Molecular cloning and characterization of UDP-GlcNAc:lactosylceramide beta 1,3-N-acetylglucosaminyltransferase (beta 3Gn-T5), an essential enzyme for the expression of HNK-1 and Lewis X epitopes on glycolipids.

A new member of the UDP-N-acetylglucosamine:beta-galactose beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T) family having the beta3Gn-T motifs was cloned from rat and human cDNA libraries and named beta3Gn-T5 based on its position in a phylogenetic tree. We concluded that beta3Gn-T5 is the most feasible candidate for lactotriaosylceramide (Lc(3)Cer) synthase, an important enzyme which plays a key role in the synthesis of lacto- or neolacto-series carbohydrate chains on glycolipids. beta3Gn-T5 exhibited strong activity to transfer GlcNAc to glycolipid substrates, such as lactosylceramide (LacCer) and neolactotetraosylceramide (nLc(4)Cer; paragloboside), resulting in the synthesis of Lc(3)Cer and neolactopentaosylceramide (nLc(5)Cer), respectively. A marked decrease in LacCer and increase in nLc(4)Cer was detected in Namalwa cells stably expressing beta3Gn-T5. This indicated that beta3Gn-T5 exerted activity to synthesize Lc(3)Cer and decrease LacCer, followed by conversion to nLc(4)Cer via endogenous galactosylation. The following four findings further supported that beta3Gn-T5 is Lc(3)Cer synthase. 1) The beta3Gn-T5 transcript levels in various cells were consistent with the activity levels of Lc(3)Cer synthase in those cells. 2) The beta3Gn-T5 transcript was presented in various tissues and cultured cells. 3) The beta3Gn-T5 expression was up-regulated by stimulation with retinoic acid and down-regulated with 12-O-tetradecanoylphorbol-13-acetate in HL-60 cells. 4) The changes in beta3Gn-T5 transcript levels during the rat brain development were determined. Points 2, 3, and 4 were consistent with the Lc(3)Cer synthase activity reported previously.

Stem cell growth factor: in situ hybridization analysis on the gene expression, molecular characterization and in vitro proliferative activity of a recombinant preparation on primitive hematopoietic progenitor cells.

INTRODUCTION: In situ hybridization of whole mouse fetuses and their tibias with a stem cell growth factor (SCGF) antisense probe demonstrated specific expression of SCGF mRNA around skeletal tissues, particularly in bone marrow cells, proliferating chondrocytes, the perichondrium and periosteum, but little expression in resting or hypertrophic chondrocytes. METHODS: Recombinant human (rh) SCGF-alpha was purified from a conditioned medium of SCGF-alpha gene-transfected CHO cells. The molecular mass of rhSCGF-alpha, 45 kDa, was shifted down to 40 kDa by digestion with endo-O-glycosidase and sialidase, suggesting O-glycosylation of rhSCGF-alpha with sialic acids. RESULTS: For human bone marrow CD34+Lin- cells, rhSCGF-alpha alone did not stimulate colony-formation, but small cluster-formation (10.3 +/- 2.5/1 x 10(3) CD34+Lin- cells). It promoted growth of erythroid and granulocyte/macrophage (GM) colonies in the primary culture with erythropoietin and GM colony-stimulating factor (CSF) or G-CSF, respectively, and further supported GM progenitor cells in a short-term liquid culture. In contrast, rhSCGF-alpha suppressed stem cell factor (SCF)-stimulated erythroid bursts, indicating some competitive interaction between SCGF and SCF. rhSCGF-alpha was synergistic with interleukin-3 and the flt3 ligand to enhance GM colony-growth, but not synergistic with those inducing ex vivo expansion of GM progenitor cells. CONCLUSION: SCGF is selectively produced by osseous and hematopoietic stromal cells, and can mediate their proliferative activity on primitive hematopoietic progenitor cells.

Isolation and characterization of a cDNA for human mouse, and rat full-length stem cell growth factor, a new member of C-type lectin superfamily.

cDNA encoding stem cell growth factor (SCGF; 245 aa), a novel human growth factor for primitive hematopoietic progenitor cells, has been previously reported (Hiraoka, A., Sugimura, A., Seki, T., Nagasawa, T., Ohta, N., Shimonishi, M., Hagiya, M. and Shimizu, S. Proc. Natl. Acad. Sci. USA 94, 7577-7582, 1997). Here we report the cloning and characterization of a full-length SCGF cDNA. This protein consists of 323, 328 and 328 aa in the human, murine and rat forms, the latter two of which share 85.1% and 83.3% aa identity, and 90.4% and 90.4% aa similarity to the human protein, respectively. Because the newly identified human clone encodes the protein longer by 78 aa than that previously identified, we term the longer clone as hSCGF-alpha and the shorter one as hSCGF-beta. The computer-assisted homology search reveals that SCGF is a new member of the C-type lectin superfamily, and that SCGF shows the greatest homology to tetranectin among the members of the family (27.2-33.7% aa identity and 46.0-53.6% aa similarity). SCGF transcripts are detected in spleen, thymus, appendix, bone marrow and fetal liver. Fluorescent in situ hybridization mapping indicates that the SCGF gene is located on chromosome 19 at position q13.3 for human form and on chromosome 7 at position B3-B5 for murine form, which are close to flk-2/flt3 ligand and interleukin-11 genes of both human and murine species.

Generation of a monoclonal antibody that induces apoptosis of hematopoietic cells.

Here, we developed anti-murine myeloid cell (NFS-60 cell) monoclonal antibodies in order to characterize the further mechanism of cell-to-cell interaction between the stromal cells and the hematopoietic progenitor cells. The established antibody, designated mAb UNF, inhibited proliferation of NFS-60 cells and induced apoptosis. Incubation of NFS-60 cells with mAb UNF initiated the cell aggregation within 3 hours. This phenomena did not require newly synthesized proteins, since the pretreatment of 1 mM cycloheximide failed to prevent the agglutination. Fifty-eight percent of mouse hematopoietic stem cells, Lin-, c-Kit+, Sca-1+ bone marrow cells, were shown to express UNF antigen. The glycolipid prepared from NFS-60 cells was specifically immunostained by mAb UNF.

First performance assessment of the small-angle X-ray scattering beamline at ELETTRA.

The double-focusing high-flux wiggler beamline dedicated to small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) at ELETTRA has gone into user operation recently. It has been designed specifically for time-resolved studies of non-crystalline and fibrous materials in the submillisecond time scale, and has been optimized for small-angle scattering measurements. An overview of the beamline status and of some representative results, highlighting the performance of the SAXS beamline, are given.

[Clinical effects of postoperative immunochemotherapy with a combination of 5-FU, CDDP and lentinan for stage IVb gastric carcinoma and long-term pharmacokinetic studies on CDDP and 5-FU].

Eleven patients with postoperative-stage IVb gastric cancer were treated by combined administration of CDDP, 5-FU and Lentinan. CDDP was given intravenously at 20 mg/body on days one to five. At the same time, 5-FU was continuously administered 250 or 500 mg/body/day via the central venous catheter for more than two weeks, while Lentinan was given at the dose of 2 mg per week. Two courses were repeated every 3 weeks. The mean response duration and survival period for the eleven patients were 13 and 21 months, respectively, and one- and two year survival rates were 82% and 27%. This combination therapy was thus considered to be very effective. Next, the pharmacokinetics of long-term CDDP and 5-FU was studied. Our method could maintain the optimum serum concentration of total CDDP. Furthermore, the continuous infusion of 5-FU at a daily dose of 250 mg/day could sufficiently maintain the effective plasma concentration, although its peak fluctuated over time. Thus, in the plasma level of 5-FU in patients in poor condition requires periodic measurement.

[Postoperative immunochemotherapy for gastric carcinoma with peritoneal dissemination--the effects with the combination of CDDP, 5-FU and lentinan].

Three cases of postoperative gastric cancer with peritoneal dissemination were treated by combined administration of CDDP, 5-FU and Lentinan. CDDP was given intravenously at 20 mg/body on days one to five. At the same time, 5-FU was infused continuously 250-500 mg/body per day for more than two weeks, and Lentinan was given at a dose of 2 mg per week. Two courses were repeated every 3 weeks. This combination treatment was very effective because the median survival rate for the three patients was 13 months with no sign of recurrence, and the quality of life has been good.

[Changes in coagulation and fibrinolytic factors observed during heparin-urokinase-pulse combined therapy for nephritis resistant to conventional treatment in children].

Coagulation and fibrinolytic factors in the blood were measured during heparin-urokinase (UK)-pulse combined therapy in order to investigate the background for the availability of the therapy. Five patients with nephritis resistant to conventional treatment were treated with this combined therapy (heparin: 350-450 U/kg day, continuously i.v. during the therapy; UK: 5000 IU/kg/2 hrs, i.v., two times a day, for 3 days = 1 Kur; methylprednisolone 20 mg/kg/2hrs, d.i.v., for 3 days = 1 Kur; 3 Kurs of UK and 3 Kurs of pulse were alternately administered). 1) Blood levels of alpha 2-plasmin inhibitor (alpha 2-PI) antigen were decreased and those of alpha 2-plasmin inhibitor.plasmin complex (alpha 2-PI. PmC) were elevated during 3 Kurs of UK administration. Accordingly, activation of the fibrinolytic system was confirmed during the combined therapy, suggesting that both alpha 2-PI and alpha 2-PI.PmC were relevant in monitoring the fibrinolytic state in blood. 2) Both tissue-plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) levels were sustained continuously in the elevated levels in the blood during both UK administration and pulse therapy. This movement of t-PA and PAI-1 was independent of that of the other fibrinolytic factors, such as alpha 2-PI,alpha 2-PI.PmC and plasminogen. 3) Inflammatory reactants such as fibrinogen, alpha 2-PI,alpha 2-macroglobulin and alpha 1-antitrypsin decreased more significantly during this heparin-urokinase-pulse combined therapy than during our previous combined therapy consisting of only heparin and urokinase. Therefore, we conclude that the anti-inflammatory effect was reinforced by adding the pulse therapy and that the combined therapy had some effect on the release of t-PA from vascular endothelial cells.

[Analysis of the electrical charge of albumin in idiopathic nephrotic syndrome by isoelectric focusing].

The state of the electrical charge of serum and urinary albumin was investigated in both the nephrotic and remission stage of idiopathic nephrotic syndrome (INS), using isoelectric focusing (IEF). 1) Both a b2 band (a main albumin band appearing at the site of isoelectric point: pI 4.7), and a b3 band (a more anionic albumin band than the b2 band) were detected commonly in all samples of urine and serum of INS patients in nephrotic and remission stages and of healthy volunteer controls even if the applied albumin in urine and serum amounted to 20 micrograms or 100 micrograms. 2) When applied albumin amounted to 20 micrograms, b1 bands (less anionic albumin bands than the b2 band) were detected between pI 4.7 and pI 6.5 in urine and in both serum and purified albumin fractionated from serum of INS patients in the nephrotic stage. However, b1 bands were not detected at all in the urine and serum of either INS patients in remission stage and healthy volunteer controls. 3) When applied albumin amounted to 100 micrograms, b1 bands were detected also in serum of healthy volunteer controls. From these results, it was confirmed that less anionic albumin existed also in the serum of healthy volunteer controls although the amount was extremely small.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term follow-up of a paediatric case of lipoprotein glomerulopathy.

A paediatric case of lipoprotein glomerulopathy, a new kidney disease characterized by glomerular lipoprotein thrombi, is reported. The patient had massive proteinuria from the age of 8 years, when the nephrotic syndrome was first detected. This was resistant to conventional treatment for more than 10 years. During the course of the disease, the hyperlipidaemia characteristic of hyper-pre-beta-lipoproteinaemia and elevation of apoprotein E persisted, and renal function gradually deteriorated. The renal histopathological findings from four biopsies were essentially the same, with storage of beta-lipoprotein in dilated, balloon-like glomerular capillary lumina. However, the number of glomeruli showing global sclerosis increased and tubulo-interstitial changes progressed in parallel with the gradual clinical deterioration. As in other cases reported in Japan some familial involvement has been noted.

[Malignant fibrous histiocytoma of the thoracic region].

A clinicopathological investigation of five malignant fibrous histiocytoma (MFH) in the thoracic region was performed. They were two cases of metastasis to the lung and 1 each of the primary lesion in the lung, chest wall and sternal region. This tumor usually occurs on the extremities, in the abdominal cavity or the retroperitoneal areas, so instances of its arising in the thoracic region, except for metastatic episodes, are considered to be rare. Tumors in 4 cases were excised surgically, while one was inoperable. On light microscopy, the lesions were found to be composed of two types of neoplastic cells, fibroblast-like cells and histiocyte-like cells, showing the characteristic histologic pattern of storiform and pleomorphic. Electron-microscopically, these two types of cells were discriminated according to the quantitative and quantitative differences of the various intracytoplasmic organelles. The histogenesis of malignant fibrous histiocytoma has been commonly considered to be the histiocytic cell derived from the bone marrow. However, more detailed pathological investigations are necessary.