of rhodamine 123, cinchonine was found to be considerably more effective than quinine. The results with cinchonine suggest that there may be quinine derivatives with a similar capacity to inhibit drug transport by P-gp. Additionally, the G2/M phase cell population in resistant cells is increased by doxorubicin/cinchonine treatment. Exposure of resistant cells to 1 microM doxorubicin and 10 microM cinchonine resulted in the expression of Fas (APO-1/CD95) in cells after 6 h. These studies demonstrate that the cell killing effects of doxorubicin and cinchonine in resistant cells

Identification of myelodysplastic syndrome-specific genes by DNA microarray analysis with purified hematopoietic stem cell fraction.

Myelodysplastic syndrome (MDS) is a slowly progressing hematologic malignancy associated with a poor outcome. Despite the relatively high incidence of MDS in the elderly, differentiation of MDS from de novo acute myeloid leukemia (AML) still remains problematic. Identification of genes expressed in an MDS-specific manner would allow the molecular diagnosis of MDS. Toward this goal, AC133 surface marker-positive hematopoietic stem cell (HSC)-like fractions have been collected from a variety of leukemias in a large-scale and long-term genomics project, referred to as "Blast Bank," and transcriptome of these purified blasts from the patients with MDS were then compared with those from AML through the use of oligonucleotide microarrays. A number of genes were shown to be expressed in a disease-specific manner either to MDS or AML. Among the former found was the gene encoding the protein Delta-like (Dlk) that is distantly related to the Delta-Notch family of signaling proteins. Because overexpression of Dlk may play a role in the pathogenesis of MDS, the disease specificity of Dlk expression was tested by a quantitative "real-time" polymerase chain reaction analysis. Examination of the Blast Bank samples from 22 patients with MDS, 31 with AML, and 8 with chronic myeloid leukemia confirmed the highly selective expression of the Dlk gene in the individuals with MDS. Dlk could be the first candidate molecule to differentiate MDS from AML. The proposal is made that microarray analysis with the Blast Bank samples is an efficient approach to extract transcriptome data of clinical relevance for a wide range of hematologic disorders.

Characterization and expression of three forms of cDNA encoding chicken platelet-derived growth factor-A chain.

Platelet-derived growth factor (PDGF) affects cell proliferation and differentiation during mammalian embryogenesis. In a number of avian species, PDGF-alpha receptors and PDGF-A chain (PDGF-A) are present during chicken limb and lens development. However, little is understood about the chicken PDGF-A gene. The present study identified short form type 1 (S1), long form (L) and short form type 2 (S2) cDNA clones encoding chicken PDGF-A chain (PDGF-A). These clones were isolated from a chicken hepatoma cell line (LMH) mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) and cDNA library cloning. Genomic sequencing and Southern blotting revealed that these forms were generated by alternative splicing. The mRNAs of S1 and L contained two transcription start sites on one exon. At the amino acid level, the mature protein encoded by the L clone showed 90 and 85% homology with the processed coding regions of the long form of human and Xenopus PDGF-A, respectively. The putative mature peptides of all forms of chicken PDGF-A encompassed the eight cysteine residues conserved in all known forms of PDGF. We examined the expression of the three forms in chicken tissues and cells using RT-PCR. Expression of these forms varied among tissues and cells. Levels of PDGF mRNAs were very low in chicken thrombocytes, which are analogous to mammalian platelets. However, the level of PDGF-A chain mRNA expression in chicken thrombocytes peaked 4 h after exposure to type 1 collagen or thrombin, and then decreased gradually with continued incubation. These results suggest that chicken PDGF in thrombocytes plays an important role in the vascular system and in healing damaged tissue.

Autoantibody against prothrombin aberrantly alters the proenzyme to facilitate formation of a complex with its physiological inhibitor antithrombin III without thrombin conversion.

Acquired coagulation factor inhibitors include pathologic immunoglobulins that specifically bind to coagulation factors and either neutralize their procoagulant activity, accelerate their clearance from the circulation, or have proteolytic activity to degrade them into inactive polypeptides. Here, an autoantibody against prothrombin is described in a patient with serious hemorrhagic diatheses. The autoantibody exerts its influence by a previously unknown mechanism in which it inhibits coagulation through aberrant activation of the proenzyme in a catalytic manner. The antibody-bound prothrombin formed a stable stoichiometric complex with antithrombin III, consisting of intact prothrombin and an antithrombin III molecule cleaved at the (393)Arg-(394)Ser bond. The antibody dissociated from prothrombin after the complex formation with antithrombin III. Although the bound antibody elicited protease activity from prothrombin, the complex was not able to convert fibrinogen to fibrin or to activate protein C. Thus, this is the first description of an autoantibody that induces protease-like activity from a human proenzyme, permitting subsequent neutralization by its physiological inhibitor. (Blood. 2001;97:3783-3789)

Reversal of vinblastine resistance in human leukemic cells by haloperidol and dihydrohaloperidol.

Haloperidol, an antipsychotic, was investigated in cells overexpressing P-glycoprotein to detemine whether it was a clinically effective drug to reverse for reversing multidrug resistance (MDR) mediated by P-glycoprotein. A nontoxic concentration of haloperidol (1-30 microM) enhanced the cytotoxic effects of vinblastine (VBL) concentration-dependently in VBL-resistant human leukemia (K562/VBL) cells, but had no effect in the parent cells. Haloperidol also enhanced the cytotoxicities of epirubicin, doxorubicin and actinomycin D in the K562/VBL cells, but not those of idarubicin or cisplatin; this enhancement was less than that of the VBL toxicity in the VBL-resistant tumor line. Haloperidol increased the intracellular accumulation of VBL in the K562/VBL cells, and the binding of [3H]-azidopine to the cell-surface protein, P-glycoprotein, was inhibited by haloperidol in a concentration-dependent manner. Haloperidol was less potent than verapamil. Thus, haloperidol appeared to potentiate anticancer agents through the reversal of MDR by competitively inhibiting drug-binding to P-glycoprotein. In contrast, the main metabolite of haloperidol, dihydrohaloperidol, without antipsychotic activity, had less of an effect. Therefore, haloperidol might be useful in reversing drug-resistance.

Role of bursin in the development of B lymphocytes in chicken embryonic Bursa of Fabricius.

Localization and role of bursin during Bursa of Fabricius (BF) ontogeny were examined by immunohistochemical staining and by in ovo injection with anti-bursin antibody. Mouse monoclonal anti-bursin antibody HU2 was generated by immunization with synthetic bursin. It recognized reticular cells (REC), follicular associated epithelium (FAE), FAE-supporting cells, and the basal layer of interfollicular epithelium (IFE) in the mature BF. Bu-1(+) cells were first detectable in the mesenchyme area at 13 days of embryogenesis (E13) before bud formation, then lined up along the bud, and homed into the bud at around E15. IgM(+) cells were detected in the bud after E13. Bursin was first observed at the under edge of the bud. Injection of HU2 into embryonal vein at E13 suppressed the appearance of IgM(+) cells in the Bursa at E17. These results indicate that bursin exists beneath the bud and may act on the appearance of IgM(+) cells during BF ontogeny.

Low frequency of BCL10 gene mutations in B-cell non-Hodgkin's lymphoma.

The BCL10 gene was identified at the breakpoint region of the t(1;14)(p22;q32) translocation in mucosa-associated lymphoid tissue lymphoma. Initially, mutations in the BCL10 gene were reported to occur at a high frequency in various types of lymphomas and solid tumors. However, subsequent studies showed that the mutations were rarely recognized. To evaluate the frequency and spectrum of its mutations in B-cell non-Hodgkin's lymphoma (B-NHL), we screened 56 cases with B-NHL by mutation analysis of exons 2 and 3 of the gene. In addition to 2 polymorphisms, a frame-shift mutation and a missense mutation were identified in 2 cases (3.6%): 1 with diffuse large B-cell lymphoma and the other with mantle cell lymphoma. Both cases showed mutations within exon 3, resulting in a C-terminal truncation in the former and a C-terminal amino acid substitution in the latter. Reverse transcriptase-polymerase chain reaction analysis of the former case revealed that both the mutated and the wild-type alleles were transcribed with or without a sequence modification. Our results, together with recent reports, indicate that BCL10 gene mutations take place in a small population of B-NHL and are not associated with specific histological subtypes.

Morphological diagnoses of the Japan adult leukemia study group acute myeloid leukemia protocols: central review.

A morphological review system of the Japan Adult Leukemia Study Group has developed from the AML-87 through the AML-92 experience. We reviewed 1427 (90%) of 1592 cases enrolled in the AML-87, -89, or -92 protocols for morphology; 1408 (88%) were eligible. The rate of diagnostic concordance between each institute and the Committee on Morphological Diagnosis ranged from 76% to 80%. Acute myeloid leukemia (AML) subtypes were as follows: AML M0, 27 (2%); M1, 179 (13%); M2, 472 (34%); M3, 358 (25%); M4, 265 (19%); M5, 57 (4%); M6, 39 (3%); and M7, 11 (1%). The reason for the high number of patients with AML M3 is that many M3 patients were enrolled in the AML-92 protocol, which contained all-trans-retinoic acid. AML M0, M6 and M7 belonged to the poor prognostic groups. Auer bodies were found in 284 (53%) of 538 patients who survived significantly longer than those without Auer bodies in AML-87/-89. In AML-92 except for AML M3, 259 (43%) of 602 cases were Auer+ and also showed better survival rates. The survival of patients with >50% myeloperoxidase (MPO)-positive blast cells was better than those with < or =50% MPO+ blast cells in AML-87/-89. This trend was also seen in AML-92 excluding M3. AML with trilineage dysplasia (AML/TLD) is characterized as a subtype of de novo AML that shows morphological dysplasia of mature hematopoietic cells on a background of leukemic blast cells The number of patients with AML/TLD was 89 (16.5%) of 545 patients reviewed in AML-87/-89. AML-92, except for M3, showed a higher rate of patients with TLD (161 cases; 27.6%) because there were no patients with TLD in the AML M3 group. Survival rates for AML/TLD were worse than those for AML/non-TLD in both the AML-87/-89 and -92 protocols. Eighty percent of all cases (793/986) entered in AML-92 were analyzed cytogenetically. Fifty-one cases were not available for karyotyping because of a lack of mitoses or inappropriate preparations. The most frequent karyotype was normal, which accounted for 34.2%. The t(15;17), t(8;21), and inv(16) karyotypes, which are regarded as good risk factors, were 23.8%, 9.2%, and 1.6%, respectively. Abnormal chromosomes 5, 7, t(9;22), and t(6;9) were considered to be poor or intermediate risk factors As a new system of karyotyping begins in the ongoing AML protocol, useful chromosomal data will be obtained in the near future.

Mechanism of resistance to oxidative stress in doxorubicin resistant cells.

Doxorubicin (DOX) is an anthracycline drug widely used in chemotherapy for cancer patients, but it often gives rise to multidrug resistance in cancer cells. The purpose of this work was to study the effect of hydrogen peroxide in DOX-sensitive mouse P388/S leukemia cells and in the DOX-resistant cell line. Hydrogen peroxide induced a significant increase in dose- and time-response cell death in cultured P388/S cells. The degree of cell death in P388/DOX cells induced by hydrogen peroxide was less than that in P388/S cells treated with hydrogen peroxide. Parent cells exposed to 3 mM of hydrogen peroxide showed a loss of mitochondrial membrane potential correlated with cell death. Hydrogen peroxide at a concentration greater than 0.3 mM increased the intracellular Ca2+ of P388/S cells dose-dependently; however, no change following addition of hydrogen peroxide (0.3-1 mM) was observed in the resistant cells. Hydrogen peroxide (0.1 and 1 mM) treatment also induced the production of intracellular ROS in P388/S cells, while no such increase was produced by this substance in P388/DOX cells. Resistant cells also showed a significant level of glutathione (GSH) compared with the parent cells. In addition, N-acetyl-L-cysteine and reduced GSH antioxidants abolished death of P388/S cells caused by hydrogen peroxide. Therefore, it is believed that the reduced effect of oxidative stress towards the resistant cells may be related to an increase in intracellular GSH level.

[Primary renal non-Hodgkin's lymphoma presenting as immune thrombocytopenia].

A 25-year-old man was admitted to our hospital because of hematuria, anemia and thrombocytopenia. Laboratory examinations revealed an increased number of bone marrow megakaryocytes and an increased level of platelet-associated immunoglobulin G, suggesting immune thrombocytopenia. Computed tomography of the abdomen showed enlargement of the bilateral kidneys with multiple low-density areas, although neither lymphadenopathy nor hepatosplenomegaly was evident. After amelioration of the thrombocytopenia by prednisolone therapy, open renal biopsy was performed and a diagnosis of diffuse large B-cell non-Hodgkin's lymphoma was made. The patient achieved complete remission after CHOP therapy. This was thought to be a rare case of primary renal non-Hodgkin's lymphoma initially presenting as immune thrombocytopenia, which was treated successfully by chemotherapy.

Effects of N(G)-nitro-L-arginine methyl ester on endotoxin-induced leakage of lactate dehydrogenase and cytotoxicity in J774A.1 cells.

In the present study, we investigated the effects of the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine-methyl ester (L-NAME) on tissue injury or cytotoxicity caused by endotoxin challenge by assaying lactate dehydrogenase (LDH) isozymes and cell viability in J774A.1 cells. In mice treated with L-NAME (10 mg kg(-1), i.v.), the activity of LDH in serum 18 h after endotoxin (6 mg kg(-1), i.p.) injection was not significantly different from that in mice treated with endotoxin alone. Mice injected with endotoxin exhibited leakage of LDH isozymes 3 and 5, but L-NAME did not protect against endotoxin-induced acute leakage of LDH isozymes. Treatment with L-NAME (10-1000 microM) significantly inhibited NO generation by endotoxin (1 microg ml(-1))-activated J774A.1 cells. However, L-NAME (10-1000 microM) did not affect endotoxin-induced cytotoxicity in J774A.1 cells. These findings suggested that endotoxin-induced NO formation may not contribute to tissue injury or cytotoxicity caused by endotoxin.

Localization of immunoglobulin G gamma-chain mRNA-expressing cells in the oviduct of laying and diethylstilbestrol-treated immature hens.

The purpose of this study was to determine the synthetic sites of IgG in the chicken oviduct by localizing IgG gamma-chain mRNA (IgGgamma mRNA)-expressing cells and the effects of estrogen on their population. Paraffin sections of oviducal tissues from laying hens (approximately 57 weeks old) and immature hens (approximately 16 weeks old) with or without diethylstilbestrol (DES) treatment were hybridized by digoxigenin-labeled IgG riboprobes or immunostained for IgG gamma-chain (IgG). Immunoreactive IgG was present in some of the mucosal epithelial cells and the plasma cell-like cells in the stromal connective tissue in all segments of the oviduct. In contrast, IgGgamma mRNA expression was observed only in the plasma cell-like cells in the stromal connective tissues, but not in the cells of mucosal epithelium. In laying hens, the lower end of the oviduct, namely the vagina and uterovaginal junction, contained more IgGgamma mRNA-expressing cells than the other segments. Treatment of immature hens with DES for 3 or 6 days increased the population of both IgGgamma mRNA-expressing cells and IgG-containing cells in the oviducal stroma. These results indicate that IgG is locally produced by plasma cell-like cells in the stroma, but not by the cells of the mucosal epithelium, and estrogen may stimulate the infiltration of IgG-producing plasma cell-like cells into the oviducal stroma.

Modification of tumor necrosis factor-induced acute toxicity D-galactosamine challenge by polymyxin B, an anti-endotoxin.

Polymyxin B (PMB), an antibiotic with anti-endotoxin activity, was used to examine the participation of endogenously produced endotoxin in the enhancement of recombinant human tumor necrosis factor (rhTNF)-induced toxicity in D-galactosamine (GalN)-sensitized mice. GalN-sensitized mice (700 mg/kg, intraperitoneally (i.p.)) injected together with rhTNF (1x10(4) U/mouse, intravenously (i.v.)) exhibited severe symptoms, with 100% mortality at 18 h. However, mice pretreated with PMB (20 mg/kg, i.p.) showed protection against the rhTNF-induced lethality following GalN sensitization. Little or no effects were observed on alanine aminotransferase (ALT) activity or lactate dehydrogenase (LDH) isozyme leakage in serum in mice 7 h after administration of rhTNF alone. Administration of rhTNF to GalN-sensitized mice resulted in marked increases in ALT activity and LDH isozyme leakage relative to those in mice treated with rhTNF alone. In mice pretreated with PMB, the levels of ALT and LDH isozyme leakage 7 h after rhTNF/GalN injection were significant decreased as compared with those in mice treated with rhTNF/GalN. Similarly, injection of PMB markedly decreased lipid peroxide formation in the liver of the GalN-sensitized mice treated with rhTNF. The injection of a low endotoxin dose (0.1 mg/kg, i.p.) markedly increased the lethality in mice treated with rhTNF (5x10(3) U/mouse, i.v.) and GalN, and these animals showed 100% mortality at 8 h. These findings suggested that the extent of TNF-induced toxicity caused by GalN administration may be a result of synergism between TNF and gut-derived endotoxin. It is likely that endogenously produced endotoxin play a significant role in rhTNF/GalN-hypersensitized mice.

[CD19-positive acute myeloblastic leukemia developed 12 years after the onset of hypereosinophilic syndrome].

We report a rare case of hypereosinophilic syndrome (HES) that developed to acute myeloblastic leukemia (AML). The patient, a 34-year-old man, presented with eosinophilia of unknown origin (white blood cells 38,200/microliter with 74% eosinophils) and pericardial effusion, and was diagnosed as having HES with a normal karyotype. He received four cycles of combination chemotherapy including cyclophosphamide, cytosine arabinoside and vindesine, and thereafter remained in remission. After 12 years, he was referred to our hospital because of fever and malaise. On admission, CBC showed white blood cells 3,000/microliter with 70% myeloblasts and 3% eosinophils. The bone marrow was hypercellular with 95% blasts, which were negative for myeloperoxidase (MPO) staining. Immunophenotype analysis revealed that the cells were positive for CD13, CD19, CD34, HLA-DR and cytoplasmic MPO. CD19-positive AML was diagnosed. Cytogenetic analysis showed 46, XY, t(6;21)(q13;q22), add(7)(q11) in 19 of 20 metaphase spreads. Rearrangement of the AML1 gene at 21q22 and fusion of the BCR/ABL gene could not be detected by fluorescence in situ hybridization analysis. The patient received combination chemotherapy and achieved a complete remission. Chromosome aberrations involving 7q as well as 21q22 suggested that the initial chemotherapy for HES might have been implicated in the pathogenesis of acute leukemia in this case.