Mitsuo Nishikawa

PHOSPHOFRUCTOKINASE DEFICIENCY IN SKELETAL MUSCLE. A NEW TYPE OF GLYCOGENOSIS.

Abstract A new type of glycogenosis due to deficiency of muscle phosphofructokinase is described. It occurred in three siblings from a single family, who had suffered from intolerance of exercise since childhood. This disease is characterized by the marked accumulation of hexose monophosphates and moderate glycogen deposition in skeletal muscles. Erythrocyte phosphofructokinase is only partially affected in this disease in contrast to the almost complete lack of muscle phosphofructokinase.

Effects of leptomycin B on the cell cycle of fibroblasts and fission yeast cells

An antifungal antibiotic, leptomycin B (LMB), which induced cell elongation of fission yeast, Schizosaccharomyces pombe, was found to be a unique inhibitor of the cell cycle of mammalian and fission yeast cells. Proliferation of rat 3Y1 fibroblasts was reversibly blocked by LMB in both the G1 and G2 phases and the treated cells were presumably introduced into the resting state (GO). After removal of LMB, proliferative tetraploid cells were produced from the cells which had been arrested by LMB at the G2 phase, as a result of DNA replication without passage through the M phase. LMB also inhibited the proliferation of S. pombe in both the G1 and G2 phases. These results suggest that the molecular target of LMB is one of the components necessary for progression of both G1 and G2 in the eukaryotic cell cycle.

Highly Efficient Ex Vivo Expansion of Human Hematopoietic Stem Cells Using Delta1‐Fc Chimeric Protein

Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy, and clinical transplantation. Here, we demonstrate efficient ex vivo expansion of HSCs measured by long‐term severe combined immunodeficient (SCID) repopulating cells (SRCs) from human cord blood CD133‐sorted cells using a soluble form of Delta1. After a 3‐week culture on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, Flt‐3 ligand, interleukin (IL)‐3, and IL‐6/soluble IL‐6 receptor chimeric protein (FP6) in a serum‐ and stromal cell‐free condition, we achieved approximately sixfold expansion of SRCs when evaluated by limiting dilution/transplantation assays. The maintenance of full multipotency and self‐renewal capacity during culture was confirmed by transplantation to nonobese diabetic/SCID/γcnull mice, which showed myeloid, B, T, and natural killer cells as well as CD133+CD34+ cells, and hematopoietic reconstitution in the secondary recipients. Interestingly, the CD133‐sorted cells contained approximately 4.5 times more SRCs than the CD34‐sorted cells. The present study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.

A leptomycin B resistance gene of Schizosaccharomyces pombe encodes a protein similar to the mammalian P-glycoproteins.

Screening for leptomycin B (LMB)‐resistant transformants in a gene library constructed in Schizosaccharomyces pombe with the chromosomal DNA of an LMB‐resistant mutant of S. pombe and with multicopy plasmid pDB248′ as the vector led to the isolation of a gene, named pmd1+, encoding a 1362‐amino‐acid protein. This protein showed great similarity in amino acid sequence to the mammalian P‐glycoprotein encoded by the multidrug resistance gene, mdr, and the Saccharomyces cerevisiae a‐factor transporter encoded by STE6. In addition, computer analyses predicted that the protein encoded by pmd1+ formed an intramolecular duplicated structure and each of the halves contained six transmembrane regions as well as two ATP‐binding domains, as observed with the P‐glycoproteins and the STE6 product. Consistent with this was that S. pombe cells containing the pmd1+ gene on a multicopy plasmid showed resistance not only to LMB but also to several cytotoxic agents. The pmd1 null mutants derived by gene disruption were viable and hypersensitive to these agents. All these data suggest that the pmd1+ gene encodes a protein that is a structural and functional counterpart of mammalian mdr proteins.

Studies on the Etiology of “Brittle Diabetes”: Relationship Between Diabetic Instability and Insulinogenic Reserve

To clarify possible etiologie mechanisms for brittleness of diabetic control, a relationship between the degree of diabetic instability and insulinogenic reserve or insulin-binding capacity of plasma IgG was studied in 46 insulin-treated diabetics attending the outpatient clinic. Evaluation of insulinogenic reserve was based on elevations of plasma C-peptide immunoreactivity (CPR) during the oral glucose tolerance test (OGTT). The degree of instability was quantified by the standard deviations (S.D.) of 10 values of fasting blood glucose, which were determined for the last six months while subjects were attending a hospital as outpatients. An inverse correlation was evident between residual B-cell secretory capacity and blood glucose regulatory instability (r = −0.69, p < 0.005), but there was no consistent relationship of the insulin-binding capacity to the degree of diabetic instability (r = 0.15, p > 0.05). Furthermore, pancreatic A-cell functions were investigated in seven unstable and seven stable diabetics, classified according to their S.D. values. Although immunoreactive glucagon (IRG) responses to an infusion of arginine were observed, plasma IRG did not rise in unstable diabetics during insulin-induced hypoglycemia, in which condition IRG in stable diabetics rose significantly. In contrast, plasma cortisol responses to the insulin-induced hypoglycemia were demonstrated in both diabetic groups. Plasma CPR did not decrease in unstable but did in stable diabetics fol towing the insulin injection. The comparison of unstable diabetic patients with more stable ones on the basis of clinical data, such as means of age, duration of diabetes mellitus, duration of insulin therapy, and dose of insulin, revealed no significant difference. The total lack of insulinogenic reserve results inevitably in loss of the automatic regulation of circulating insulin levels, which seems to be one of the essential factors for causing the hyperla-biUty of diabetic control. The pancreatic A-cell dysfunction is also attributable in part to the metabolic variability in brittle diabetes.

Involvement of a Novel Q-SNARE, D12, in Quality Control of the Endomembrane System

The cellular endomembrane system requires the proper kinetic balance of synthesis and degradation of its individual components, which is maintained in part by a specific membrane fusion apparatus. In this study, we describe the molecular properties of D12, which was identified from a mouse expression library. This C-terminal anchored membrane protein has sequence similarity to both a yeast soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE), Use1p/Slt1p, and a recently identified human syntaxin 18-binding protein, p31. D12 formed a tight complex with syntaxin 18 as well as Sec22b and bound to α-SNAP, indicating that D12 is a SNARE protein. Although the majority of D12 is located in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments at steady state, overexpression or knockdown of D12 had no obvious effects on membrane trafficking in the early secretory pathway. However, suppression of D12 expression caused rapid appearance of lipofuscin granules, accompanied by apoptotic cell death without the apparent activation of the unfolded protein response. The typical cause of lipofuscin formation is the impaired degradation of mitochondria by lysosomal degradative enzymes, and, consistent with this, we found that proper post-Golgi maturation of cathepsin D was impaired in D12-deficient cells. This unexpected observation was supported by evidence that D12 associates with VAMP7, a SNARE in the endosomal-lysosomal pathway. Hence, we suggest that D12 participates in the degradative function of lysosomes.

Pure red cell aplasia caused by parvovirus B19 infection in a renal transplant recipient

To the Editor: Human parvovirus B 19, which is the etiologic agent of transient aplastic crisis in hemolytic anemia, is known to cause chronic bone marrow failure in immunocompromised hosts (1-4). To our knowledge, there have been 2 previous case reports describing parvovirus B 19 infection in renal transplant recipients (5 ,6 ) . We report here another case of severe anemia caused by parvovirus B 19 infection in a renal transplant recipient. Although humoral immune response to the virus was absent for a while due to immunosuppressive therapy, treatment with a regimen of intravenous commercial immunoglobulin resulted in rapid elevation of the reticulocyte count and resolution of the anemia. A 48-year-old man with chronic renal failure secondary to diabetic nephropathy, who had been undergoing chronic hemodialysis and administration of recombinant human erythropoietin (rhEPO) for the treatment of renal anemia, was hospitalized to receive a cadaveric kidney transplant. On the 1st hospital day (d 0), he underwent cadaver donor kidney transplantation, and immunosuppressive therapy with cyclosporin (3 mg/kg/d), prednisolone (70 mg/d), mizoribine (200 mg/d), and antilymphocyte globulin (1000 mg/d) was started. The donor was a 52-year-old man who had died of subarachnoid hemorrhage. Laboratory examination of the recipient on admission showed a white blood cell count of 5300/mm3, a hemoglobin level of 10.6 g/dl, and a platelet count of 204 000/mm3. After transplantation, his anemia began to progress without improvement after rhEPO treatment. On d 18, his hemoglobin level dropped to 5.0 g/dl, reticulocyte count was 0.08 %, white blood cell count was 2500/mm3, and platelet count was 172000/mm3. Bone marrow aspiration revealed severe erythroid hypoplasia with the appearance of many giant proerythroblasts, suggesting the presence of parvovirus B 19 infection. Thereafter, rhEPO was discontinued and the patient was treated with a 7-d course of intravenous commercial immunoglobulin preparations ( 5 g/d) and red cell transfusion. Thereafter, the reticulocyte count increased and his anemia improved. Normal hematopoiesis was observed on bone marrow examination on d 38. The serial serum specimens during the course of illness were subjected to enzyme-linked immunosorbent assay (ELISA) for detection of virus antigen, and polymerase chain reaction (PCR) for detection of virus DNA. B19 antigen became positive on d 2, reached a peak on d 5 , and lasted 18 d. However, viral DNA was detected by PCR in the serum specimen taken just before transplantation, and was negative or faintly positive on d 48. Interestingly, the amount of viral DNA was transiently elevated on d 153 (Fig. 1). The presence of a replicative form of B 19 virus was demonstrated by Southern blot analysis in the bone marrow specimen (Fig. 2) (7). In the serum specimen from the donor, viral DNA was not detected. The assay of virusspecific antibody revealed that IgM and IgG response was not observed until d 153 while the appearance of IgG response was detected in the sample

Notch ligand Delta-1 differentially modulates the effects of gp130 activation on interleukin-6 receptor α-positive and -negative human hematopoietic progenitors

Interleukin (IL)‐6 plays pleiotropic roles in human hematopoiesis and immune responses by acting on not only the IL‐6 receptor‐α subunit (IL‐6Rα)+ but also IL‐6Rα− hematopoietic progenitors via soluble IL‐6R. The Notch ligand Delta‐1 has been identified as an important modulator of the differentiation and proliferation of human hematopoietic progenitors. Here, it was investigated whether these actions of IL‐6 are influenced by Delta‐1. When CD34+CD38− hematopoietic progenitors were cultured with stem cell factor, flt3 ligand, thrombopoietin and IL‐3, Delta‐1, in combination with the IL‐6R/IL‐6 fusion protein FP6, increased the generation of glycophorin A+ erythroid cells but counteracted the effects of IL‐6 and FP6 on the generation of CD14+ monocytic and CD15+ granulocytic cells. Although freshly isolated CD34+CD38− cells expressed no or only low levels of IL‐6Rα, its expression was increased in myeloid progenitors after culture but remained negative in erythroid progenitors. It was found that Delta‐1 acted in synergy with FP6 to enhance the generation of erythroid cells from the IL‐6Rα− erythroid progenitors. In contrast, Delta‐1 antagonized the effects of IL‐6 and FP6 on the development of monocytic and granulocytic cells, as well as CD14−CD1a+ dendritic cells, from the IL‐6Rα+ myeloid progenitors. These results indicate that Delta‐1 interacts differentially with gp130 activation in IL‐6Rα− erythroid and IL‐6Rα+ myeloid progenitors. The present data suggest a divergent interaction between Delta‐1 and gp130 activation in human hematopoiesis. (Cancer Sci 2007; 98: 1597–1603)

Enhanced degradation of MDM2 by a nuclear envelope component, mouse germ cell-less

A mouse homologue of Drosophila germ cell less, mouse germ cell less-1 (mgcl-1), encodes a nuclear envelope component essential for nuclear integrity. To analyze the molecular function of mGCL-1, we carried out two hybrid screening and found that mGCL-1 bound to the gene product of tumor susceptibility gene 101 (tsg101). Effects of mGCL-1 on the expression of MDM2-p53 axis were examined, since TSG101 has been shown to elevate the amount of MDM2 by inhibiting the ubiquitination. mGCL-1 significantly reduced the amount of MDM2 probably by changing the sub-cellular localization of the MDM2 and facilitating the ubiquitination of MDM2. In addition, the amount of p53 was increased and transactivation by p53 was enhanced by mGCL-1. Thus, mGCL-1 turned out to be a factor modulating MDM2-p53 axis by enhanced degradation of MDM2.

Thrombopoietin Inhibits Murine Mast Cell Differentiation

We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin or addition of this growth factor to bone marrow‐derived mast cell cultures severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target antiapoptotic gene Bcl2.