Biomedical subjects

I Kojima

Publications and source records attributed to I Kojima.

At least 19 recordsLinked to original sources

PubMed1999

Impaired differentiation of endocrine and exocrine cells of the pancreas in transgenic mouse expressing the truncated type II activin receptor.

Activin A is expressed in endocrine precursor cells of the fetal pancreatic anlage. To determine the physiological significance of activins in the pancreas, a transgenic mouse line expressing the truncated type II activin receptor under the control of beta-actin promoter was developed. Histological analyses of the pancreas revealed that the pancreatic islets of the transgenic mouse were small in size and were located mainly along the pancreatic ducts. Immunoreactive insulin was detected in islets, some acinar cells, and in some epithelial cells in the duct. In addition, there were abnormal endocrine cells outside the islets. The shape and the size of the endocrine cells varied and some of them were larger than islets. These cells expressed immunoreactive insulin and glucagon. In the exocrine portion, there were morphologically abnormal exocrine cells, which did not form a typical acinar structure. The cells lacked spatial polarity characteristics of acinar cells but expressed immunoreactive amylase, which was distributed diffusely in the cytoplasm. Plasma glucose concentration was normal in the transgenic mouse before and after the administration of glucose. The insulin content of the pancreas in transgenic and normal mice was nearly identical. These results suggest that activins or related ligands regulate the differentiation of the pancreatic endocrine and exocrine cells.

Activin Receptors, Type II↗

PubMed1999

Processing and juxtacrine activity of membrane-anchored betacellulin.

Betacellulin (BTC) was originally isolated as a secreted growth factor from a mouse pancreatic beta-tumor cell line, whereas the cDNA sequence predicts that BTC is synthesized as a larger transmembrane protein. In the present study, we have characterized the membrane-anchored forms of BTC, using Chinese hamster ovary (CHO) cells, mouse fibroblast A9 cells, and a human breast cancer cell line MCF-7, all of which were stably transfected with human BTC cDNA. A9 and MCF-7 transfectants produced membrane-anchored BTC isoforms of 21, 25, 29, and 40 kDa on the cell surface, as well as a secreted BTC isoform. CHO transfectants secreted little BTC but accumulated the membrane-anchored isoforms. The cleavage of the membrane-anchored forms to release a secreted from of BTC was not enhanced by biological mediators such as a phorbol ester, which stimulates the cleavage of other membrane-anchored growth factors. The membrane-anchored forms of BTC expressed on the transfected cells induced the insulin production and/or promoted the growth in subclones of AR42J rat pancreatic cells. These results suggest that the membrane-anchored BTC can function as a juxtacrine factor in regulating the growth and differentiation of pancreatic endocrine cells.

3T3 Cells↗

PubMed1999

Role of mitogen-activated protein kinase and phosphoinositide 3-kinase in the differentiation of rat pancreatic AR42J cells induced by hepatocyte growth factor.

AIMS/HYPOTHESIS: Pancreatic AR42J cells express both exocrine and neuroendocrine properties. When exposed to activin A, approximately 50 % of the cells die within 3 days by apoptosis. Addition of hepatocyte growth factor prevents apoptosis induced by activin A and induces differentiation into insulin-producing cells. The present study was conducted to examine the role of mitogen-activated protein kinase and phosphoinositide 3-kinase in the action of hepatocyte growth factor. METHODS: The role of mitogen-activated protein kinase was assessed by using 2-(2'-amino-3 '-methoxyphenol)-oxanaphthalen-4-one (PD098059). Cells were also transfected with cDNA for mitogen-activated protein kinase phosphatase and constitutively active mutant of mitogen-activated protein kinase kinase. RESULTS: Hepatocyte growth factor induced sustained activation of the mitogen-activated protein kinase, which was inhibited by PD098059. PD098059 completely blocked the differentiation and also blocked the prevention of apoptosis. Transfection of the cells with cDNA for mitogen-activated protein kinase phosphatase reproduced the effect of PD098059. Conversely, transfection with cDNA for the constitutively active mutant of mitogen-activated protein kinase kinase reproduced the effect of hepatocyte growth factor. In contrast, addition of wortmannin or transfection of the dominantly negative form of the p85 subunit of the phosphoinositide 3-kinase did not affect differentiation induced by hepatocyte growth factor. Instead, wortmannin enhanced the increase in the insulin content of the differentiated AR42J cells. CONCLUSION/INTERPRETATION: The MAP kinase pathway is necessary and sufficient for the action of HGF on differentiation of AR42J cells.

Activins↗

PubMed1998

Studies on the betacellulin receptor in pancreatic AR42J cells.

Betacellulin is a member of the epidermal growth factor family and converts pancreatic AR42J cells into insulin-producing cells. This study was conducted to characterise the receptor for betacellulin in AR42J cells. AR42J cells expressed two classes of binding sites for radioactive iodine labelled betacellulin, with Kd values of 4.6 x 10(-11) mol/l and 3.0 x 10(-10) mol/l. The binding of [125I]betacellulin was inhibited by unlabelled betacellulin in a dose-dependent manner, but epidermal growth factor was 50 fold less effective than betacellulin. Affinity cross-linking showed a [125I]betacellulin-binding protein with a molecular weight of approximately 180 KDa. When this protein was immunoprecipitated with antibody against epidermal growth factor receptors ErbB-1, ErbB-2, ErbB-3 or ErbB-4, it was immunoprecipitated only by the anti-ErbB-1 antibody. When the [125I]betacellulin-labelled proteins were immunoprecipitated with a combination of the four ErbB antibodies, and the unprecipitated proteins were then immunoprecipitated with anti-phosphotyrosine antibody, a 190 KDa protein was observed. Betacellulin induced the tyrosine phosphorylation of ErbB-1, ErbB-2 and ErbB-4. Finally, while 100 pmol/1 betacellulin converted all of the AR42J into insulin-producing cells in the presence of activin A, 10 nmol/l epidermal growth factor induced differentiation in only about 30 % of the cells. Higher concentrations of epidermal growth factor were less effective. Neu differentiation factor in the presence or absence of epidermal growth factor was ineffective. These results indicate that betacellulin binds to ErbB-1 and possibly another protein with a molecular weight of 190 KDa. The latter betacellulin-binding protein may be involved in the differentiation-inducing activity of betacellulin.

Animals↗

PubMed1998

Immediate onset of DNA synthesis in remnant rat liver after 90% hepatectomy by an administration of follistatin.

BACKGROUND/AIM: Follistatin is an antagonist of activins and is effective in promoting liver regeneration after 70% hepatectomy. To examine its efficacy under more critical conditions, we studied the effect of follistatin on liver regeneration in 90% hepatectomized rat. METHODS: Human recombinant follistatin was infused into the portal vein immediately after 90%, hepatectomy in 24-h-starved rats, and changes in the liver regeneration rate and nuclear bromodeoxyuridine labeling were measured. RESULTS: In control rats, nuclear labeling was first detected at 11 h of hepatectomy. In follistatin-treated rats, nuclear labeling was markedly increased at 3 h, and was significantly higher than that in control rats at 24, 72, 96, 120 and 144 h. The liver regeneration rate was significantly higher in follistatin-treated rats at 48, 72, 96, 120, 144 and 168 h. To determine the reason for the accelerated growth in starved rats, we compared the expression of mRNA for c-myc, p53, p21CIP1, p15INK4B, p27KIP1, and subunits of activins in fed and starved rats. mRNA for p21CIP1 and p15INK4B, but not p27KIP1 were decreased in 24 h-starved rats compared to the fed rats. mRNA for betaA subunit of activin was not detected in either fed or 24-h-starved rats, whereas that for betaC subunit was increased in starved rats. CONCLUSION: These results indicate that follistatin induces immediate onset of DNA synthesis in 90% hepatectomized rats and is quite effective in promoting liver regeneration.

Animals↗

PubMed1998

Activation of calcium-permeable cation channel by insulin in Chinese hamster ovary cells expressing human insulin receptors.

The present study was conducted to examine the ability of insulin receptor to activate the calcium signaling system in Chinese hamster ovary (CHO) cells expressing human insulin receptor (CHO-IR cells). In these cells, insulin evoked the elevation of cytoplasmic free calcium concentration, [Ca2+]c, measured by using fura-2. Insulin-induced increase in [Ca2+]c was blocked by reducing the extracellular calcium concentration to 1 microM or by adding nickel chloride, an inorganic inhibitor of calcium entry. Insulin did not elevate [Ca2+]c in parental CHO cells or in CHO cells expressing mutant insulin receptor lacking an ATP-binding site. When the transmembrane calcium current was measured by perforated whole-cell patch clamp, adding insulin to the bath solution markedly augmented the inward calcium current. In a cell-attached patch, a single channel activity appeared when insulin was included in the pipette. In contrast, insulin added outside the patch was ineffective. The current/voltage relationship demonstrated that insulin activated a voltage-independent calcium-permeable cation channel with a single-channel conductance of 10 pS. Exposing CHO-IR cells to pertussis toxin abolished the subsequent insulin effect on [Ca2+]c and activation of the calcium-permeable channel. Mastoparan activated the 10-pS calcium-permeable cation channel. In an inside-out patch, insulin activated the calcium-permeable channel when the bath solution contained both GTP and ATP. Nonhydrolyzable ATP could substitute for ATP. These results indicate that in CHO-IR cells, insulin elevates [Ca2+]c by activating the 10-pS calcium-permeable cation channel. Activation by the insulin receptor involves pertussis toxin-sensitive G protein.

Adenosine Triphosphate↗

PubMed1998

Antifungal activities of rapamycin and its derivatives, prolylrapamycin, 32-desmethylrapamycin, and 32-desmethoxyrapamycin.

The antifungal agent rapamycin is highly effective in inhibiting growth of yeast and mold strains. This study demonstrates that in liquid medium, rapamycin is more active than its derivatives (prolylrapamycin, 32-desmethylrapamycin, 32-desmethoxyrapamycin) against Candida albicans, Saccharomyces cerevisiae, and Fusarium oxysporum. All the rapamycins were more active than amphotericin B. Although four other molds were not inhibited in liquid medium, they were very sensitive to rapamycin and its derivatives when tested on agar. The latter assay showed that rapamycin is the most active and 32-desmethylrapamycin is more active than prolylrapamycin and 32-desmethoxyrapamycin. The conclusion of this study is that rapamycin is the most active antifungal agent of the compounds examined. The unexpected finding of high activity of rapamycin and its derivatives against filamentous fungi when assayed by the agar diffusion assay suggests that rapamycin or a derivative may hold promise for chemotherapy against pathogenic molds as well as yeasts.

Amphotericin B↗

PubMed1998

[Recent advances in growth factor receptor research].

Growth factors regulate cell growth, differentiation, migration, and apoptosis by stimulating the intrinsic protein kinase activities of their specific cell surface receptors. Most growth factors such as PDGF, EGF, FGF and IGF-I, bind to receptors with tyrosine kinase activity but members of the TGF-beta supergene family bine to those containing serine/threonine kinase activity. Recently, there has been substantial progress in the research field of growth factor receptors and their signaling mechanisms. This review briefly summerizes the tyrosine kinase receptors and also describes recent works in receptors for the TGF-beta supergene family and their signaling mechanisms.

Animals↗

PubMed1997

Subcellular distribution of GLUT4 in Chinese hamster ovary cells overexpressing mutant dynamin: evidence that dynamin is a regulatory GTPase in GLUT4 endocytosis.

We have investigated the subcellular distribution of GLUT4 by immunofluorescence microscopy after transfection with wild-type or mutant dynamin cDNA into Chinese hamster ovary cells expressing insulin receptor and GLUT4. In the basal state, GLUT4 was distributed exclusively within the cells in the cells overexpressing wild-type dynamin (CHOIR-GLUT4-WT) but was located at the cell surface in the cells overexpressing mutant dynamin (CHOIR-GLUT4-K44E). Insulin induced subcellular shift of GLUT4 to the cell surface in CHOIR-GLUT4-WT cells but had little effect in CHOIR-GLUT4-K44E cells. When insulin-stimulated cells were treated with wortmannin, GLUT4 was redistributed within the cells in CHOIR-GLUT4-WT cell, whereas it remained at the cell surface in CHOIR-GLUT4-K44E cell. These results suggest that dynamin is a regulatory GTPase in endocytosis of GLUT4.

Animals↗

PubMed1997

Regulation of the expression of follistatin in rat hepatocytes.

To elucidate the regulation of follistatin production in the liver, we studied changes in steady-state follistatin mRNA levels in cultured rat hepatocytes. Activin A stimulated follistatin mRNA levels in a time- and concentration-dependent manner. The stimulatory effect of activin A on follistatin mRNA was significant at 2 h, maximal at 6 h and declined thereafter. Incubating the cells with EGF increased follistatin mRNA levels at 48 h and later. The EGF-induced increase in follistatin mRNA was markedly inhibited by exogenous follistatin in the culture medium, which blocks the action of activin A synthesized in hepatocytes, suggesting that endogenous activin A at least partly mediated the effect of EGF. We also examined the effects of transforming growth factor-beta (TGF-beta), glucagon and alpha-adrenergic agonist, phenylephrine, on follistatin mRNA levels. TGF-beta increased the follistatin mRNA to levels similar to those caused by activin A. Phenylephrine and glucagon also increased follistatin mRNA levels but the effects were transient and weaker than those caused by activin A. Finally, follistatin mRNA levels were markedly increased in remnant liver 3 h after 70% hepatectomy. The mRNA remained elevated for up to 72 h. These results indicate that the expression of mRNA for follistatin is positively controlled by activin A, TGF-beta and other hormones or neurotransmitters. The stimulatory effect of EGF on follistatin mRNA is mediated by activin A released from hepatocytes.

Activins↗

PubMed1997

Insulin stimulates guanine nucleotide exchange on Rab4 via a wortmannin-sensitive signaling pathway in rat adipocytes.

Rab4, a member of the Rab family of Ras-related small GTP-binding proteins, has been shown to be associated with GLUT4-containing vesicles and implicated in the insulin action on glucose transport in rat adipocytes. In the present study, we investigated the insulin effects on the guanine nucleotide exchange on Rab4. In electrically permeabilized rat adipocytes, the amount of [35S]guanosine 5'-O-(3-thiotrisphosphate) (GTPgammaS) bound to Rab4 increased in a time-dependent manner during 45 min of the incubation period. Addition of insulin resulted in about a 2-fold stimulation of the binding of [35S]GTPgammaS to Rab4, indicating that insulin stimulated the guanine nucleotide exchange on the GTPase. Pretreatment of the cells with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, completely abolished the stimulatory effect of insulin on [35S]GTPgammaS binding to Rab4. Wortmannin also attenuated the nucleotide binding to Rab4 in the basal cells, suggesting that phosphatidylinositol 3-kinase activity may be essential for regulation of guanine nucleotide exchange on the GTPase and insulin may up-regulate the exchange activity by stimulating the lipid kinase. Insulin-induced subcellular redistribution of Rab4 from the microsomal fraction to the soluble fraction was also inhibited by wortmannin. These results suggest that insulin stimulates the guanine nucleotide exchange on Rab4 via a phosphatidylinositol 3-kinase-dependent signaling pathway and that Rab4 is one of possible targets of insulin action on intracellular vesicle traffic in rat adipocytes.

3-O-Methylglucose↗

PubMed1997

Activation of the calcium-permeable cation channel CD20 by alpha subunits of the Gi protein.

When the calcium-permeable cation channel CD20 is expressed in Balb/c 3T3 cells, it is activated by insulin-like growth factor-I (IGF-I) via the IGF-I receptor (Kanzaki, M., Nie, L., Shibata, H., and Kojima, I. (1997) J. Biol. Chem. 272, 4964-4969). The present study was conducted to investigate the role of G proteins in the regulation of the CD20 channel. In the excised patch clamp mode, activation of the CD20 channel by IGF-I required GTP, Mg2+, and ATP in the bath solution, and removal of either GTP or ATP attenuated the activation. Non-hydrolyzable ATP could substitute for ATP, and guanyl-5'-yl thiophosphate blocked the activation of the channel by IGF-I. The CD20 channel was also activated by guanosine 5'-3-O-(thio)triphosphate, and ATP was not required for the activation. Addition of a preparation of Gi/Go holoprotein purified from bovine brain activated the CD20, and the beta-adrenergic receptor kinase peptide did not affect the number of channel openings induced by the G protein. The CD20 channel was stimulated by the GTP-bound form of recombinant Gi2 alpha subunit purified from Sf9 cells. The Gi3 alpha subunit was less effective, and the Gi1 alpha subunit had no effect. Purified recombinant beta1gamma2 subunits did not affect the activity of the channel. Finally, IGF-I-induced activation of CD20 was inhibited by an antibody against Gi2 alpha subunit. These findings indicate that the CD20 channel expressed in Balb/c 3T3 cells is activated by the IGF-I receptor via the alpha subunits of heterotrimeric G proteins.

3T3 Cells↗

PubMed1997

Activation of a calcium-permeable cation channel CD20 expressed in Balb/c 3T3 cells by insulin-like growth factor-I.

CD20 functions as a calcium-permeable cation channel. When expressed in Balb/c 3T3 cells, CD20 accelerates the G1 progression induced by insulin-like growth factor-I (IGF-I). To further characterize how CD20 modulates the action of IGF-I, we investigated whether the activity of CD20 channel was affected by IGF-I. In quiescent cells expressing CD20, IGF-I increased cytoplasmic free calcium concentration, [Ca2+]c, which was reversed by the removal of extracellular calcium. In contrast, IGF-I did not increase [Ca2+]c in cells that did not express CD20. In perforated patch clamp recordings, addition of IGF-I to the bath solution augmented the Ca2+ permeability, which was reversed by anti-CD20 antibody. In cell-attached patch, calcium-permeable channel activity with unitary conductance of 7 picosiemens was detected, which was abolished by anti-CD20 antibody. The single channel activities were markedly enhanced when IGF-I was included in the pipette solution, whereas IGF-I added to the bath solution was ineffective. When cells were first exposed to pertussis toxin, activation of the channel by IGF-I was blocked. Transfection of cDNA for Gip2, a constitutive active form of alphai2, activated the CD20 channel. These results indicate that the CD20 channel is regulated by the IGF-I receptor by a mechanism involving pertussis toxin-sensitive G protein.

3T3 Cells↗

PubMed1997

Assessment of the role of activin A and transforming growth factor beta in the regulation of AML12 cell growth.

The present study was conducted to determine the role of two autocrine factors, activin A and transforming growth factor beta (TGF-beta), in the growth regulation of AML12 hepatocytes. We overexpressed truncated type II activin and/or TGF-beta receptors in AML12 cells. In AML12 cells overexpressing truncated type II activin receptors (AML-tAR cells), the inhibitory effect of activin A on DNA synthesis was completely blocked. AML-tAR cells proliferated faster than parental cells, both in the presence and absence of epidermal growth factor (EGF). However, AML-tAR cells could not grow in soft agar. Follistatin augmented EGF-induced DNA synthesis in AML12 cells, whereas it was ineffective in AML-tAR cells. In AML12 cells overexpressing truncated type II TGF-beta receptor (AML-tTR cells), the inhibitory effect of TGF-beta on DNA synthesis was blocked. AML-tTR cells proliferated faster than parental cells, both in the presence and absence of EGF, but at a slower rate than that of AML-tAR cells. AML-tTR cells did not grow in soft agar. The growth rate of cells overexpressing both types of truncated receptors was identical to that of AML-tAR cells, and these cells did not grow in soft agar. These results indicate that both activin A and TGF-beta act as autocrine inhibitors of DNA synthesis in AML12 cells, and that the blocking of the actions of two factors does not lead to transformation. Activin A is a predominant autocrine factor in these cells.

Activin Receptors↗

PubMed1997

Inhibition of proliferation of MCF-7 breast cancer cells by a blocker of Ca(2+)-permeable channel.

In MCF-7 breast cancer cells, insulin-like growth factor-1 (IGF-1) increased the calcium-permeability of the cells by activating a voltage-independent calcium-permeable channel. IGF-1 also induced oscillatory elevation of cytoplasmic free calcium concentration in these cells. An anti-allergic compound, tranilast, reduced the calcium-permeability augmented by IGF-1 in a dose-dependent manner and blocked the oscillatory elevation of cytoplasmic free calcium concentration. Tranilast did not affect early intracellular signals activated by IGF-1, including receptor autophosphorylation, activations of Ras, mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Tranilast inhibited increases in [3H]-thymidine incorporation, DNA content and cell number induced by IGF-1. The ID50 for [3H]-thymidine incorporation and DNA content were about 10 microM. The inhibitory effect of tranilast was reversible, and cell viability was not affected. Treatment with tranilast increased the number of cells in the G1 phase suggesting that this compound induced G1 arrest. Tranilast also reduced the phosphorylation of the retinoblastoma protein. These results indicate that tranilast inhibits the IGF-1-induced cell growth in MCF-7 cells by blocking calcium entry.

Breast Neoplasms↗

PubMed1997

Effect of nitrogen source on biosynthesis of rapamycin by Streptomyces hygroscopicus.

Six non-amino acid nitrogen compounds were examined as nitrogen source for growth of Streptomyces hygroscopicus and biosynthesis of rapamycin. Of the nitrogen sources studied, ammonium sulfate was the best with respect to formation of rapamycin, and supported cell growth comparable to the organic nitrogen sources used in the control chemically defined medium, i.e., aspartate, arginine plus histidine. In the new chemically defined medium, which is buffered with 200 mM 2-(N-morpholino)ethanesulfonic acid to prevent decline of pH during fermentation, an ammonium sulfate concentration of 40 mM was optimal for biosynthesis of rapamycin. Rapamycin production increased by more than 30% on both volumetric and specific bases as compared to the previous medium containing the three amino acids as nitrogen source.

Ammonium Sulfate↗

PubMed1997

Reciprocal expression of mRNA for inhibin betaC and betaA subunits in hepatocytes.

Messenger RNA expression of activin betaC subunit in the liver was compared with that for betaA subunit before and after 70% hepatectomy. mRNA for betaC was abundantly expressed in the liver but decreased at 12 h and later after 70% hepatectomy, whereas that for betaA was increased 12 h after the hepatectomy. We also compared the expression of mRNA for betaA and betaC in cultured rat hepatocytes. mRNA for betaC subunit was abundantly expressed in the beginning of the culture but was reduced gradually after stimulation with epidermal growth factor. In contrast, mRNA for betaA subunit was undetectable before stimulation and was increased 24 to 48 h after stimulation with the mitogen. These results indicate that expression of mRNA for betaC and betaA is regulated differently. The role of activin C may be different from that of activin A in the liver.

Animals↗

PubMed1996

A natural sulfated polysaccharide, calcium spirulan, isolated from Spirulina platensis: in vitro and ex vivo evaluation of anti-herpes simplex virus and anti-human immunodeficiency virus activities.

A sulfated polysaccharide named calcium spirulan (Ca-SP) has been isolated from a sea alga, Spirulina platensis, as an antiviral component. The anti-human immunodeficiency virus type 1 (HIV-1) and anti-herpes simplex virus type 1 (HSV-1) activities of Ca-SP were compared with those of dextran sulfate (DS) as a representative sulfated polysaccharide. Anti-HIV-1 activities of these agents were measured by three different assays: viability of acutely infected CD4-positive cells, or a cytopathology assay; determination of HIV-1 p24 antigen released into culture supernatants; and inhibition of HIV-induced syncytium formation. Anti-HSV-1 activity was assessed by plaque yield reduction. In addition, their effects on the blood coagulation processes and stability in the blood were evaluated. These data indicate that Ca-SP is a potent antiviral agent against both HIV-1 and HSV-1. Furthermore, Ca-SP is quite promising as an anti-HIV agent because even at low concentrations of Ca-SP an enhancement of virus-induced syncytium formation was not observed, as was observed in DS-treated cultures, Ca-SP had very low anticoagulant activity, and showed a much longer half-life in the blood of mice when compared with that of DS. Thus, Ca-SP can be a candidate agent for an anti-HIV therapeutic drug that might overcome the disadvantages observed in many sulfated polysaccharides. When the role of chelation of calcium ion with sulfate groups was examined by removing calcium or its replacement by sodium, the presence of calcium ion in the molecule was shown to be essential for the dose-dependent inhibition of cytopathic effect and syncytium formation induced by HIV-1.

Animals↗