Identification of a novel osteoblastic gene, inducible by C-type natriuretic peptide, whose transcript might function in mineralization as a noncoding RNA.

We reported previously that C-type natriuretic peptide (CNP) promotes the differentiation and mineralization of osteoblast-like cells. However, little information is available about the mechanism of action of CNP in differentiating osteoblastic cells. In this study, using the technique known as differential display-polymerase chain reaction, we identified a novel cDNA fragment that corresponded to a transcript whose level was increased by CNP in mouse clonal preosteoblastic MC3T3-E1 cells. Northern blotting analysis revealed transcripts of 1.3 kb and 2.3 kb in MC3T3-E1 cells. Both these transcripts were also expressed at high levels in the heart and stomach. We isolated a full-length cDNA (2,135 bp) from a cDNA library derived from MC3T3-E1 cells using the original cDNA fragment. Analysis of the sequence and of products of transcription and translation in vitro indicated that the transcript of the gene did not include any extensive open reading frames. Enhanced expression, after transfection, of transcript in MC3T3-E1 cells stimulated the deposition of calcium of these cells and the formation of mineralized nodules, but did not affect the activity of alkaline phosphatase. Our results suggest that CNP promotes the expression of a novel transcript, which might stimulate the mineralization of MC3T3-E1 cells.

Structure, properties and enhanced expression of galactose-binding C-type lectins in mucous cells of gills from freshwater Japanese eels (Anguilla japonica).

Using a Japanese-eel (Anguilla japonica) gill cDNA subtraction library, two novel beta-d-galactose-binding lectins were identified that belong to group VII of the animal C-type lectin family. The eel C-type lectins, termed eCL-1 and eCL-2, are simple lectins composed of 163 amino acid residues, including a 22-residue signal peptide for secretion and a single carbohydrate-recognition domain (CRD) of approximately 130 residues typical of C-type lectins. The galactose specificity of the CRD was suggested by the presence of a QPD motif and confirmed by a competitive binding assay. Using Ruthenium Red staining, the lectins were shown to bind Ca(2+) ions. SDS/PAGE showed that native eCL-1 and eCL-2 have an SDS-resistant octameric structure (a tetramer of disulphide-linked dimers). Northern and Western blot analyses demonstrated high-level expression of eCL-1 and eCL-2 mRNAs and their protein products in gills from freshwater eels, which decreased markedly when the eels were transferred from freshwater to seawater. Immunohistochemistry showed that the eel lectins are localized in the exocrine mucous cells of the gill.

Effects of L-carnitine supplementation on renal anemia in poor responders to erythropoietin.

While renal anemia can be successfully treated by use of erythropoietin (EPO) in most hemodialysis (HD) patients, some patients have anemia that is refractory to treatment with a high dose of EPO. We examined whether L-carnitine treatment could raise hematocrit (Hct) levels in such patients. Fourteen HD patients who showed a poor response to EPO and no evident factors which inhibit a response to EPO were selected to receive oral L-carnitine (500 mg/day) in a 3-month trial. During the study, 36% of the patients showed Hct increases of more than 2%. Statistical analysis revealed significant increases of Hct (p = 0.003) and total iron-binding capacity (TIBC) (p = 0.050) and a significant decrease of ferritin (p = 0.005). In addition, we found that red blood cells (RBCs) in HD patients contained a comparable level of carnitine to normal controls, despite the presence of serum carnitine deficiency, and that RBC carnitine was not removed through HD, in contrast to serum carnitine. These results suggest that RBC carnitine may be essential for RBCs to perform their metabolic function in renal anemia and that oral L-carnitine treatment could improve anemia in poor responders to EPO.

The transcript for a novel protein with a zinc finger motif is expressed at specific stages of mouse spermatogenesis.

The cDNA for an RNA that is expressed predominantly in mouse spermatogenic cells was cloned and characterized. It was found to encode novel zinc finger protein. We first generated a cDNA fragment from mouse osteoblastic cells by the differential display method. To our surprise, Northern blot analysis revealed that the corresponding transcript was expressed at high levels in the testis rather than in osteoblastic cells. Therefore, using this fragment as a probe, we isolated the full-length cDNA (3340 bp) from a mouse testis cDNA library. Analysis of the open reading frame of the cDNA indicated that the encoded protein was a polypeptide of 942 amino acids residues that included three distinct domains, namely, a zinc finger domain of the Cys(2)-His(2) type, four basic amino acid-rich domains, and a myosin II-homology domain. In situ hybridization indicated that the transcript was present in seminiferous tubules of adult mice. Elevated expression of the transcript during testicular development in mice was restricted to spermatocytes at the pachytene stage of meiotic prophase and to round and elongated spermatids, as indicated by Northern blot analysis and RT-PCR. Our results suggest that this novel zinc finger protein might act as a transcriptional regulator during spermatogenesis and, in particular, during meiotic division.

Identification by differential display of a hypertonicity-inducible inward rectifier potassium channel highly expressed in chloride cells.

By using differential mRNA display to monitor the molecular alterations associated with adaptation of euryhaline eels to different salinities, we identified a cDNA fragment strongly induced in seawater eel gills. Cloning of a full-length cDNA and its expression in COS-7 cells indicated that the clone codes for an inward rectifier K+ channel (eKir) of 372 amino acid residues, which has two transmembrane segments and a typical pore-forming region (H5). Only low sequence similarities are present, except the H5 region, compared with other members of the inward rectifier K+ channel family (Kir). Consistent with this divergence in the amino acid sequence, a phylogenetic analysis indicated early divergence and independent evolution of eKir from other members; it is only distantly related to the Kir5.0 subfamily members. RNase protection analysis showed that eKir is highly expressed in the seawater eel gill, kidney, and posterior intestine but very weakly in freshwater eels. Immunohistochemistry of gill sections revealed dense localization of eKir in the chloride cells. Immunoelectron microscopy indicated that eKir is mainly present in the microtubular system in the chloride cell. This location and its salt-inducible nature suggest that the eKir channel cloned here is a novel member of the Kir5.0 subfamily of the Kir family and is implicated in osmoregulation.

Neuropeptide specificity and inhibition of recombinant isoforms of the endopeptidase 3.4.24.16 family: comparison with the related recombinant endopeptidase 3.4.24.15.

Endopeptidase EC 3.4.24.16 (EP24.16c, neurolysin) and thimet oligopeptidase EC 3.4.24.15 are close related members of a large family of metalloproteases. Besides their cytosolic and membrane bound form, endopeptidase EC 3.4.24.16 appears to be present in the inner membrane of the mitochondria (EP24.16m). We have overexpressed two porcine EP24.16 isoforms in E. coli and purified the recombinant proteins to homogeneity. We show here that these peptidases hydrolyse a series of neuropeptides with similar rates and at sites reminiscent of those elicited by classically purified human brain EP24.16c. All neuropeptides, except neurotensin, were similarly cleaved by recombinant endopeptidase 3.4.24.15 (EP24.15, thimet oligopeptidase), another zinc-containing metalloenzyme structurally related to EP24.16. These two EP24.16 isoforms were drastically inhibited by Pro-Ile and dithiothreitol and remained unaffected by a specific carboalkyl inhibitor (CFP-AAY-pAb) directed toward the related EP24.15. The present purification procedure of EP24.16 should allow to establish, by mutagenesis analysis, the mechanistic properties of the enzyme.

Valproate therapy does not deplete carnitine levels in otherwise healthy children.

OBJECTIVE: To determine whether children with epilepsy undergoing valproate (VPA) antiepileptic therapy and who are otherwise healthy have a lower serum level of carnitine (CAR) and a higher plasma level of plasma ammonia than do normal children. METHODOLOGY: A total of 45 children with epilepsy, 6.3 to 21.7 years of age, who were treated solely with VPA and were free of abnormal neurologic findings or nutritional problems were randomly selected (VPA-treated group). An age-matched control group (n = 45) was selected from subjects without epilepsy (control group). Total (T) and free (F) serum CAR, serum VPA concentration, and the plasma ammonia level were measured and analyzed. RESULTS: Serum VPA concentration exhibited a weak negative correlation with both T- (r = -0.34) and F-CAR (r = -0.41). The T-CAR levels were 55.7 +/- 12.4 and 57.6 +/- 12.1 mM, and the F-CAR levels 42.7 +/- 9.9 and 44.4 +/- 9.9 mM in the VPA-treated and control groups, respectively. Thus, there was no significant difference in T- or F-CAR levels between the VPA-treated and control groups. Plasma ammonia levels were the same in the two groups: 26 +/- 9.2 and 29.4 +/- 11.8 mM in the VPA-treated and control groups, respectively. There was no significant correlation between blood ammonia and either T- (r = +0.024) or F-CAR (r = -0. 026). CONCLUSION: Children on a regular diet ingest a sufficient amount of CAR that more than meets their daily CAR requirement. The level of neither T- nor F-CAR in patients with epilepsy and without severe neurologic or nutritional problems being treated with VPA appeared to be affected by VPA therapy. Because the blood CAR level depends on nutritional condition rather than on blood VPA concentration, CAR deficiency caused by VPA is not likely to occur in this population. The usefulness of supplementation of CAR for this type of patient with epilepsy, therefore, must be reevaluated carefully.

[Effects of folic acid supplementation on hyperhomocysteinemia in CAPD patients: effects on unsaturated fatty acids].

Hyperhomocysteinemia has been recognized as one of the risk factors for atherosclerosis and premature vascular disease. Patients on dialysis and end-stage renal disease also manifest high plasma concentrations of homocysteine. We performed this study to evaluate the effects of folic acid supplementation on hyperhomocysteinemia in CAPD patients. Twenty-three CAPD patients (8 males, 15 females, 49.1 +/- 14.2-years-old) dialyzed for 22.7 +/- 19.2 months participated in the study. Daily 5-mg doses of folic acid supplementation for 4 weeks significantly reduced plasma concentrations of total homocysteine (p < 0.01) and serine (p < 0.001). This observation suggests that the reduction of plasma concentrations of total homocysteine results from activation of homocysteine remethylation to methionine. On the other hand, folic acid supplementation also revealed significant correlations between changes in serum concentrations of both dihomo-gamma-linolenic acid and arachidonic acid and changes in plasma concentrations of total homocysteine (r = -0.517, p < 0.05, r = -0.451, p < 0.05, respectively). In addition, serum concentrations of both dihomo-gamma-linolenic acid and arachidonic acid in 11 CAPD patients with hyperhomocysteinemia (> or = 35 micromol/litter) were significantly lower than those of 12 CAPD patients with normohomocysteinemia (< 35 micromol/litter) (p < 0.05, p < 0.05, respectively). Serum concentrations of both dihomo-gamma-linolenic acid and arachidonic acid in CAPD patients with hyperhomocysteinemia increased significantly (p < 0.01, p < 0.05, respectively) and reached similar levels of CAPD patients with normohomocysteinemia, while plasma concentrations of total homocysteine decreased after folic acid supplementation. These findings suggest that correction of hyperhomocysteinemia in patients on dialysis produces an increase in unsaturated fatty acids.

Partial cloning of the hormone-binding domain of the cortisol receptor in tilapia, Oreochromis mossambicus, and changes in the mRNA level during embryonic development.

Cortisol is one of the central hormones in osmoregulation in fish, especially in seawater adaptation. A cDNA of 453 bp was cloned from liver mRNA of freshwater-reared tilapia (Oreochromis mossambicus), by reverse transcription polymerase chain reaction (RT-PCR) with primers designed for the hormone-binding domain of glucocorticoid receptors (GRs) in mammals and rainbow trout. The sequence of PCR product has 83% homology to the trout GR at the nucleotide level and 92% at the amino acid level. The PCR product of tilapia showed highest homology (74% at the amino acid level) to GR among human steroid hormone receptors, including mineralocorticoid receptor. The length of the receptor mRNA of tilapia was about 6.5 kb as determined by Northern blot hybridization. The mRNA concentration in the gills was relatively higher among various organs, the highest concentration being observed in blood cells. Signal intensity of the receptor message in the gills was stronger in fish reared in freshwater than in those reared in seawater or in concentrated (160%) seawater. During early development of tilapia, the highest concentration of receptor mRNA in the total RNA extracted from the whole egg was found just after fertilization, and its concentration decreased steadily toward hatching. The absolute amount of receptor mRNA per egg increased gradually before the initiation of cortisol production by the embryo. When embryos were transferred from fresh water to seawater 2 days before hatching, no difference was observed in the signal intensity of the receptor mRNA among embryos after 1, 2 (the day of hatching), 4, and 7 days.

Carnitine depletion during total parenteral nutrition despite oral L-carnitine supplementation.

Carnitine CAR) plays an important role in the beta-oxidation of fatty acids. Less attention. however, has been paid to CAR compared to other nutrients even in total parenteral nutrition (TPN). To examine CAR metabolism during TPN and the effect of simultaneous oral L-CAR supplementation on CAR levels, the blood CAR level was measured in a 3-year-old boy receiving long-term TPN because of short bowel syndrome. Both the total and acyl CAR in the serum were evaluated under various nutritional conditions including oral supplementation of L-CAR. Low CAR concentrations were observed especially when lipid containing TPN regimens were in place. Oral L-CAR supplementation was not sufficient to restore the low CAR levels in the present index patient even when the dose was increased to 120 mg/kg in accordance with the result of the L-CAR absorption test that revealed poor intestinal absorption of this nutrient. Moreover, a markedly low CAR level was measured during the onset of sepsis in the patient, and the blood CAR was depleted when lipid metabolism was activated by lipid loading or sepsis. To date, the late effects of CAR depletion on child growth have not been well examined. It is recommended that the blood CAR level be maintained at normal levels before any prominent manifestations of the deficiency have developed. The intravenous administration of CAR appears to be necessary to supply a sufficient amount of CAR for patients with severe malabsorption.

Cloning, characterization, and tissue distribution of porcine SPAI, a protein with a transglutaminase substrate domain and the WAP motif.

The primary and gene structures and tissue distribution of porcine SPAI-2, a protein that belongs to the WAP protein superfamily and has a sodium-potassium ATPase inhibitory activity, were determined by molecular cloning and Northern analysis. A full-length cDNA clone was isolated from a porcine duodenum cDNA library. The cDNA insert encoded a polypeptide of 187 amino acids, which is composed of three domains: a hydrophobic presequence of 21 amino acids, a prosegment of 105 amino acids ending with Asp126, and the mature SPAI-2 sequence of 61 amino acids beginning with Pro127. The prosegment contained 16 repeats of a hexapeptide that is highly homologous to the repetitive sequence found in the transglutaminase domain of the human elafin, an elastase-specific inhibitor that also belongs to the WAP superfamily. The repetitive sequence was demonstrated to be a good substrate of transglutaminase using a recombinant preparation produced in Escherichia coli. A porcine genomic library was then screened for the SPAI gene. Characterization and sequencing of positive clones indicated that the gene is similar to the elafin gene, having 3 exons encoding the 5'-untranslated region and signal sequence, proSPAI, and 3'-untranslated region, respectively. Northern blot analysis revealed intestine-specific expression of SPAI mRNA; the message was especially abundant in the small intestine. ProSPAI was also found in the circulation. The similarity of proSPAI to elafin in the domain structure, the acid-labile nature of the cleavage site (Asp126-Pro127), and the fact that the major form of SPAI in the plasma is proSPAI strongly suggest that proSPAI is not the precursor but rather it is the native form of SPAI. Like elafin, therefore, SPAI appears to be a new type of biologically active substance with a transglutaminase substrate domain that acts as an anchoring sequence.

Intermittent expression of BmFTZ-F1, a member of the nuclear hormone receptor superfamily during development of the silkworm Bombyx mori.

BmFTZ-F1 is a sequence-specific DNA-binding factor in the silkworm Bombyx mori sharing similar biochemical characteristics with Drosophila FTZ-F1, a member of the nuclear hormone receptor superfamily. Using DNA sequence homology with FTZ-F1 and information on tryptic peptide sequences of BmFTZ-F1, we isolated a cDNA encoding for BmFTZ-F1. Amino acid sequences in the zinc finger DNA-binding region and the putative ligand-binding domain of BmFTZ-F1 showed strong similarity to not only FTZ-F1 but also its mammalian homologues, LRH-1, ELP, and Ad4BP, suggesting the importance of each region for the function of these proteins. Northern blot analyses of RNA isolated from the middle and posterior silk glands and fat bodies showed that a 6.1-kb BmFTZ-F1 mRNA is present in all tissues so far examined. Expression of BmFTZ-F1 mRNA is intermittent, being high during larval molting and both the larval-pupal and the pupal-adult transformations. Injection of 20-hydroxyecdysone at the third day of the 5th instar larvae induced BmFTZ-F1 mRNA in the posterior silk gland after 24 hr. When 5th instar silk glands were cultured in vitro, BmFTZ-F1 mRNA was induced by a 6-hr exposure to 20-hydroxyecdysone followed by 6 hr in hormone-free medium. These results suggest that BmFTZ-F1 is inducible by decline in the ecdysteroid titer and may play an important role in the development of the silkworm as a transcription factor.

Anemia and neutropenia due to copper deficiency in enteral nutrition.

Copper deficiency has been regarded a rare complication of total parenteral nutrition. This report describes the first known case of anemia and neutropenia caused by copper deficiency in a patient receiving long-term enteral nutrition. A 34-year-old man presented with bulbar palsy and tetraplegia after an operation for cerebellar hemorrhage on June 7, 1989. Nasopharyngeal tube feeding with a defined-formula diet (Besvion) was instituted on June 19, 1989. He developed normocytic anemia and neutropenia approximately 19 months after the initiation of the tube feeding. Serum copper and ceruloplasmin levels were markedly below normal. There were anisocytosis and erythrocyte deformities in the peripheral blood smear. Bone marrow aspirates revealed a hypocellular marrow with numerous myeloid and erythroid cells with cytoplasmic vacuoles. Because the amount of copper administered was estimated to be 2.6 to 5.1 mumol/d during the tube feeding, copper deficiency was suspected to be the cause of the hematologic disorders. The patient's daily oral copper intake was increased to 34 mumol/d. His serum copper and ceruloplasmin concentrations reached a normal level after 16 days and 23 days of copper supplementation, respectively. A marked reticulocytosis occurred after 10 days of copper supplementation, and his anemia gradually improved over the next 3 months. His blood neutrophil count also returned to normal within 2 weeks.

Affected paroxysmal nocturnal hemoglobinuria T lymphocytes harbor a common defect in assembly of N-acetyl-D-glucosamine inositol phospholipid corresponding to that in class A Thy-1- murine lymphoma mutants.

Deficient expression of glycoinositol phospholipid (GPI) anchored proteins in affected paroxysmal nocturnal hemoglobinuria (PNH) cells has been traced to a defect in GPI anchor assembly. In a previous study (Schubert, J., Schmidt, R. E., and Medof, M. E. (1993) J. Biol. Chem., in press) we characterized the biosynthesis of putative Man-containing GPI anchor precursors in normal peripheral blood lymphocytes and investigated assembly of these intracellular GPI intermediates in CD48- affected and CD48+ unaffected T and natural killer cell lines of PNH patients. We found that affected T cells from five patients exhibited a uniform defect in which dolichol-phosphoryl-Man was synthesized but no GPI mannolipids were expressed. In this study, membranes of patients' affected T cells were labeled with UDP-[3H]GlcNAc to evaluate earlier steps in GPI synthesis, and intact cells were fused to Thy-1- murine lymphoma mutants harboring different defects in early GPI assembly to test for the presence of corresponding or complementary lesions. In all cases, affected cell membranes failed to assemble GlcNAc-inositol phospholipid, the initial precursor of GPI anchor structures, and the intact cells failed to complement class A mutants while complementing other classes. Affected polymorphonuclear leukocytes from three additional patients of different origin were then labeled with [3H]Man and the labeling patterns found to correspond to those obtained with the T lymphocytes. Taken together the data indicate that the genetic lesion in PNH cells resides in a DNA element which: 1) encodes a product required for the synthesis of GlcNAc-inositol phospholipid, 2) corresponds to that altered in class A Thy-1- murine lymphoma mutants, and 3) is commonly affected in different patients.

The effect of nifedipine and dipyridamole on the Doppler blood flow waveforms of umbilical and uterine arteries in hypertensive pregnant women.

In a study of 45 hypertensive pregnant women, the systolic velocity/diastolic velocity ratio and pulsatility index of the umbilical and uterine arteries showed good correlation with the maternal blood pressure, and they appeared to provide a good parameter for the fetoplacental condition. Using the pulse Doppler method, we studied the effects of the antihypertensive agent nifedipine and of dipyridamole (an agent used to treat proteinuria) on the blood flow of the umbilical and uterine arteries in 16 hypertensive pregnant women. The results proved that both drugs caused a decrease in the vascular resistance of the umbilical artery and suggested that they increased the blood flow volume of this artery and were useful in the treatment of hypertension during pregnancy.

Comparative experimental study of antiepileptics--regional specificity of antiepileptic action of carbamazepine, phenobarbital and valproate sodium.

The antiepileptic action of carbamazepine (CBZ), phenobarbital (PB), and valproate sodium (VPA) was examined in 109 adult male rabbits during steady state serum levels produced on the basis of the pharmacokinetics of each drug. The duration and discharge pattern of electrically induced focal and secondarily generalized after-discharges (ADs) in the motor, visual cortices and hippocampus were compared before and after administration of doses of each drug. A CBZ level of 3-30 micrograms/ml shortened the duration of both neocortical and hippocampal ADs. CBZ at levels above 5 micrograms/ml suppressed the spread in the secondarily generalized ADs originating from the neocortical areas, but not that originating from the hippocampus. PB levels of 10-80 micrograms/ml shortened the duration of both neocortical ADs, especially motor ADs, whereas they had little or no effect on the hippocampal ADs. VPA showed inhibitory effects on both the neocortical and hippocampal AD durations only at high levels of 400-1,100 micrograms/ml. However, the inhibitory effects were more marked on the latter than the former, and further, the effects were enhanced as time elapsed. These results suggest that the action of each drug on focal seizures is region-specific.

Dietary fat and immune function. I. Antibody responses, lymphocyte and accessory cell function in (NZB x NZW)F1 mice.

The influence of dietary fat on autoimmunity in lupus-prone (NZB x NZW)F1 mice has been demonstrated. In defining further the effects of dietary lipid on the immune system of this strain, female weanling mice were placed on four diets differing in quantity and type of fat. Their immunologic response was then studied by a variety of tests at 4 and 7 mo of age. Few differences were seen among the four groups at 4 mo of age. At 7 mo of age, however, the mice receiving diets high in saturated and unsaturated fats had a reduced mitogenic response to T cell mitogens and an enhanced response to the B cell mitogen LPS. Immunoglobulin levels and delayed hypersensitivity responses did not show any consistent differences among the diet groups. At 7 mo, however, mice receiving diets high in unsaturated fat demonstrated hyperresponsiveness to injected sheep red blood cells as measured by the hemolytic plaque technique. In addition, peritoneal leukocytes from the same diet group exhibited an increased response to bromelain-treated autologous erythrocytes which was decreased after treatment with anti-Thy-1 antiserum and complement. Phagocytosis by peritoneal macrophages was significantly decreased in the animals fed high-fat diets, particular high saturated fat. Similarly, natural killer cell activity was markedly reduced in the mice with a high intake of saturated lipid, a finding which correlated with the in vitro production of interferon. These results indicate that diets high in fat influence immune responses and thus can affect the onset and severity of autoimmune disease. A low-fat diet can reduce the development of disease by maintaining normal immune responses. The data also suggest that unsaturated fat may influence T helper cell activity and therefore antibody production, whereas saturated fats may affect cellular immune responses which are dependent on membrane contact.

The use of xanthine oxidase for a specific and sensitive fluorimetric determination of 6-mercaptopurine in serum.

A specific and highly sensitive fluorimetric method was developed for the determination of 6-mercaptopurine (6MP) in serum. The method is based on the enzymatic oxidation of 6MP with xanthine oxidase to the oxypurine, followed by oxidation with acidic chromate to the corresponding 6-sulfonate. The fluorescent product has excitation and emission maxima at 330 and 400 nm, respectively. The limit of sensitivity was approximately 22 pg/ml for 6MP in water. The sensitivity limit for 6MP in serum containing azathioprine was approximately 2.2 ng/ml. The rate constants for conversion of 6MP into the final product (6-thiouric acid) and the apparent Michaelis constant were also determined by a nonlinear regression analysis based on the integrated Michaelis-Menten equation using the ultraviolet absorbance data and the simplified complementary tristimulus colorimetry.

[Adjuvant chemotherapy after surgery in advanced gastric cancer].

Two hundred and fifty-four patients with advanced gastric cancer underwent radical surgery and ftorafur (FT) or 5-fluorouracil (5-FU) was orally administered as the adjuvant chemotherapy (ACT). Recurrence after ACT were analyzed by the quantification method II. As a result, it was found that: 1) ACT over 2 years could lower the recurrence rate, and careful follow-up is still necessary up to 4 years, and 2) after the remission induction therapy with mitomycin C and/or 5-FU and cytarabine, 12 mg/kg/day of FT for the first year and 8 mg for the second year are advisable.