Biomedical subjects

N Takemoto

Publications and source records attributed to N Takemoto.

At least 37 records · Page 2Linked to original sources

PubMed1997

Isolation and identification of hematopoietic stem cell-stimulating substances from Kampo (Japanese herbal) medicine, Juzen-taiho-to.

We have previously found that TJ-48 has the capacity to accelerate recovery from hematopoietic injury induced by radiation and the anti-cancer drug mitomycin C (MMC). The effects are found to be due to its stimulation of spleen colony-forming unit (CFU-S) counts on day 14. In the present study, we attempt to isolate and purify the active components in TJ-48 extracts using a new in vitro hematopoietic stem cell (HSC) assay method. n-Hexane extract from TJ-48 shows a significant stimulatory activity. The extract is further fractionated by silica gel chromatography and HPLC in order to identify its active components. 1H-NMR and GC-EI-MS indicate that the active fraction is composed of free fatty acids (oleic acid and linolenic acid). When 27 kinds of free fatty acids (commercially available) are tested using the HSC proliferating assay, oleic acid, elaidic acid, and linolenic acid are found to have potent activity. The administration of oleic acid to MMC-treated mice enhances CFU-S counts on days 8 and 14 to twice the control group. These findings strongly suggest that fatty acids contained in TJ-48 actively promote the proliferation of HSCs. Although many mechanisms seem to be involved in the stimulation of HSC proliferation, we speculate that at least one of the signals is mediated by stromal cells, rather than any direct interaction with the HSCs.

Animals↗

PubMed1997

Ethyl nitrosourea and methyl methanesulfonate mutagenicity in sperm and testicular germ cells of lacZ transgenic mice (Muta Mouse).

The germ cell mutagens ethyl nitrosourea (ENU) and methyl methanesulfonate (MMS), were tested for their genotoxicity in sperm cells and testicular germ cells using lacZ transgenic mice (Muta Mouse). Eight- to 10-week-old Muta mice were treated with ENU (150 mg/kg) or MMS (40 mg/kg) by intraperitoneal injection. Three and 14 days after treatment, testes and sperm were collected for lacZ mutation analysis. Sperm were isolated from the epididymis and vas deferens by washing out the minced tissue. Germ cell DNA was isolated from testicular germ cells and sperm with the help of 2-mercaptoethanol, and the target lacZ gene, which is integrated into a lambda shuttle vector, was recovered by in vitro packaging. The resultant phages were allowed to infect to E. coli C (galE), and the lacZ mutant plaques were dominantly selected on a plate containing phenyl-beta-D-galactoside. Spontaneous mutant frequencies (MF) in vehicle-treated control mice were approximately 1 x 10(-5) and 3 x 10(-5) in testicular germ cells and sperm, respectively, at both sampling times. ENU treatment increased the MF in the testicular germ cells to 5 x 10(-5) on days 3 and 14, but did not affect sperm MF. MMS was not mutagenic in either tissue. The peripheral blood micronucleus assay was performed on the same animals 48 h after treatment, and strong inductions of micronucleated reticulocytes (MNRETs) were observed in both ENU- and MMS-treated mice. These data suggest that agents mutagenic to premeiotic germ cells, e.g., ENU, can be detected by transgenic mutation assay system using germ cells isolated from the testis. On the other hand, those mutagenic to postmeiotic cells, e.g., MMS, are insensitive in the assay system.

Animals↗

PubMed1997

Suppression for the proliferation of fibroblasts by external DNA.

Phase-contrast and fluorescence microscopic observation showed that DNA added in the cell-culture medium for fibroblasts localized just on the surface of fibroblasts. The DNA bound to fibroblasts was found to be eluted by treating with collagenase. The suppression for the proliferation of fibroblasts by external DNA was confirmed with microscopic observation for the cells cultured in the presence and absence of DNA. Proliferation of the cells decreased from 412 to 155% by the addition of DNA. These results indicate that DNA has an affinity for collagen, the most major extracellular-matrix produced by fibroblasts, and suppresses the growth of fibroblasts.

Cell Division↗

PubMed1997

Four P-like elements are required for optimal transcription of the mouse IL-4 gene: involvement of a distinct set of nuclear factor of activated T cells and activator protein-1 family proteins.

We previously identified the P sequence as a critical regulatory element of the human IL-4 promoter. In the mouse IL-4 promoter, there are five elements homologous to the human P sequence designated conserved lymphokine element 0 (CLE0), P, P2, P3 and P4. To characterize the role of these P-like elements and their binding factors in the native promoter, we did transient transfection and electrophoretic mobility shift assays (EMSA). Transfection of EL-4 cells with the IL-4 promoter-reporter constructs carrying mutated P-like elements showed that four P-like elements, CLE0, P, P2 and P4, but not P3, were required for optimal activation of the IL-4 promoter. EMSA showed that both constitutive and inducible complexes bound to CLE0, P, P2 and P4, whereas only a constitutive complex bound to P3. In competition and antibody supershift assays in EMSA, complexes formed with P or P2 proved to contain nuclear factor of activated T cells (NFAT) family proteins as major components. Activator protein (AP)-1 family proteins interacted with CLE0, P, P2 and P4. NFAT/AP-1 complex formed only with P and P2. Cross-competition assays among the P-like elements revealed element-specific and common complexes. Six tandem repeats of the P element linked to the SV40 promoter responded to phorbol 12-myristate 13-acetate, while that of other elements did not. It would thus appear that components of each P-like element-binding complexes are not identical and may coordinately contribute to transcriptional activity.

Animals↗

PubMed1996

Neurotrophic effect of hepatocyte growth factor on central nervous system neurons in vitro.

Although the expression of hepatocyte growth factor (HGF) and its receptor, proto-oncogene c-met, has been demonstrated in the central nervous system (CNS), the function of HGF in the CNS was not fully understood. In the present studies, we determined the effects of HGF on neuronal development in neocortical explant and mesencephalic neurons obtained from embryonic rat brain. HGF clearly enhanced neurite outgrowth in neocortical explants. In the mesencephalic culture, the number of tyrosine hydroxylase (TH)-positive neurons was significantly higher in the HGF-treated wells and the neurites of the TH-positive neurons appear to be more developed. Moreover, the dopamine uptake into mesencephalic neurons was also enhanced by HGF treatment, indicating that HGF promotes the survival and/or maturation of mesencephalic dopaminergic neurons. In both neocortical explants and mesencephalic neurons, c-met autophosphorylation was induced by HGF and MAP kinase activation was also detected in the neocortical explant. Furthermore, Western blot analysis of the cultured CNS cells revealed that HGF was expressed mainly in microglia. These results suggest that HGF from microglia has neurotrophic activity on the CNS neurons and plays significant roles in the development of the CNS.

Animals↗

PubMed1996

Effects of calcium in continuous cardioplegia on myocardial protection.

The effects of calcium (Ca) on a hyperkalemic cardioplegic solution for continuous cardioplegia were examined in an isolated perfused working rat heart model. The coronary arteries were perfused with a modified Krebs-Henseleit bicarbonate buffer (K-H) solution, containing various concentrations of Ca (0.1, 0.6, 1.2, and 2.5 mmol/l) and a high concentration of potassium (20 mmol/l), for 180 min, after which cardiac arrest was induced at 37 degrees C for 180 min. Cardiac function and creatine kinase (CK) were measured. In the control group, K-H solution was infused in place of the cardioplegic solution, and cardiac arrest was not induced. No significant differences were observed between the groups infused with the K-H solution containing Ca concentrations of 0.6, 1.2, and 2.5 mmol/l in the percent recovery of aortic flow (82.1 +/- 2.9%, 80.6 +/- 2.0%, and 71.5 +/- 3.7% (mean +/- SEM) respectively) or in the recovery of other indices of cardiac function, or in CK leakage. There were also no significant differences in the recovery of cardiac function and CK leakage between these groups and the control group. In the Ca 0.1 mmol/l group, however, the characteristic Ca paradox was observed. These findings suggest that if the Ca concentration in a cardioplegic solution is higher than 0.6 mmol/l during continuous cardioplegia, excellent cardioprotective effects will be achieved.

Animals↗

PubMed1996

Pectic polysaccharides from roots of Glycyrrhiza uralensis: possible contribution of neutral oligosaccharides in the galacturonase-resistant region to anti-complementary and mitogenic activities.

Digestion with endo-alpha-(1-->4)-polygalacturonase liberated the enzyme-resistant region (PG-1c) as an active site of the anti-complementary and mitogenic pectic polysaccharide (GR-2IIc) from Glycyrrhiza uralensis. Partial acid hydrolysis of PG-1c resulted in acidic oligosaccharides, and methylation analysis and GC-MS analysis of the acidic oligosaccharides suggested that PG-1c comprised a rhamnogalacturonan core such as -->2)-Rha-(1-->4)-GalA-(1-->2)-Rha-(1-->4)-GalA-(1-->-->4)-GalA-(1-->4) as the acidic moiety. Degradation of uronic acids by lithium decreased the anti-complementary and mitogenic activities of PG-1c. Although the products from PG-1c were still active, the methylglycoside of alpha-L-Rha-(1-->4)-alpha-D-GalA-(1-->2)-alpha-L-Rha-(1-->4)-alpha-D-Gal A did not show both activities. The products obtained by the lithium degradation from PG-1c gave fractions containing various neutral oligosaccharide-alditols. Among these fractions the longest and the short oligosaccharide-alditol fractions had relatively potent anti-complementary activity, whereas all oligosaccharide-alditol fractions expressed weak but significant mitogenic activity. GC-MS analysis indicated that the short oligosaccharide-alditol fraction contained various kinds of di- to tetrasaccharide-alditols. However, malto-oligosaccharide-alditols, and malto-, isomalto-, and laminari-oligosaccharides did not show anti-complementary and/or mitogenic activities, and these results suggested that certain neutral carbohydrate chains in PG-1c were responsible for the expression of mitogenic activity as well as anti-complementary activity of PG-1c.

Animals↗

PubMed1996

[Sleeve pulmonary arterial resection for bronchogenic carcinoma].

Two cases of bronchogenic carcinoma undergone left upper lobectomy (R 3) with bronchoplasty and sleeve pulmonary arterial resection via mid-sternotomy were reported. Both cases were squamous cell carcinoma originated in the orifice of the left upper lobe. Case 1 was stage IIIB (T2N3M0) bronchogenic carcinoma, its postoperative course was uneventful and died of distant lymphatic metastasis thirty-three months after operation. Case 2 was stage II (T2N1M0) bronchogenic carcinoma and its postoperative management was laborious because of hard expectoration of the sputum but is doing well fifteen months after operation. In order to preserve adequate pulmonary function and to maintain reasonable quality of life (QOL) for the patients with impaired pulmonary function, this angioplastic procedure seems to be acceptable. It is still under discussion to perform this procedure for the patients who would be able to withstand undergoing pneumonectomy, therefore we adopt this method only for every patient for whom it is difficult to maintain desirable QOL after pneumonectomy. Namely, for the patient whose predicted one second forced expiratory volume (FEV1.0) after pneumonectomy is less than 900 ml/m2, we'll be likely to try this angioplastic procedure at first.

Aged↗

PubMed1996

[A case of a patient with post-operative empyema due to Mycobacterium chelonae].

We reported a case of a thoracic empyema due to M. chelonae (Atypical Mycobacterium, group IV) after thoracic surgery. A 49-year-old male underwent right middle lobectomy for pulmonary metastasis of esophageal cancer. Postoperative course was complicated for intractable air leakage, and several procedures were tried before successful re-thoractomy. Seven days after re-thoractomy, mycobacterium was proved in pleural effusion. And later on M. chelonae was identified by DNA hybridization method. Therefore, open window thoracotomy was performed at once. M. chelonae disappeared 7 days after operation and the patient discharged on 40 postoperative days. Thoracic empyema by M. chelonae is rare, and only one case was reported in Japan so far. Present case was not combined with infectious pulmonary disease by some mycobacterium. Therefore it is most reasonable to suppose this intrathoracic infection developed through the thoracic drain. In conclusion, because of the M. chelonae toleranced for almost all anti-biotics including anti-tuberculous agents, except clarithromycin, the timing of surgical approach is important for the treatment of this infectious disease.

Carcinoma, Squamous Cell↗

PubMed1995

Effect of oral administration of a pectic polysaccharide fraction from a kampo (Japanese herbal) medicine "juzen-taiho-to" on antibody response of mice.

A dried decoction of a kampo (Japanese herbal) medicine, Juzen-Taiho-To (TJ-48), has been fractionated into five fractions and tested for their effects on antibody response of mice. An intraperitoneal injection (300 mg/kg) of TJ-48 stimulated anti-sheep red blood cell (SRBC) antibody response of normal Balb/c mice, but only the polysaccharide fraction (F-5, 300 mg/kg) enhanced the antibody response among the fractions from TJ-48. When F-5 (0.5 or 1.0 g/kg/day) was orally administered to normal Balb/c mice (7-weeks-old) from 7 days before to 4 days after immunization with SRBC, the number of anti-SRBC-IgM-PFC in spleen and the titer of anti-SRBC-IgM in plasma were increased significantly. However, a lower dose (0.1 g/kg/day) of F-5 did not show a significant stimulative activity on the anti-SRBC-response. Although aged Balb/c mice (6-months-old) produced a lower level of anti-SRBC-IgG in comparison with young Balb/c mice (8-weeks-old), the anti-SRBC-IgG response of the aged mice was stimulated significantly when F-5 (0.13 g/kg/day) or TJ-48 (1.0 g/kg/day) was orally administered to the aged mice from 6 days before immunization. Intraperitoneal injections of i-carrageenan (2.5 mg/kg/day) at 3 and 1 days before the immunization with SRBC increased the level of anti-SRBC antibody response compared with normal mice. Oral administrations of TJ-48 (1.0 g/kg/day) or F-5 (0.5 g/kg/day) to the i-carageenan-treated mice reduced the level of the anti-SRBC-antibody response near to that of normal mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗

PubMed1995

Purification and characterization of the Pseudomonas aeruginosa NfxB protein, the negative regulator of the nfxB gene.

The protein NfxB, involved in conferring resistance to quinolones in Pseudomonas aeruginosa, has a helix-turn-helix motif which is similar to that of other DNA-binding proteins. It appears to affect the membrane-associated energy-driven efflux of some antibiotics (H. Nikaido, Science 264:382-388, 1994). We constructed a plasmid that overproduced NfxB in Escherichia coli and purified the protein. Two species of NfxB (23 and 21 kDa), which are probably translated from different initiation codons, were isolated. Both proteins are also expressed in vivo in P. aeruginosa, with the 23-kDa NfxB being the major species. NfxB specifically binds upstream of the nfxB coding region as demonstrated by gel retardation and DNase I footprinting. Expression of the phi (nfxB'-lacZ+) (Hyb) gene was repressed in the presence of the nfxB gene product provided by a second compatible plasmid in E. coli. In the P. aeruginosa wild-type strain (PAO2142), NfxB was undetectable by immunoblotting; however, it was detected in the nfxB missense mutant (PK1013E). These results suggested that NfxB negatively autoregulates the expression of nfxB itself. Since the 54-kDa outer membrane protein (OprJ) (N. Masuda, E. Sakagawa, and S. Ohya, Antimicrob. Agents Chemother. 39:645-649, 1995) was overproduced in nfxB mutants, NfxB may also regulate the expression of membrane proteins that are involved in the drug efflux machinery of P. aeruginosa.

Amino Acid Sequence↗

PubMed1994

Glial conditioned medium alters the expression of amyloid precursor protein in SH-SY5Y neuroblastoma cells.

The effect of conditioned medium from astroglia or microglia cultures on the mRNA expression of amyloid precursor protein (APP) in SH-SY5Y human neuroblastoma cells was examined. When incubated with conditioned medium of glial cells, SH-SY5Y cells expressed a larger amount of APP mRNA than the control cells. Reverse transcription followed by the polymerase chain reaction showed marked induction of expression of APP isoforms containing a Kunitz-type protease inhibitor domain. Our results suggest that glial cells may contribute to the regulation of expression of APP in neurons.

Amyloid beta-Protein Precursor↗

PubMed1994

Effect of magnesium and calcium on myocardial protection by cardioplegic solutions.

The cardioprotective effect of the magnesium and calcium content of hyperkalemic cardioplegic solutions was investigated using isolated rat hearts subjected to hypothermic ischemic arrest. Hearts were arrested for 180 minutes at 20 degrees C by administering a 3-minute infusion of cardioplegic solution containing various concentrations of magnesium and calcium. Treatment groups received solution with either 0, 8, or 16 mmol/L magnesium. For each of these magnesium concentrations, 0.1, 0.6, or 1.2 mmol/L calcium was also present in the solution. At each concentration of magnesium, the percentage recovery of aortic flow was dependent on the calcium concentration. The maximum percentage recovery of aortic flow was 67.9% +/- 2.3% (mean +/- standard error of the mean) in the Mg-free, 0.1 mmol/L Ca group, whereas it was 65.1% +/- 2.7% in the 8 mmol/L Mg, 0.1 mmol/L Ca group and 70.0% +/- 3.5% in the 16 mmol/L Mg, 0.6 mmol/L Ca group. No significant differences in the recovery of cardiac function and creatine kinase leakage were observed between the three groups. The findings suggested that the cardioprotective effect was dependent on the relative concentration of both magnesium and calcium, and that it is important to maintain an appropriate ionic balance in cardioplegic solutions.

Animals↗

PubMed1994

A novel type of B-cell mitogen isolated from juzen-taiho-to (TJ-48), a Japanese traditional medicine.

Juzen-taiho-to (TJ-48), a Japanese traditional medicine, is known to have various immunological activities including the induction of B-cell proliferation. We investigated the properties of the pectic polysaccharide fraction of TJ-48 (F-5-2) which is most active in the proliferation of spleen cells. To an extent equal to that of TJ-48, F-5-2 induced the proliferation of B-cells, particularly those holding both sIgM and sIgD. The proliferation induced by F-5-2 was T-cell independent and macrophage dependent. The macrophages could be substituted for a soluble factor(s) secreted from the macrophages but not for IL-1. Generally, B-cell mitogens are known to induce the proliferation of B-cells and subsequently differentiation into plasma cells. However, although F-5-2 induced the B-cell differentiation, it arrested their development in the intermediate stage of the B-cell differentiation. The B-cells induced by F-5-2 produced IgM antibody in response to IL-6 and an antigen (SRBC) but not IgG antibody. F-5-2 induced the expression of IL-6R not only on IgM+ and IgG+ B-cells but also on IgD+ B-cells. These results suggest that F-5-2 is a new type of B-cell mitogen.

Animals↗

PubMed1994

Plasminogen binds specifically to alpha-enolase on rat neuronal plasma membrane.

Plasminogen (PGn) that we identified in microglial-conditioned medium has a neurotrophic factor-like effect on cultured neurons. We have also shown that PGn binds specifically to a protein with a molecular mass of 45 kDa in the neuronal plasma membrane. As a candidate PGn receptor-like molecule on the neuronal surface, this 45-kDa protein was purified from the plasma membrane of embryonic rat brain. Amino acid sequence analysis of polypeptides derived from the cleavage of the protein with cyanogen bromide and V8 protease revealed that the 45-kDa protein is identical to rat alpha-enolase. In fact, PGn was found to bind to purified rat alpha-enolase and also to a synthetic peptide (30 residues) that corresponds to the carboxyl terminal region of rat alpha-enolase. Physical properties of the 45-kDa protein, such as molecular mass, isoelectric point, and the ability to form dimers, are quite similar to those of alpha-enolase. The 45-kDa PGn-binding protein in the plasma membrane was also recognized by anti-rat alpha-enolase antibody, and pretreatment with alpha-enolase antibody markedly diminished the PGn-binding to the plasma membrane. In addition, immunocytochemical staining of the cultured cells under the nonpermeable condition showed that alpha-enolase is present on the cell surface of a certain population of neurons. These results suggest that alpha-enolase may function as a PGn-binding molecule on the neuronal cell surface.

Amino Acid Sequence↗

PubMed1993

Plasminogen-binding protein associated with the plasma membrane of cultured embryonic rat neocortical neurons.

To investigate the receptor-like molecule(s) for plasminogen (PGn) on the neuronal surface, the properties of binding of PGn to the plasma membrane of cultured embryonic rat neocortical neurons were investigated. [125I]PGn was found to specifically bind to the plasma membrane depending on the incubation temperature and time. The binding was also affected strongly by ionic strength and slightly by Ca2+. Furthermore, ligand blotting analysis revealed that [125I]PGn binds to a major protein with an apparent molecular weight of 45 kDa among plasma membrane proteins. These results suggest that the 45-kDa protein is a PGn receptor-like molecule on the neuronal surface.

Animals↗

PubMed1993

Microglia-derived plasminogen enhances neurite outgrowth from explant cultures of rat brain.

Recently, we reported the production and secretion of plasminogen (Pg) in cultured rat brain microglia [Nakajima et al., (1992) Fedn. Eur. Biochem. Socs Lett. 308, 179-182]. To investigate the physiological significance of Pg, we determined the effect of Pg on neurite outgrowth of cultured neocortical explants of an embryonic rat brain in serum-free chemically defined medium. Pg markedly enhanced the neurite outgrowth. Although plasmin, which is derived from Pg by activation by urokinase (UK), had a similar effect in this explant culture system, UK itself did not show any effect. Furthermore, we studied the characteristics of Pg binding to cultured neocortical neurons dissociated from an embryonic 16-day-old rat brain by using 125I-Pg. Specific binding of Pg to neocortical neurons was detected and Scatchard plot analysis revealed high- and low-affinity binding sites on the neurons. The estimated dissociation constants of high- and low-affinity binding sites were approximately 16.1 and 124.2 nM, respectively. These results suggest that microglia-derived Pg plays certain roles in the regulation of neurite extension through binding to the surface of neocortical neurons.

Animals↗

PubMed1993

Microglia-derived elastase produces a low-molecular-weight plasminogen that enhances neurite outgrowth in rat neocortical explant cultures.

In the course of analysis of plasminogen in microglial conditioned medium (Mic-CM), novel low-molecular-weight (LMW) zymogen with a molecular mass of approximately 36 kDa was detected by casein-urokinase zymography. Because this form was produced when rat native plasminogen was incubated with Mic-CM, a specific protease in the Mic-CM was thought to be responsible for the production of LMW plasminogen. The production of LMW plasminogen was strongly inhibited by elastase inhibitors. Furthermore, elastase (pancreatic or leukocyte) was also found to produce LMW zymogen from native plasminogen. These results indicate that LMW plasminogen is produced through limited proteolysis by an elastase-like protease in Mic-CM. To determine the biochemical characteristics of LMW plasminogen, rat native plasminogen was cleaved by pancreatic elastase, and the fragments (LMW plasminogen and nonzymogen fragments) were purified by several kinds of column chromatography. Amino acid sequence analysis revealed that LMW plasminogen is a carboxy-terminal region that contains the fifth kringle domain and a protease active site, and the amino acid sequence is identical to that of LMW plasminogen produced by Mic-CM. On the other hand, the nonzymogen fragment was the amino-terminal region containing four kringle domains. The effects of native plasminogen and the fragments on neurite outgrowth of rat brain explant were examined. LMW plasminogen promoted neurite outgrowth as well as did native plasminogen, whereas nonzymogen fragments did not. These results suggest that LMW plasminogen, which is produced from native plasminogen by elastase, may be a physiologically active molecule that mediates the intercellular interaction between microglia and neurons.

Amino Acid Sequence↗