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The Wilms’ tumor gene WT1is overexpressed in leukemia and solid tumors and has an oncogenic role in leukemogenesis and tumorigenesis. However, precise regulatory mechanisms of WT1 overexpression remain undetermined. In the present study, microRNA-125a (miR-125a) was identified as a miRNA that suppressed WT1 expression via binding to the WT1-3’UTR. MiR-125a knockout mice overexpressed WT1, developed myeloproliferative disorder (MPD) characterized by expansion of myeloid cells in bone marrow (BM), spleen and peripheral blood, and displayed urogenital abnormalities. Silencing of WT1 expression in hematopoietic stem/progenitor cells of miR-125a knockout MPD mice by short-hairpin RNA inhibited myeloid colony formation in vitro. Furthermore, the incidence and severity of MPD were lower in miR-125a (−/−) mice than in miR-125a (+/−) mice, indicating the operation of compensatory mechanisms for the complete loss of miR-125a. To elucidate the compensatory mechanisms, miRNA array was performed. MiR-486 was occasionally induced in compete loss of miR-125a and inhibited WT1 expression instead of miR-125a, resulting in the cancellation of MPD occurrence. These results showed for the first time the post-transcriptional regulatory mechanisms of WT1 by both miR-125a and miR-486 and should contribute to the elucidation of mechanisms of normal hematopoiesis and kidney development.

An efficient synthesis of Ro 28-2653, a selective matrix metalloproteinase inhibitor, has been developed. The title compound was prepared in four steps and 76% overall yield from 4-biphenylacetic acid. The key, barbituric acid formation step was significantly improved by using 2-propanol and potassium tert-butoxide as the solvent and base, respectively, instead of the typical ethanol and sodium ethoxide combination.

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The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12 to 24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to the difficulty in visualizing pronuclei, the incidence of successful injection of linear DNA was higher when zygotes were injected between 20 and 24 h, as compared with an early period between 12 and 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP expression rate were not affected by the injection time. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5% of morulae that developed from the NT eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of nonintegrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.

Abstract: A 64-year-old man with neurofibromatosis type 1 (NF1) developed a primary malignant melanoma of the anus. Genetic analysis of the resected tumor confirmed loss of heterozygosity (LOH) of the NF1 gene. Anorectal malignant melanoma in NF1 is extremely rare, and genetic studies of the NF1 gene in such patients have not been reported. The allelic loss detected in the present patient supports the previously raised idea that NF1 can function as a tumor suppressor gene in the development of malignant melanoma in patients with NF1.

Information that originates from peripheral sensory organs is conveyed by axons of cephalic sensory cranial ganglia connecting the sensory organs to appropriate central targets in the brain. Thus, the establishment of correct axonal projections by sensory afferents is one of the most important issues in neural development. Previously, we examined the development of the vestibular nerve that originates from the VIIIth ganglion using a flat whole-mount preparation of the rat hindbrain and developed an in vitro, culture preparation that can recapitulate vestibular nerve development (Tashiro, Y., Endo, T., Shirasaki, R., Miyahara, M., Heizmann, C. W. and Murakami, F. (2000) J. Comp. Neurol. 417, 491-500). Both in vivo and in vitro, the ascending branch of the VIIIth ganglion projecting to the cerebellum reaches the base of the cerebellar primordium and starts to splay out towards the rhombic lip, apparently avoiding the ventral metencephalon. We now examine the nature of cues that guide vestibulocerebellar axons by applying various manipulations to the flat whole-mount in vitro preparation. Our observations suggest that local nonpermissive cues and oriented cues play a pivotal role in the guidance of vestibular axons to their central target.

Transgenic mice expressing enhanced green fluorescent protein under acrosin promoterwere used to study the role of the Golgi complex and of the cytoskeleton during early development of the acrosomic system in exactly defined stages of the seminiferous epithelial cycle during in vitro differentiation. First acrosin expression was found uniformly in the cytoplasm of stage IV pachytene spermatocytes. The steady-state level increased up to stage X pachytene spermatocytes, and in diakinetic primary spermatocytes, acrosin started to accumulate into the Golgi complex. During step 2 of spermiogenesis, several small fluorescent proacrosomic granules were seen in various parts of the Golgi complex, and they fused to a solid acrosomic system at step 3. In cultured stage I–III seminiferous tubule segments, nocodazole slowed down acrosin incorporation and increased the distance of the acrosomic system from the nucleus. Follicle stimulating hormone had an opposite effect by increasing density of the acrosomic system together with activation of the surrounding microtubule network. The observations suggest that microtubules have an important function during the early differentiation of the acrosomic system.

This study was undertaken in order to investigate the effect of zinc (Zn) administration on induction of Zn-binding metallothionein in rat liver with thioacetamide-induced cirrhosis, and the localization of metallothionein in the liver. Normal and cirrhotic rats received intraperitoneal injections with or without Zn. Subsequently, metal analyses, purification of metallothionein by gel filtration and immunohistochemical assessments of metallothionein were carried out. Although in Zn-injected cirrhotic rats, the Zn contents in the liver and plasma increased significantly depending upon the dose of Zn, the Zn contents in the liver and plasma of the cirrhotic rats were lower than those of normal rats after the same dose of Zn. The results of gel filtration also showed that the levels of Zn-metallothionein in the cirrhotic liver were reduced in comparison with those of the normal liver. By the immunohistochemical method, the presence of metallothionein in the parenchymal areas but not in the fibrotic areas of the cirrhotic liver was confirmed. These results suggested that the induced metallothionein was only located in the parenchymal areas. The metallothionein induced in the parenchymal areas was considered to play a role in protecting the parenchymal cells against the progression of fibrosis, because metallothionein has been thought to be involved in the cellular defense against oxidative stress.

To elucidate the function of Bcl10, recently cloned as an apoptosis-associated gene mutated in MALT lymphoma, we identified its binding partner TRAF2, which mediates signaling via tumor necrosis factor receptors. In mammalian cells, low levels of Bcl10 expression promoted the binding of TRAF2 and c-IAPs. Conversely, excessive expression inhibited complex formation. Overexpressed Bcl10 reduced c-Jun N-terminal kinase activation and induced nuclear factor kappaB activation downstream of TRAF2. To determine whether overexpression of Bcl10 could perturb the regulation of apoptosis in vivo, we generated Bcl10 transgenic mice. In these transgenic mice, atrophy of the thymus and spleen was observed at postnatal stages. The morphological changes in these tissues were caused by acceleration of apoptosis in T cells and B cells. The phenotype of Bcl10 transgenic mice was similar to that of TRAF2-deficient mice reported previously, indicating that excessive expression of Bcl10 might deplete the TRAF2 function. In contrast, in the other organs such as the brain, where Bcl10 was expressed at high levels, no apoptosis was detected. The altered sensitivities to overexpressed Bcl10 may have been due to differences in signal responses to Bcl10 among cell types. Thus, Bcl10 was suggested to play crucial roles in the modulation of apoptosis associated with TRAF2.

To examine what causes increased viscosity in culture broth in Streptomyces fradiae culture, various natural nitrogen sources were investigated. Extracellular protease activity increased with culture time and decomposed the natural nitrogen source into amino acids. In the case of gluten meal, after a culture time of 5 d, concentrations of glutamic acid and aspartic acid had increased to 600 and 200 mg/L, respectively, which were about 3- and 2-fold as high as levels in cultures under similar conditions using Pharmamedia. For various amino acids tested, the addition of glutamic acid or aspartic acid mixture to the culture medium raised the apparent viscosity to its highest demonstrated value, 260 mPa·s after 5 d of culture, which was 3-fold higher than without amino acids. Consumption of the decomposed glutamic acid and aspartic acid was dependent on the activities of glutamate dehydrogenase and aspartate aminotransferase, respectively. When ammonium ion was used as the nitrogen source, cell concentration reached 1.75 g/L measured as an intracellular nucleic acid concentration, which was about 2.3-fold higher than that with any other natural nitrogen source. However, apparent viscosity was only 75 mPa·s, a value one-third that of the amino acid mixture, and 70% of the pellets were bigger than 1.2 × 10⁴ μm². In the case of gluten meal or the amino acid mixture, pellets bigger than 1.2 × 10⁴ μm² comprised only 8%. This demonstrates that consumption of some amino acids affected the formation of filamentous morphology, which caused an increase in the apparent viscosity of the culture broth, and the apparent viscosity was not caused by the mycelial concentration but the mycelial morphology.

Notch family genes encode transmembrane proteins involved in cell-fate determination. Using gene targeting procedures, we disrupted the mouse Notch2 gene by replacing all but one of the ankyrin repeat sequences in the cytoplasmic domain with the E. coli (beta)-galactosidase gene. The mutant Notch2 gene encodes a 380 kDa Notch2-(beta)-gal fusion protein with (beta)-galactosidase activity. Notch2 homozygous mutant mice die prior to embryonic day 11.5, whereas heterozygotes show no apparent abnormalities and are fully viable. Analysis of Notch2 expression patterns, revealed by X-gal staining, demonstrated that the Notch2 gene is expressed in a wide variety of tissues including neuroepithelia, somites, optic vesicles, otic vesicles, and branchial arches, but not heart. Histological studies, including in situ nick end labeling procedures, showed earlier onset and higher incidence of apoptosis in homozygous mutant mice than in heterozygotes or wild type mice. Dying cells were particularly evident in neural tissues, where they were seen as early as embryonic day 9.5 in Notch2-deficient mice. Cells from Notch2 mutant mice attach and grow normally in culture, demonstrating that Notch2 deficiency does not interfere with cell proliferation and that expression of the Notch2-(beta)-gal fusion protein is not toxic per se. In contrast to Notch1-deficient mice, Notch2 mutant mice did not show disorganized somitogenesis, nor did they fail to properly regulate the expression of neurogenic genes such as Hes-5 or Mash1. In situ hybridization studies show no indication of altered Notch1 expression patterns in Notch2 mutant mice. The results indicate that Notch2 plays an essential role in postimplantation development in mice, probably in some aspect of cell specification and/or differentiation, and that the ankyrin repeats are indispensable for its function.

Extracellular factors such as FGF and EGF control various aspects of morphogenesis, patterning and cellular proliferation in both invertebrates and vertebrates. In most systems, it is primarily the distribution of these factors that controls the differential behavior of the responding cells. Here we describe the role of Sprouty in eye development. Sprouty is an extracellular protein that has been shown to antagonize FGF signaling during tracheal branching in Drosophila. It is a novel type of protein with a highly conserved cysteine-rich region. In addition to the embryonic tracheal system, sprouty is also expressed in other tissues including the developing eye imaginal disc, embryonic chordotonal organ precursors and the midline glia. In each of these tissues, EGF receptor signaling is known to participate in the control of the correct number of neurons or glia. We show that, in all three tissues, the loss of sprouty results in supernumerary neurons or glia, respectively. Furthermore, overexpression of sprouty in wing veins and ovarian follicle cells, two other tissues where EGF signaling is required for patterning, results in phenotypes that resemble the loss-of-function phenotypes of Egf receptor. These results suggest that Sprouty acts as an antagonist of EGF as well as FGF signaling pathways. These receptor tyrosine kinase-mediated pathways may share not only intracellular signaling components but also extracellular factors that modulate the strength of the signal.

A series of 3-substituted A-ring pyrrole compounds of duocarmycin were synthesized and evaluated for in vitro anticellular activity against HeLa S3 cells and in vivo antitumor activity against murine sarcoma 180 in mice. These compounds were evaluated on the peripheral blood toxicity and delayed lethal toxicity. Further, to expand our investigation of their peripheral blood toxicity, the toxicity to bone marrow cells (CFU-GM, CFU-Meg) was investigated. Among 3-substituted A-ring pyrrole compounds of duocarmycin bearing a 5‘,6‘,7‘-trimethoxy-2‘-indolecarboxyl group as segment-B (Seg-B), several analogues showed remarkably potent antitumor activity with low peripheral blood toxicity. The 3-formyl compound 12h, one of such analogues, showed stronger antitumor activity with lower toxicity to bone marrow cells compared to DU-86 (2a), an active metabolite of KW-2189 (2b). However, compound 12h caused delayed death. On the other hand, the 3-bromo compound 15f, one of the 3-substituted A-ring pyrrole derivatives bearing a 4‘-methoxycinnamoyl group as Seg-B, showed the most potent antitumor activity among the 4‘-methoxycinnamate analogues with low toxicity to bone marrow cells. Furthermore, compound 15f did not cause delayed death similarly to 2d. These results would indicate the importance of the C-3 substituents of A-ring pyrrole duocarmycin derivatives for exhibiting antitumor activity and decreasing toxicity.

The focal adhesion kinase (FAK) protein-tyrosine kinase plays important roles in cell adhesion in vertebrates. Using polymerase chain reaction-based cloning strategy, we cloned a Drosophila gene that is homologous to the vertebrate FAK family of protein-tyrosine kinases. We designated this gene Dfak56 and characterized its gene product. The overall protein structure and deduced amino acid sequence of Dfak56 show significant similarity to those of FAK and PYK2. Dfak56 has in vitro autophosphorylation activity at tyrosine residues. Expression of the Dfak56 mRNA and the protein was observed in the central nervous system and the muscle-epidermis attachment site in the embryo, where Drosophila position-specific integrins are localized. The results suggest that like FAK in vertebrates, Dfak56 functions downstream of integrins. Dfak56 was tyrosine-phosphorylated upon integrin-dependent attachment of the cell to the extracellular matrix. We conclude that the Dfak56 tyrosine kinase is involved in integrin-mediated cell adhesion signaling and thus is a functional homolog of vertebrate FAK.

Three mAb (R2/60, S7 and 1B11) were used to study the expression of murine CD43 on peripheral T cells and intestinal intraepithelial lymphocytes (IEL) from normal mice, and from mice during acute graft versus host disease (GVHD). In the spleen, essentially all T cells expressed the R2/60 and S7 antigens, whereas the 1B11 antigen was expressed on about half of the CD8⁺ cells and ~15% of CD4⁺ T cells. Interestingly, a significant proportion of resting splenic B cells expressed the 1B11 and R2/60 antigens, but not the S7 antigen. The majority of IEL expressed R2/60 antigen; however, the S7 and 1B11 markers were differentially expressed on CD8α, CD8β, TCRαβ and TCRγδ cells. Immunoprecipitation and Western blotting analyses identified characteristic 115 and 130 kDa reactive components from IEL lysates with mAb S7 and 1B11 respectively, and reactivity to both molecular entities by mAb R2/60. During acute intestinal GVHD induced by injecting CB6F₁ athymic nude mice with spleen cells from C57BL/6 enhanced-green fluorescent protein transgenic mice, 80-90% of donor T cells in the intestine epithelium expressed all CD43 isoforms; however, the level of expression of the 130 kDa CD43 antigen increased significantly and the level of the 115 kDa antigen decreased on GVHD donor T cells compared to cells at the time of transfer. Using EL4 cells, a similar shift in the expression of CD43 isoforms occurred experimentally following treatment with neuraminidase, suggesting that the type of CD43 isoform expressed on T cells is strongly influenced by conditions which affect membrane charge. The significance of these findings for intestinal immunopathology is discussed.

Three mAb (R2/60, S7 and 1B11) were used to study the expression of murine CD43 on peripheral T cells and intestinal intraepithelial lymphocytes (IEL) from normal mice, and from mice during acute graft versus host disease (GVHD). In the spleen, essentially all T cells expressed the R2/60 and S7 antigens, whereas the 1B11 antigen was expressed on about half of the CD8(+) cells and approximately 15% of CD4(+) T cells. Interestingly, a significant proportion of resting splenic B cells expressed the 1B11 and R2/60 antigens, but not the S7 antigen. The majority of IEL expressed R2/60 antigen; however, the S7 and 1B11 markers were differentially expressed on CD8alpha, CD8beta, TCRalphabeta and TCRgammadelta cells. Immunoprecipitation and Western blotting analyses identified characteristic 115 and 130 kDa reactive components from IEL lysates with mAb S7 and 1B11 respectively, and reactivity to both molecular entities by mAb R2/60. During acute intestinal GVHD induced by injecting CB6F(1) athymic nude mice with spleen cells from C57BL/6 enhanced-green fluorescent protein transgenic mice, 80-90% of donor T cells in the intestine epithelium expressed all CD43 isoforms; however, the level of expression of the 130 kDa CD43 antigen increased significantly and the level of the 115 kDa antigen decreased on GVHD donor T cells compared to cells at the time of transfer. Using EL4 cells, a similar shift in the expression of CD43 isoforms occurred experimentally following treatment with neuraminidase, suggesting that the type of CD43 isoform expressed on T cells is strongly influenced by conditions which affect membrane charge. The significance of these findings for intestinal immunopathology is discussed.

Generation of Chinese Hamster Ovary (CHO) cell lines stably expressing green fluorescent protein (GFP) was achieved using a plasmid vector that encoded the red-shifted pCX-xGFP under the control of a strong hybrid promoter composed of a CMV enhancer and a β-actin/β-globin gene promoter. Cotransfection of the promoter-less pSV2-Neo helper plasmid transmitting neomycin resistance was followed by selection with the antibiotic G418. Constitutive GFP expression could be visualized in living and fixed cells using fluorescence spectroscopy, fluorescence microscopy, and flow cytometry. DNA repair-proficient (AA8) and deficient (UV5) CHO strains were used for survival tests after UVC irradiation. Cells carrying the GFP construct (AA8-pGFP, UV5-pGFP) show the same response to UV irradiation (colony forming ability) as their nontransformed parental cell lines (AA8, UV5). Using GFP as a marker for cell viability, cells were harvested after certain postirradiation growth periods and the numbers of GFP expressing cells and fluorescence intensities were determined by FACS analysis. Generally, GFP fluorescence in irradiated cells is not seen when cell membranes are damaged (leak-out of the soluble GFP). Irradiated cells without membrane damage express GFP continuously (leading to a dose-dependent increase in GFP contents).

About 100 derivatives of human recombinant granulocyte colony- stimulating factor (rhG-CSF) were created by various gene-mutagenic techniques, and KW-2228, in which amino acids were replaced at five positions of N-terminal region of intact rhG-CSF, was picked up and evaluated for its biologic and physicochemical properties in comparison with intact rhG-CSF. KW-2228 showed two to four times higher specific activity than that of intact rhG-CSF in mouse and/or human bone marrow progenitor cells by colony-forming unit assay in soft agar, and by cell- proliferation assay in liquid culture. KW-2228 showed a potency to increase peripheral neutrophil counts when it was administered to normal C3H/He mice by single intravenous injection. Increase of total leukocyte count and neutrophils was observed, with peak level at 8 to 12 hours at low doses (0.5 to 1.0 micrograms/mouse), and the highest level was maintained for 24 to 30 hours at high doses (5 to 10 micrograms/mouse). The granulopoietic effect of KW-2228 was examined by several doses of single course (once daily for 10 days) or multiple courses (twice daily injection for 5 days followed by cessation for 9 days on one cycle, 3 cycles in total) of treatment. KW-2228 showed higher activity than that of rhG-CSF, especially at sub-optimal doses of multiple courses of treatment. Furthermore, KW-2228 was found to be more stable physicochemically and biologically than intact rhG-CSF, especially under thermal conditions at 56 degrees C and in the human plasma at 37 degrees C, suggesting a protease resistancy. Pharmacokinetic study showed that plasma concentration of KW-2228 assayed for its bioactivity maintained a higher level than that of intact rhG-CSF for 60 minutes after intravenous injection of this protein to normal mice. Those results suggest that KW-2228 might show a superior in vivo hematopoietic effect to intact rhG-CSF due to its high specific activity to progenitor cells, and also due to its improved physicochemical, biologic, and pharmacokinetic stability in host animals.

Itraconazole is a triazole antifungal agent that inhibits cell membrane serol biosynthesis. Currently, itraconazole is a potent candidate for in vivouse to revert multidrug resistance in acute leukemias, with the added benefit of its antifungal effect. As previously reported, itraconazole, as well as verapamil, reversed adriamycin-resistant K562 cells (K562/ADR) and HL60 cells (HL60/ADR) in dosages compatible to the plasma levels achieved by the therapeutic dosages used for the treatment of fungal infections. By RT-PCR analysis of mdr1, mdr3, and mrp mRNA, these adriamycin-resistant cells showed a higher expression of the transcript of these genes than those of the parent cells. By FACS analysis, both the adriamycin-resistant cells showed a higher expression of P-glycoprotein on their cell surfaces. These results suggested the involvement of itraconazole in the mdr gene and/or mrp gene product-associated resistance. Furthermore, itraconazole partially reversed etoposide resistance in both the K562 and K562/ADR cells. The present study suggests that itraconazole may reverse multidrug resistance, at least in part, via a classical MDR-associated mechanism.

In order to determine the relevance of the p53 tumor suppressor gene mutations in acute myelogenous leukemia (AML), we analyzed the p53 gene in genomic DNA of 18 unselected cases of AML by polymerase chain reaction–single-strand conformation polymorphism (PCR–SSCP) and direct sequencing. We detected three cases (16.7%) with the p53 gene mutations showing only the mutant alleles; the high incidence in cases with loss of a whole chromosome 17 (two of three) contrasted with the low incidence in cases without abnormalities of chromosome 17 (one of 15). These cases containing the mutations of the p53 gene showed a poor prognosis. Although we analyzed a rather small series of patients, these findings suggest that the p53 gene mutations might be involved in the progression and prognosis of at least some cases of AML.

Summary We investigated the breakpoints of thebcr gene in 46 Ph1-positive CML cases by Southern blot analysis ofbcr rearrangement, and in 17 CML cases by a combination of Southern blot analysis and RT-PCR. By Southern blot, the breakpoint was not identified on M-bcr in three CML cases, of which one case showed the P210-typebcr/abl transcript and two cases showed the ALL-type (P190-type)bcr/abl transcript with or without P210 transcript. Later two cases showed unique hematological profiles such as thrombocytosis, mild myelofibrosis, and relative resistance to alkylating agents. Therefore, the present study suggests that expression of the P190-type transcript may affect clinical and hematological findings in CML.

To evaluate whether only 20 cysteine residues at invariant positions are needed to bind and coordinate the metals in metallothioneins (MTs), and whether changing the positions of cysteine residues in the sequence affects the metal-binding capacity and the coordination of MTs, we examined the cadmium-binding affinities of seven mutant MTs using an Escherichia coli expression system. Five mutant MTs in which the constitutive amino acid residues other than cysteines of the -fragment were replaced with glycine residues, and the remaining two mutant MTs in which the invariant positions of the cysteine residues of the -fragment were shifted, were analysed for their ability to be expressed as cadmium-binding forms and for their biochemical properties. The results showed that extreme alteration of the constitutive amino acid residues other than cysteines in the MT -fragment leads to disruption of their cadmium-binding abilities and of their structure. However, mutant MTs containing changes of the invariant positions of the cysteine residues were expressed in a cadmium-binding form in Escherichia coli, although the invariant positions of 20 cysteine residues in the MTs are thought to be important for their metal-binding abilities. These results suggest that the position of cysteine residues and the chemical nature of the other amino acids in the amino acid sequence of MTs are less critical than expected.

The c-kit proto-oncogene encodes a receptor tyrosine kinase that is crucial to hematopoiesis, melanogenesis, and gametogeneis. Although the enzymatic activity of the c-kit product (KIT) is regulated by its ligand, both the Val559-->Gly (G559) mutation in the juxtamembrane domain and the Asp814-->Val (V814) mutation in the phosphotransferase domain lead to constitutive activation of KIT. By retroviral infection of hematopoietic progenitor cells with KIT(G559) or KIT(V814), KIT(G559) induced development of granulocyte/macrophage and mast-cell colonies in vitro without the addition of exogenous growth factors. KIT(V814) induced factor-independent growth of various types of hematopoietic progenitor cells, resulting in the development of mixed erythroid/myeloid colonies in addition to granulocyte/macrophage and mast-cell colonies. Furthermore, transplantation of KIT(G559) and KIT(V814)-infected bone marrow cells led to development of acute leukemia in one of 10 and six of 10 transplanted mice, respectively. No mice developed hematologic malignancies after transplantation of wild- type KIT-infected cells. Furthermore, transgenic mice expressing KIT(V814) developed acute leukemia or malignant lymphoma. These results demonstrate a direct role of the mutant KITs, particularly KIT(V814), in tumorigenesis of hematopoietic cells and suggest that similar mutations may contribute to the development of human hematologic malignancies.

Herbimycin A, a benzoquinoid ansamycin antibiotic, was demonstrated to decrease intracellular phosphorylation by protein tyrosine kinase (PTK). In Philadelphia chromosome (Ph1)-positive leukemias such as chronic myelogenous leukemia (CML) and Ph1-positive acute lymphoblastic leukemia (ALL), both of which express bcr-abl fused gene products (P210bcr-abl or P190bcr-abl protein kinase) with augmented tyrosine kinase activities, herbimycin A markedly inhibited the in vitro growth of the Ph1-positive ALL cells and the leukemic cells derived from CML blast crisis. However, the same dose of herbimycin A did not inhibit in vitro growth of a broad spectrum of Ph1-negative human leukemia cells, and several other protein kinase antagonists also displayed no preferential inhibition. Furthermore, we demonstrated that herbimycin A has an antagonizing effect on the growth of transformed cells by a transfection of retroviral amphotrophic vector expressing P210bcr/abl into a murine interleukin (IL)-3-dependent myeloid FDC-P2 cell line. This inhibition was abrogated by the addition of sulfhydryl compounds, similar to the reaction previously described for Rous sarcoma virus transformation. The inhibitory effect of herbimycin A on the growth of Ph1-positive cells was associated with decreased bcr/abl tyrosine kinase activity, but no decrease of bcr-abl mRNA and protein, suggesting that the inactivation of bcr-abl tyrosine kinase activity by herbimycin A may be induced by its binding to the bcr-abl protein portion that is rich with sulfhydryl groups. The present study indicates that herbimycin A is a beneficial agent for the investigation of the role of the bcr-abl gene in Ph1-positive leukemias and further suggests that the development of agents inhibiting the bcr-abl gene product may offer a new therapeutic potential for Ph1-positive leukemias.

The serum of mice hyperimmune to L1210 leukemia was cytotoxic to L1210 cells and, to a much lesser extent, to P388 cells in the presence of complement. However, it did not suppress in vitro growth of L1210 cells, nor did it endow a recipient mouse with immunity to inoculated L1210 cells. This indicates that the serum did not play a significant role, if any, in immune protection of hyperimmune mice.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by complement-mediated hemolysis. Abnormal hematopoietic cells from patients with PNH are deficient in glycosylphosphatidylinositol (GPI)-anchored proteins and clonally dominate various hematopoietic lineages in the bone marrow and the peripheral blood. Analysis of many patients with PNH has showed that somatic mutation in the X-linked gene PIG-A is responsible for the GPI- anchor deficiency in PNH. The PIG-A mutation must also be relevant to the clonal dominance of GPI-anchor deficient (GPI-) blood cells because two or more PIG-A mutant clones become dominant in many patients. However, whether the PIG-A mutation alone is sufficient for clonal dominance is not known. To address this question, we generated chimeric mice using Pig-a (the murine homologue of PIG-A) disrupted embryonic stem (ES) cells, in which the animals are chimeric with respect to the surface expression of GPI-anchored proteins. The chimerism of hematopoietic and nonhematopoietic tissues in such mice was always low, suggesting that the higher contribution of Pig-a disrupted GPI- cells had a lethal effect on the chimera. GPI- cells appeared in the peripheral blood of some of the chimeric mice. However, the percentage of GPI- erythrocytes did not increase for 10 months after birth, implying that the Pig-a mutation alone does not immediately cause the clonal dominance of GPI- blood cells; another pathologic or physiologic change(s) in the hematopoietic environments or in the clone itself may be necessary.

A new Philadelphia chromosome (Ph1)-positive cell line, designated TOM- 1, was derived from bone marrow cells of a patient with Ph1-positive acute lymphocytic leukemia (ALL). The TOM-1 cells were positive for Ia and B1 antigens and terminal deoxynucleotidyl transferase (TdT) but negative for common ALL antigen. Although neither surface Ig nor cytoplasmic Ig was detected, the TOM-1 cells contained rearranged immunoglobulin-H chain genes but retained germ-line kappa chain and germ-line T cell receptor beta-chain genes. These results indicate that the TOM-1 cells reside as the progenitor of pre-B cells. We have investigated the chromosome 22 breakpoint and c-abl gene expression in the TOM-1 cells. We found that the breakpoint on chromosome 22 was within the breakpoint cluster region (bcr) in the TOM-1 cells. We also found the breakpoints within or near bcr in four of six Ph1-positive ALL cases, similar to the findings in Ph1-positive CML cases. Amplification of the c-abl gene was not detected in the TOM-1 cells. The leukemic cells isolated from a patient with CML in myeloid crisis contained a novel 8-kilobase (kb) abl-related messenger RNA (mRNA), but the TOM-1 cells contained c-abl transcripts of only normal sizes, despite the fact that they showed the bcr gene rearrangement.

Distributions of norepinephrine (NE), dopamine (DA) and its metabolites in the cerebral cortex in Long-Evans Cinnamon (LEC) rats, an animal model of Wilson's disease, aged 4, 10 (in the early stage of the disease) and 20 weeks (in the advanced stage of the disease) were determined to elucidate the effect of the abnormal copper (Cu) metabolism observed in the LEC rat on the brain catecholamine metabolism. NE depression as well as excessive accumulations of DA and facilitation of the DA catabolic pathway to its acidic metabolites were observed in the cerebral cortex of the LEC rat at 4 and 10 weeks of age. Furthermore, immunohistochemical analysis of the cuproenzyme dopamine-@b-hydroxylase (DBH) showed lower antigenicity of DBH in the cortical neurons in the cerebral cortex of LEC rats aged 4 weeks than in control rats. These results suggests that neurochemical disturbances involved in an abnormal catecholamine metabolism may occur in the cerebral cortex of the LEC rat before excessive Cu accumulation.

We report on the histochemistry of copper-metallothionein (Cu-MT) in the kidneys of Long Evans Cinnamon (LEC) rats. We used the visualization principle of histochemistry based on the autofluorescence emission from the fluorophore of Cu(+)-thiolate clusters in proteins. Intense autofluorescence signals were observed with a ring at the outer stripe of the outer medulla. Orange fluorescence signals were observed in the nuclei and cytoplasm of proximal straight tubular (PST) cells of segment 3 (S3) at the outer stripe of the outer medulla, and yellow-orange signals were detected in lysosome-like organelles in the proximal convoluted tubule (PCT) cells of segments 1 and 2 (S1 and S2) adjacent to the glomeruli in the cortex. These fluorescent materials were identified as Cu-MT because both signals were quenched by withdrawing Cu+ or by blocking cysteine residues, the distributions of cysteine residues and immunoreactive MT showed identical patterns to the localization of the fluorescence signals, and the fluorescent proteins containing Cu were eluted at the same Kd value of purified Cu-MT by gel filtration chromatography. However, a high level of MT mRNA was detected only in the outer stripe of the outer medulla where the orange fluorescence signals were detected, but not in the cortex. This difference in localization between the protein and the mRNA suggested that synthesis of renal MT occurs do novo in the outer stripe of the outer medulla. The yellow-orange fluorescent Cu-MT located in the lysosomal organelles at S1 and S2 of the PCT cells in the cortex could be Cu-MT of nonrenal origin, i.e., Cu-MT transported from other organs.