Biomedical subjects

S Iijima

Publications and source records attributed to S Iijima.

At least 55 records · Page 3Linked to original sources

PubMed1999

Simultaneous analysis of serum immunoglobulins in patients with M protein using cellulose acetate membrane isoelectric focusing.

We developed a method for the simultaneous analysis of microheterogeneity of human serum IgG, IgA, IgM, IgD, and IgE, and serum protein pattern using cellulose acetate membrane isoelectric focusing, and analyzed in 11 healthy subjects and 67 patients with M protein (17 cases of multiple myeloma [MM] and 50 cases of monoclonal gammopathy of undetermined significance [MGUS]). Using this method, bands indicating the microheterogeneity of each immunoglobulin could clearly be detected.Among healthy subjects, the detected IgG, IgA, and IgM bands did not vary, but the detected IgE and IgD bands did vary. Therefore, IgA, IgM, and IgG were selected for comparison of serum immunoglobulins in MM and in MGUS. In the IgA-type M protein group, normal IgM and IgG bands were decreased in MM patients compared to MGUS patients, while the M band and other bands were increased in MM patients compared to MGUS patients, but the differences between the two groups were not significant. In the IgG-type M protein group, normal IgM, IgA, and IgG were significantly decreased in MM patients compared to MGUS patients. We examined the changes in electrophoretic pattern in six MM patients and eight MGUS patients with IgA-type M protein after neuraminidase treatment. The width of the M band in MM patients with IgA-type M protein decreased with neuraminidase treatment. On the other hand, the width of the M band in MGUS patients with IgA-type M protein increased with neuraminidase treatment. We concluded that the decrease of the normal immunoglobulins in MM patients with IgG type M protein could be detected by this method, and IgA type of M protein binding sugar chain were different between MM and MGUS patients.

Blood Proteins↗

PubMed1999

New detection method after cellulose acetate membrane isoelectric focusing: activity staining for lactate dehydrogenase, detection for sugar chains using lectin and simple western blotting.

To extend the clinical applications for cellulose acetate (CA) membrane isoelectric focusing, we designed two detection methods and two blotting methods that improve the characteristics of the CA membrane; that is, detection of the lactate dehydrogenase (LD) isozyme on the CA membrane, detection of the sugar chain in glycoprotein on the CA membrane, and simple and rapid methods for western blotting. In the detection of the LD isozyme, when 50% saturated ammonium sulfate solution was used as a fixing solution, five main LD bands and seven sub LD bands were detected clearly. By treating the CA membrane with 5% sulfosalicylic acid, glycoprotein was fixed on the CA prior to detection of the sugar chain of glycoprotein using Lens Culinaris (LCA), Phasseolus Vulgaris-L(4 )(PHA-L(4)), Maackia Amurensis (MAM), and Sambucus Siedoldiana (SSA) lectins. Using this procedure, clear electrophoretic patterns were obtained. As simple and rapid methods for blotting proteins on polyvinylidene difluoride membranes, we designed natural contact blotting and suction blotting techniques. The natural contact blotting method did not need special apparatus. The suction blotting method completed protein blotting for 10 minutes. By both methods, clear electrophoretic patterns were obtained.

Blotting, Western↗

PubMed1999

Growth induction of rat primary hepatocytes using antisense oligonucleotides.

We examined growth control of adult and fetal hepatocytes by regulating the expression of cell-cycle-related proteins using antisense S-oligonucleotides to tumor suppressors retinoblastoma (RB) protein and p53, and cyclin-dependent kinase (CDK) inhibitors p21 and p27. The protein expression in both adult and fetal hepatocytes was significantly suppressed with the addition of corresponding antisense oligonucleotides at a concentration of 2.5 microM. For the evaluation of growth, 3H-thymidine incorporation and DNA content were measured and the results demonstrated that all the antisense oligonucleotides had growth-promoting effects and the promoting potential was equivalent or slightly greater than that with the addition of hepatocyte growth factor (HGF) (10 ng/ml). The growth-promoting effect of the antisense oligonucleotides was enhanced by HGF in both adult and fetal hepatocyte cultures, and the effects on hepatocyte growth were also observed in a suspension culture.

Journal Article↗

PubMed1999

Integrase-mediated nonviral gene transfection with enhanced integration efficiency.

Retroviruses efficiently integrate their genome into the host chromosome. Two elements of the retrovirus genome are needed for the integration: long terminal repeats (LTRs) and integrase protein. We attempted to incorporate the retrovirus integration machinery in lipid vesicle-mediated gene transfection with the aim of achieving efficient stable transfection in a nonviral gene transfection system. A DNA fragment, in which a neomycin-resistant gene was flanked between partial LTR sequences derived from the Rous sarcoma virus (RSV), was constructed. This DNA fragment was transfected together with purified recombinant RSV integrase or integrase expression vectors by means of lipid vesicle-mediated gene transfection. The integrase-mediated transfection enhanced the stable transfection efficiency. The length and the end structure of the LTR sequences were important in achieving high efficiency. Under optimal conditions, the stable transfection efficiency showed a 16-fold improvement over that without integrase.

Journal Article↗

PubMed1999

Enhanced cell aggregation and liver functions using polymers modified with a cell-specific ligand in primary hepatocyte cultures.

Hepatocytes cultured as multicellular aggregates called spheroids exhibit enhanced liver functions and maintain them over a long period compared with monolayer culture. We previously reported the induction of hepatocyte spheroids using the synthetic polymer Eudragit (a copolymer of methacrylic acid and methylmethacrylate) as an artificial matrix in a cell suspension system (Yamada et al., J. Biochem., 123, 1017-1023, 1998). In this method, hepatocyte aggregation was promoted by the effects of electrostatic and hydrophobic interactions between cells and the polymer. To enhance the cell aggregation ability and cell-specificity of the polymer, in the present study, we prepared hepatocyte-targeting polymers containing lactone, a ligand of the asialoglycoprotein receptor. Addition of the lactone-modified polymers to the medium promoted cell aggregation and spheroid formation more effectively than unmodified Eudragit. The spheroids induced by the polymers exhibited enhanced liver functions, i.e., albumin secretion, ammonia removal, and urea synthesis, from early in the culture. We also investigated the induction of hetero-spheroids composed of various liver constitutive cells by this method. The hetero-spheroids induced by the polymers showed improved liver functions.

Journal Article↗

PubMed1999

Enhancement of transfection efficiency by protamine in DDAB lipid vesicle-mediated gene transfer.

We have previously developed a simple gene transfection procedure mediated by cationic lipid vesicles for animal cells, in which a commercially available cationic surfactant, dimethyldioctadecyl ammonium bromide (DDAB), was used for making lipid vesicles. In the present study, we examined enhancement of transfection efficiency for this method by adding protamine to plasmid DNA solution before the formation of DNA/lipid vesicle complexes. Both free-base protamine and protamine sulfate provided enhanced transfection efficiency and expression level, but the optimal amount of the two protamines was different. The enhancement in transfection efficiency and expression level by protamines was observed in all the cell lines (COS-7, Hela, NIH3T3, MDCK, and BHK-21C13) and all the plasmids (pCMVbeta, pmiwZ, and pCH110) tested. The enhancement in both transfection efficiency and expression level was at most 20-fold compared with that using only DDAB lipid vesicles. Protamines seemed to protect DNA from degradation by DNase and promote DNA delivery into a nucleus.

3T3 Cells↗

PubMed1999

Solitary form of infantile myofibromatosis: a histologic, immunohistochemical, and electronmicroscopic study of a regressing tumor over a 20-month period.

We present the repeated clinical, histologic, immunohistochemical, and ultrastructural observations on a cutaneous myofibromatous tumor over a 20-month period. A 6-day-old Japanese female had a solitary tumor on her left wrist at birth. A biopsy was first performed at 16 days of age, when the tumor was likely fully developed. Thereafter, the tumor gradually regressed. A second biopsy was performed at 58 days of age, when the tumor was already in a phase of early regression. Finally, the tumor was resected at 20 months of age, when it was in a phase of late regression. Our study demonstrated that undifferentiated immature histiocytic cells predominated over spindle cells in the first biopsy specimen, but thereafter the former cells decreased or disappeared in parallel with the increase in the latter cells, which showed characteristics similar to myofibroblasts, in regressing lesions. This evidence suggests that the undifferentiated immature histiocytic cells are precursors of the spindle cells. Spindle cells in the phase of early regression also showed many vacuoles and lipid-like droplets in the cytoplasm, even though they actively produced massive amounts of glycogen. These findings also suggest that tumor regression results from cytoplasmic vacuolation and disruption of spindle cells. Our results are considered to demonstrate, for the first time, the clinical and histologic features of the different developmental or regressive phases of infantile myofibromatosis.

Biomarkers, Tumor↗

PubMed1999

Molecular characterization of type-specific capsular polysaccharide biosynthesis genes of Streptococcus agalactiae type Ia.

The type-specific capsular polysaccharide (CP) of a group B streptococcus, Streptococcus agalactiae type Ia, is a high-molecular-weight polymer consisting of the pentasaccharide repeating unit 4)-[alpha-D-NeupNAc-(2-->3)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1- ->3 )]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1. Here, cloning, sequencing, and transcription of the type Ia-specific capsular polysaccharide synthesis (cps) genes and functional analysis of these gene products are described. A 26-kb DNA fragment containing 18 complete open reading frames (ORFs) was cloned. These ORFs were designated cpsIaA to cpsIaL, neu (neuraminic acid synthesis gene) A to D, orf1 and ung (uracil DNA glycosylase). The cps gene products of S. agalactiae type Ia were homologous to proteins involved in CP synthesis of S. agalactiae type III and S. pneumoniae serotype 14. Unlike the cps gene cluster of S. pneumoniae serotype 14, transcription of this operon may start from cpsIaA, cpsIaE, and orf1 because putative promoter sequences were found in front of these genes. Northern hybridization, reverse transcription-PCR, and primer extension analyses supported this hypothesis. DNA sequence analysis showed that there were two transcriptional terminators in the 3' end of this operon (downstream of orf1 and ung). The functions of CpsIaE, CpsIaG, CpsIaI, and CpsIaJ were examined by glycosyltransferase assay by using the gene products expressed in Escherichia coli JM109 harboring plasmids containing various S. agalactiae type Ia cps gene fragments. Enzyme assays suggested that the gene products of cpsIaE, cpsIaG, cpsIaI, and cpsIaJ are putative glucosyltransferase, beta-1, 4-galactosyltransferase, beta-1,3-N-acetylglucosaminyltransferase, and beta-1,4-galactosyltransferase, respectively.

Bacterial Capsules↗

PubMed1999

D-Glucose and insulin stimulate migration and tubular formation of human endothelial cells in vitro.

Effects of high D-glucose and insulin on the endothelial cell migration and tubular formation were investigated with the use of ECV304 cells, a clonal human umbilical cord endothelial cell line. Exposure of the cells to high D-glucose resulted in a marked increase in the migration, which was blocked by inhibitors of protein kinase C such as H7 (10 microM) and GF109203X (200 nM). Furthermore, a protein kinase C agonist, phorbol 12-myristate 13-acetate, had an effect similar to that of glucose on ECV304 cells. Glucose stimulation of the migration was additively enhanced by 100 nM insulin, and the insulin effect was found to be unaffected by either PD-98059 or wortmannin, a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor and a phosphatidylinositol 3-kinase inhibitor, respectively. Neither did H7 inhibit insulin stimulation of the migration. In contrast, a combination of high D-glucose and insulin, rather than either one alone, promoted tubular formation, which was inhibited by addition of 10 microM PD-98059. Stimulation of ECV304 cells by the combination of high D-glucose and insulin also caused an activation of MAPK, which was again obliterated by the same concentration of PD-98059. In conclusion, human endothelial cell migration and tubular formation are stimulated by high D-glucose and insulin in different ways. In the former reaction, either is effective, a combination of the two results in an additive effect, and activation of protein kinase C is involved. In contrast, tubular formation will only occur in the presence of a combination of high D-glucose and insulin, and MAPK plays an essential role.

Cell Line↗

PubMed1999

Hemodialysis neutropenia correlates with a decreased filterability and an increase in the number of cytoplasmic actin filaments in peripheral blood neutrophils, which is preceded by a decrease in the number of surface expression of L-selectin.

In order to clarify the precise cellular mechanism of hemodialysis neutropenia, we examined the changes in the viscoelasticity of peripheral blood neutrophils using both the micropore and the microchannel filtration methods, and the changes in the neutrophil surface expression of Mac-1, L-selectin and sialyl Lewis X and the cytoplasmic expression of the actin filaments using a flow cytometric analysis during a dialysis session. Five patients with chronic renal failure were selected who showed a nadir leukocyte count in peripheral blood at 30 min after the initiation of the dialysis session. The neutrophil count also reached a nadir at 30 min and thereafter returned to almost the predialysis level by 180 min. Both the micropore filtration time and the microchannel passage time, which reflect the viscoelasticity of the peripheral blood neutrophils, correlated inversely with the neutrophil count. At the nadir of neutropenia, the neutrophils were observed to have become both adhesive and viscoelastic. The actin filaments in the neutrophil cytoplasm gradually increased in number from the start of dialysis, reaching a peak level at 30 min, and thereafter decreasing to predialysis levels. The Mac-1 expression continuously increased up from 30 min until the end of dialysis. The L-selectin expression first decreased at 15 min, but thereafter returned to predialysis levels within 60 min. The SLex expression did not change throughout the course of the session. These results thus indicated the neutrophil counts during a dialysis session to inversely correlate with the viscoelasticity of the neutrophils expressed by the micropore filtration time or microchannel passage time, which possibly depends on the contents of cytoplasmic actin filaments. In addition, the shedding of L-selectin from neutrophil surface may also be involved in the first step of hemodialysis neutropenia.

Actin Cytoskeleton↗

PubMed1999

Growth and differentiation of cultured fetal hepatocytes isolated various developmental stages.

We examined the relationship between cell proliferation and differentiation of cultured rat fetal and newborn hepatocytes isolated from various developmental stages. The albumin production rate increased along with cell growth under in vitro culture and became maximal two days after the growth cessation. AFP was secreted by both fetal and newborn hepatocytes with growth ability. Furthermore, the responses to HGF addition in fetal hepatocyte cultures were observed in terms of growth stimulation and down-regulated of the Met receptor. We also studied the changes in RB and liver enriched transcription factors (C/EBPs) for investigating the mechanism underlying proliferation and differentiation of fetal hepatocytes. Western blot analysis of hepatocytes taken from various gestation stages of rat liver showed that the expression of RB and C/EBP beta increased as gestation stage proceeded. When RB antisense S-oligonucleotide was added to the culture medium, proliferation and AFP expression increased, while C/EBP alpha and albumin expressions decreased. These results indicated that the tumor suppressor gene product RB had a profound role not only in cell proliferation but also hepatocyte differentiation.

Albumins↗

PubMed1999

IGF-1 regulates migration and angiogenesis of human endothelial cells.

Recent studies revealed favorable para- and/or autocrine effects of IGF-1 in the pathogenesis of diabetic complications. On the other hand, hyperglycemia is a risk factor for the development of diabetic vascular complications. In this study we examined the effects of high glucose and/or IGF-1 on cell migration and angiogenesis (tubular formation) by using human endothelial cells (EC) in vitro. First we examined cell migration by the two-chamber method. Chronic treatment with a high concentration of D-glucose strongly stimulated the cell migration, which was mimicked by PMA, a protein kinase C (PKC) agonist. The cell migration was also induced by IGF-1. The glucose-induced cell migration was blocked by PKC inhibitor, H7. IGF-1-induced cell migration was not blocked by PD98059, MAPK/ERK kinase (MEK) inhibitor or wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor. Next we examined the effects of high glucose and/or IGF-1 on the tubular formation of EC. The tubular formation was induced only when the cells were exposed to a combination of high glucose and IGF-1. The tubular formation was blocked by MEK inhibitor and PI 3-kinase inhibitor but not by PKC inhibitor. These results indicate that hyperglycemia and IGF-1, respectively, stimulate the EC migration, and tubular formation is induced by a combination of IGF-1 and hyperglycemia.

Androstadienes↗

PubMed1998

Coaxial nanocable: silicon carbide and silicon oxide sheathed with boron nitride and carbon

Multielement nanotubes comprising multiple phases, with diameters of a few tens of nanometers and lengths up to 50 micrometers, were successfully synthesized by means of reactive laser ablation. The experimentally determined structure consists of a beta-phase silicon carbide core, an amorphous silicon oxide intermediate layer, and graphitic outer shells made of boron nitride and carbon layers separated in the radial direction. The structure resembles a coaxial nanocable with a semiconductor-insulator-metal (or semiconductor-insulator-semiconductor) geometry and suggests applications in nanoscale electronic devices that take advantage of this self-organization mechanism for multielement nanotube formation.

Journal Article↗

PubMed1998

No induction of chromosome aberrations in human spermatozoa exposed to extremely low frequency electromagnetic fields.

Clastogenic effects of extremely low frequency electromagnetic fields (ELF-EMFs) on human sperm chromosomes were studied using an interspecific in vitro fertilization system with zona-free golden hamster oocytes. Semen samples from healthy men were exposed to ELF-EMFs (50 Hz, 20 mT) for 2 h at 37 degreesC under 5% CO2 in air. The samples were then cryopreserved in liquid nitrogen for shipment to a cytogenetic laboratory. After thawing the samples, motile spermatozoa were collected using a continuous Percoll density gradient centrifugation and then capacitated for in vitro fertilization with hamster oocytes. Sperm-derived chromosomes were analyzed at first cleavage metaphase. The present experiment was performed twice using semen samples from two different donors. In test-1, incidence of spermatozoa that displayed structural chromosome aberrations was 17.0% (35/206) in the exposed group and 20.8% (55/264) in the control group. In test-2, structural chromosome aberrations were observed in 11.1% (13/117) of exposed spermatozoa and 13.8% (13/94) of spermatozoa in the control group. In both tests, there was no significant difference in the incidence of chromosomally abnormal spermatozoa between the exposed group and the control group. Types of aberrations observed and their incidences per spermatozoon in the exposed group were similar to those of the control group. Despite the small sample size, the present results suggest that ELF-EMFs have no clastogenic effect on human sperm chromosomes.

Animals↗

PubMed1998

Improved hatching for in vitro quail embryo culture using surrogate eggshell and artificial vessel.

The establishment of avian embryonic culture is important both for the analysis of the developmental process and the establishment of transgenic chickens that produce useful biological materials in eggs. However, the hatchability of cultured embryos has been approximately 50%. We identified that the low rate of hatchability of cultured embryos was caused by limited oxygen and calcium availability. In quail embryo culture using chicken eggshell as a culture vessel, viability in the middle stage of culture was improved and 30% of embryos were hatched by oxygen enrichment. Furthermore, hatchability increased to 80% by supplementation with calcium lactate in addition to oxygen aeration. In the present study, a fully artificial vessel for quail embryo culture was designed using a gas-permeable Teflon membrane. By the addition of fine eggshell powder and calcium lactate, quail embryos grew and developed normally, and 43% of embryos hatched. Although the hatchability was lower than that of cultures using a surrogate eggshell, we achieved in hatching an avian embryo using a fully artificial vessel.

Animals↗

PubMed1998

Efficient induction of hepatocyte spheroids in a suspension culture using a water-soluble synthetic polymer as an artificial matrix.

The preparation of hepatocyte spheroids by adding a water-soluble synthetic polymer as an artificial matrix was performed in a cell suspension system. Cell-aggregation was promoted without cytotoxicity by adding Eudragit (a copolymer of methacrylic acid and methylmethacrylate) to the culture medium. Spheroid-like cell aggregates, whose liver functions were enhanced, were effectively formed in the presence of 0.1% Eudragit, independent of the cultural substratum. Moreover, the mass preparation of spheroids could be achieved with a high production yield by means of a suspension culture in a spinner flask. In this case, the polymer protected the cells from damage due to agitation. The spheroids induced with Eudragit expressed high liver functions, such as albumin secretion, ammonia removal, and urea synthesis. On histological observation, the spheroids showed a well-developed cell adhesion apparatus and bile canaliculi. In addition, a higher calcium ion concentration in the cells of spheroids was observed compared with in monolayer cells.

Animals↗

PubMed1998

Differentiation and proliferation of primary rat hepatocytes cultured as spheroids.

We studied spheroid (multicellular aggregate) formation by hepatocytes and the expression of liver-specific functions such as albumin secretion when hepatocytes were cultured with various extracellular matrices. Hepatocytes cultured on Primaria(R) and poly-D-lysine coated dishes, and in the presence of a polymer, Eudragit, formed spheroids, and they also exhibited higher liver-specific functions and poor growth compared to monolayer cultures. The results indicated that the cell morphological change and cell-cell interaction caused by the spheroid formation were key factors promoting the expression of the liver-specific functions. To elucidate the mechanism underlying the poor growth in spheroids, we examined the HGF signaling pathway. Phosphorylation and down-regulation of the HGF receptor (c-Met proto-oncogene product) were observed for the cells from both monolayer and spheroid cultures, but Ras activation was partly blocked in spheroids. Furthermore, we found that CDK inhibitors, p21 and p27, were highly expressed in spheroids. These results suggested that the reduced Ras signaling and high expression of the CDK inhibitors might cause the lower growth in spheroids. We then examined the relationship between liver-enriched transcription factors (C/EBPalpha and beta) and liver-specific functions. The results revealed that the high expression of C/EBPalpha was maintained during cultures when hepatocytes formed spheroids. Antisense oligonucleotides of C/EBPalpha repressed albumin secretion and the expression of p21, suggesting that the transcription factor, C/EBPalpha, may play a crucial role in the growth and differentiation of hepatocytes in spheroids.

Animals↗

PubMed1998

Serum concentration of the pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP) is a useful indicator of decline and recovery of bone mineral density in lumbar spine: analysis in Japanese postmenopausal women with or without hormone replacement.

To reassess the clinical utility of serum pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP), a promising but controversial indicator of bone resorption, we evaluated its performance as a biochemical marker in a 6-month study of a strictly selected population of 76 Japanese postmenopausal and healthy women, 33 recipients of hormone replacement therapy and 43 nonrecipients. We measured bone mineral density (BMD) of the lumbar spine (L2-L4), and serum ICTP, carboxyterminal propeptide of type I procollagen (PICP) and other conventional serum biochemical markers, e.g. bone gla protein (BGP), alkaline phosphatase, calcium and phosphate at the entry and 6 months later. We calculated the percent change between the baseline and 6-month values (delta%) in lumbar BMD and the biochemical markers, individually, and compared the degree of correlations between delta% in BMD and that in the biochemical markers. Delta% in ICTP and delta% in BGP correlated with delta% in BMD negatively and significantly. Especially delta% in ICTP correlated with that in BMD to a high degree (P<0.0001). No significant correlation was observed in other biochemical markers. We concluded that serum ICTP is a sensitive and useful bone resorption marker in the postmenopausal population, which strongly correlates with the change in BMD.

Adult↗