Kenji Niiya

Hypoproduction of erythropoietin contributes to anemia in chronic cadmium intoxication: clinical study on Itai-itai disease in Japan

Itai-itai disease is a condition caused by longterm exposure of the inhabitants of Toyama prefecture, Japan, to cadmium intoxication. The characteristic clinical features of this disease include renal tubular dysfunction, osteomalacia, and anemia. In order to clarify the pathogenesis of the anemia, the red blood cell count, hemoglobin concentration, hematocrit, serum iron level, total ironbinding capacity, serum ferritin level, serum erythropoietin level, creatinine clearance, fractional excretion of β2-microglobulin, and bone marrow morphology were determined in ten patients with Itai-itai disease. Low serum iron or ferritin levels were not observed, and bone marrow aspiration did not reveal any specific hematological disorders. A close relationship was observed between the decrease in the hemoglobin level and the progression of renal dysfunction. Low serum erythropoietin levels were detected despite the presence of severe anemia. These results suggest an important role of renal damage in the anemia which develops in Itai-itai disease.

Diagnostic and prognostic significance of chromosome abnormalities in marrow and mitogen response of lymphocytes of acute nonlymphocytic leukemia

Chromosomes of bone marrow from 28 patients with acute nonlymphocytic leukemia (ANLL) (26 with AML, 2 with AMMoL), 19 of whom had chromosome abnormalities, were studied; 11 cases exhibited previously unreported karyotypic abnormalities. The marrows of two cases had 8-21 translocations associated with an iso-X chromosome in the female patient and with 9q13- and a missing Y in the male patient. Usually, AML patients with a 8-21 translocation have been considered to have a good prognosis; however, our cases had rather short survival times. Therefore, the prognosis of AML with an 8-21 translocation but associated with other abnormalities is still not clear. Centromere spreading (CS), which was originally reported in marrow cells of megaloblastic anemia (B12 and folic acid deficiency), was detected in leukemic cells, disappeared during remission, and reappeared on relapse. These findings suggest that CS may be a new type of abnormality in AML. In two patients with atypical hypoplastic anemia and hemolytic anemia, chromosome abnormalities were detected at the anemic stage. One case with CS was associated with atypical hypoplastic anemia and developed AML after 1 year; the other with 48,XY,+i(1q),+3,/12 and -14 had hemolytic anemia and developed AMMoL 3 weeks later. Interestingly, identical clones were detected both before and after the clinical diagnosis of leukemia. These cases strongly support the concept that some chromosome abnormalities precede the clinical manifestations of leukemia. The present study also revealed that lymphocytes in ANLL respond poorly to PHA in the presence of high numbers of blasts but do respond well to mitogens during remission. Therefore, the response of lymphocytes to PHA may serve as one criterion for determining remission.

Heparin cofactor II-dependent antithrombin activity of calcium spirulan

Calcium spirulan (Ca-SP), a novel sulfated polysaccharide isolated from the blue-green alga Spirulina platensis, enhanced the antithrombin activity of heparin cofactor II (HC II) more than 10000-fold. The apparent second-order rate constant of thrombin inhibition by HC II was calculated to be 4.2 x 10(4) M-1 min-1 in the absence of Ca-SP, and it increased in the presence of 50 micrograms/ml Ca-SP to 4.5 x 10(8) M-1 min-1. Ca-SP effectively induced the formation of a thrombin-HC II complex in plasma. In the presence of Ca-SP, both the recombinant HC II variants Lys173-->Leu and Arg 189-->His, which are defective in interactions with heparin and dermatan sulfate, respectively, inhibited thrombin in a manner similar to native rHC II. This result indicates that the binding site of HC II for Ca-SP is different from the heparin- or dermatan sulfate-binding site. When we removed the calcium from the Ca-SP, the compound did not exert any antithrombin activity. Furthermore, Na-SP, which was prepared by replacement of the calcium in Ca-SP with sodium, accelerated the antithrombin activity of HC II as Ca-SP did. We therefore suggest that the molecular conformation maintained by Ca or Na is indispensable to the antithrombin activity of Ca-SP. The HC II-dependent antithrombin activity of Ca-SP was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, but not by treatment with chondroitinase ABC and chondroitinase AC II, suggesting that a heparin- or dermatan sulfate-like structure is not responsible for the activation of HC II by Ca-SP. Ca-SP is therefore thought to be a unique sulfated polysaccharide which shows a strong antithrombin effect in an exclusively HC II-dependent manner.

Calcium spirulan as an inducer of tissue-type plasminogen activator in human fetal lung fibroblasts

Calcium spirulan (Ca-SP), a novel sulfated polysaccharide isolated from the blue-green alga Spirulina platensis, has been found to have antiviral and heparin cofactor II-dependent antithrombin activities. We have obtained evidence that Ca-SP is a potent inducer of tissue-type plasminogen activator (t-PA) production. The addition of Ca-SP to a culture of IMR-90 human fetal lung fibroblasts increased t-PA concentrations in the conditioned medium, in a dose- and time-dependent manner, but in the cell lysate, t-PA concentrations were unchanged, suggesting that t-PA induced by Ca-SP is easily secreted into the conditioned medium. The amount of newly synthesized t-PA in IMR-90 cells, as measured by labeling with [35S]methionine and subsequent immunoprecipitation of t-PA from conditioned medium, was significantly increased by Ca-SP-stimulation. However, Ca-SP did not increase the t-PA mRNA levels. As previously reported, thrombin stimulated t-PA gene transcription in IMR-90 cells, and the simultaneous treatment with Ca-SP and thrombin caused further enhancement of t-PA production, in a synergistic manner. It would thus appear that Ca-SP increases t-PA production through post-transcriptional processes. IMR-90 cells also produce plasminogen activator inhibitor type-1 (PAI-1), but Ca-SP showed little effect on the PAI-1 production. H-SP, which was obtained by removing the calcium from Ca-SP, had no effect on the t-PA production. Na-SP, which was prepared by replacement of the calcium with sodium, stimulated the t-PA production similarly to Ca-SP. Thus, Ca-SP specifically induces t-PA production, and the molecular conformation of Ca-SP maintained by Ca or Na may be essential for the stimulation of t-PA synthesis.

Induction of c-fos protooncogene transcription and apoptosis by Δ12-prostaglandin J2 in human Pl-21 myeloid leukemia and RC-K8 pre-B lymphoma cells

Abstract Δ 12 -prostaglandin J 2 (PGJ 2 ) is a dehydration product of PGD 2 and thought to be the most potent antitumor agent among prostaglandin compounds. We examine the cytotoxic effects of PGJ 2 on the cell growth of leukemia/lymphoma cells. PGJ 2 inhibited the growth of both human PL-21 myeloid leukemia and RC-K8 pre-B lymphoma cells in culture in a dose-dependent manner with fragmentation of nucleus and formation of apoptotic body. Agarose gel electrophoresis revealed DNA ladder formation in the cells treated with PGD 2 . Furthermore, PGJ 2 induced a rapid and transient expression of apoptosis-related protooncogene, c-fos , in both cells. The gene transcriptional rate was remarkably increased approximately 3.3-fold in PGJ 2 treated cells, but the stability of c-fos mRNA was not significantly changed. Inhibition of de novo protein synthesis with cycloheximide increased c-fos mRNA stability but not abrogated PGJ 2 -induced c-fos transcription. These data suggest that PGJ 2 can induce apoptosis of human leukemia/lymphoma cells and the rapid activation of c-fos protooncogene transcription in which de novo protein synthesis is not required.

Trisomy 4 in a case of acute nonlymphocytic leukemia (M2)

Trisomy 4 in a Japanese patient with type M2 acute nonlymphocytic leukemia is reported. Karyotypic analysis was unsuccessful at diagnosis and complete remission lasted 4 years. The cytogenetic change was detected in the first and second relapses as the only chromosome abnormality. Unusual nuclear irregularities were found in many leukemic cells.

Factor VII Morioka (FVII L-26P): a homozygous missense mutation in the signal sequence identified in a patient with factor VII deficiency.

We investigated the molecular basis of factor VII deficiency in a Japanese patient and identified a novel missense mutation in the signal sequence of the gene. Factor VII activity and antigen level measured in the patient were 10.7% and 11% of normal, respectively. All exons except 1B and the 5′‐flanking region containing promoter region were amplified by polymerase chain reaction (PCR) from genomic DNA. Sequencing analysis of the PCR fragments revealed that the patient was a homozygote for a T to C substitution at nucleotide position 38. This mutation predicts an amino acid replacement of leucine to proline at codon −26 in the hydrophobic core of the signal peptide, which probably affects translocation of the protein into endoplasmic reticulum and subsequently causes reduction in plasma factor VII level.

Selective activation of heparin cofactor II by a sulfated polysaccharide isolated from the leaves of Artemisia princeps

While checking anticoagulant activities in crude fractions from Wakan-Yakus (traditional herbal drugs), we detected antithrombin activity in the polysaccharide fraction of the leaves of Artemisia princeps Pamp. A sulfated polysaccharide purified from the crude fractions by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B potentiated the heparin cofactor II (HC II)-dependent antithrombin activity but not the antithrombin activity of antithrombin III (AT III). The polysaccharide enhanced the HC II-thrombin reaction more than 6000-fold. The apparent second-order rate constant of thrombin inhibition by HC II increased from 3.8 × 104 (in the absence of the polysaccharide) to 23 × 108 M-1 min-1 in the presence of 25–125 μ/ml of the polysaccharide. In human plasma, the poly saccharide accelerated the formation of thrombin—HC II complex. The stimulating effect on HC II-dependent antithrombin activity was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, while chondroitinase ABC or chondroitinase AC II had little or no effect. These results suggest that the polysaccharide is a glycosaminoglycan-like material with properties that are quite distinct from heparin or dermatan sulfate.

Down-regulation of urokinase secretion from a human lymphoma cell line RC-K8 by dexamethasone without inducing plasminogen activator inhibitors

The plasminogen activator (PA) activity in various cell lines is suppressed by glucocorticoids. These phenomena are attributed to either a suppression of PA biosynthesis, to an increase of PA inhibitor or to a combination of both. The regulation of urokinase (UK) production in a human pre-B cell lymphoma line, RC-K8, by dexamethasone (Dex) and phorbol myristate acetate (PMA) was investigated. RC-K8 is a cell line which is consistently producing a high molecular weight UK in the conditioned medium (Kubonishi, I., et al: Jpn. J. Cancer Res. 76, 12-15, 1985). The cells were cultured in RPMI-1640 with Dex or PMA for 1-4 days. UK activity was measured using a chromogenic substrate S-2444 and the antigen by an ELISA kit. PAI-1 and PAI-2 antigens were also measured by ELISA kits and the complex between PA and PAI was examined by SDS-PAGE fibrin-zymography. The UK secretion in RC-K8 cells was inhibited by cycloheximide and actinomycin D. PMA at 0.16-1.6 uM up-regulated the UK activity approximately two-fold, parallel with the antigen, whereas Dex at 1-10 uM decreased the UK expression approximately half. These were verified by SDS-PAGE fibrin-zymography. Neither PAI-1, PAI-2 nor PA/PAI complex was detected in the conditioned medium and in the cell lysate. These data suggest that PMA up-regulates the UK secretion without inducing PAIs and the down-regulation of the UK secretion by Dex results from the inhibition of the expression of UK itself but not from the induction of PAIs.

Ellagic acid/phospholipid-induced coagulation and dextran sulfate-induced fibrinolytic activities in β2-glycoprotein I-depleted plasma

Beta 2-glycoprotein I (beta 2-GPI) binds negatively charged substances and inhibits intrinsic blood coagulation in the presence of ellagic acid-phospholipid suspension. Beta 2-GPI is thought to be an important protein in the reaction between negatively charged phospholipids and anti-phospholipid antibodies which appear in patients with lupus anticoagulant/antiphospholipid antibody syndrome. We prepared a monoclonal antibody against beta 2-GPI purified from human plasma and obtained beta 2-GPI-depleted plasma using a monoclonal antibody-coupled column. Either partial thromboplastin time or the activation of prekallikrein induced by diluted ellagic acid-phospholipid suspension in beta 2-GPI-depleted plasma was not different from that in control plasma. Beta 2-GPI inhibited the intrinsic blood coagulation only when added to control or beta 2-GPI-depleted plasma in excess (more than physiological concentrations). The intrinsic fibrinolysis in beta 2-GPI-depleted plasma induced by dextran sulfate was not impaired and, again, beta 2-GPI inhibited the intrinsic fibrinolysis only when added to control or beta 2-GPI-depleted plasma in excess. These results indicate that both in vitro Actin-induced intrinsic coagulation and dextran sulfate-induced fibrinolytic activities are significantly inhibited by more than physiological concentrations of beta 2-GPI.