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Biomedical subjects

I Kojima

Publications and source records attributed to I Kojima.

At least 37 records · Page 2Linked to original sources
PubMed2001

Thermodynamics of the distribution of some carboxylic acids between organic solvents and a perchlorate solution.

The distribution of formic, benzoic and 4-methylbenzoic acids between a 0.1 mol dm(-3) (Na+, H+)ClO4- solution and octanol and carbon tetrachloride was studied at five different temperatures of 10 - 30 degrees C. The thermodynamic parameters for the transfer process of monomeric acid between two phases and for the dimerization of monomeric acid in carbon tetrachloride (deltaH and deltaS) were evaluated from the van't Hoff isochore. The free-energy change for the transfer of a methylene group from water to organic solvents was entropically controlled, irrespective of the organic solvents. The distribution constants of formic and benzoic acids were larger than those expected from the distribution constants of acetic and phenylacetic acids, respectively. The free-energy change for all solutes, except for benzoic and 4-methylbenzoic acids, was also entirely entropically controlled in the case of octanol as a solvent. The enthaly-entropy compensation for the dimerization constant of acids in carbon tetrachloride was observed.

Carboxylic Acids↗
PubMed2001

Changes in the expression of transcription factors in pancreatic AR42J cells during differentiation into insulin-producing cells.

Pancreatic AR42J cells possess both exocrine and neuroendocrine properties and convert to insulin-producing cells upon treatment with activin A and hepatocyte growth factor (HGF). We studied changes in the mRNA expression of various transcription factors during the course of differentiation. Among the transcription factors studied, expression levels of Pax4 and neurogenin3 changed significantly. These two factors were not detected in naive cells, whereas their mRNA levels were markedly increased after treatment with activin A and HGF. Thus, these two factors were induced by activin A. Transfection of Pax4 did not induce any changes in morphology or expression of pancreatic polypeptide (PP). Furthermore, introduction of antisense Pax4 did not affect the conversion into insulin-producing cells induced by activin A and HGF. In contrast, transfection of neurogenin3 induced morphological changes similar to those induced by activin A. In addition, transfection of neurogenin3 induced the expression of PP. Conversely, introduction of antisense neurogenin3 blocked the differentiation of AR42J cells induced by activin A and HGF. These results indicate that activin A regulates the expression of neurogenin3, which is critical for the differentiation of AR42J into endocrine cells.

Activins↗
PubMed2001

[Calcium-permeable cation channel activated by growth factors].

Calcium is required for cell-cycle progression. Growth factors which promote G(1) progression activate calcium-permeable cation channels and induce sustained increase in calcium entry. Inhibition of these channels blocks the action of growth factors. Despite of the physiological significance, little is known about the molecular nature of the growth factor-regulated channels. Recently, primary structure of GRC-1, one of growth factor-regulated channel, was identified.

English Abstract↗
PubMed2000

Regional expression of a gene encoding a neuron-specific Na(+)-dependent inorganic phosphate cotransporter (DNPI) in the rat forebrain.

We have analyzed expression of a gene encoding a brain-specific Na(+)-dependent inorganic phosphate cotransporter (DNPI), which was recently cloned from human brain, in rat forebrain using in situ hybridization. The expression of DNPI mRNA showed a widespread but highly heterogeneous pattern of distribution in the forebrain, where hybridization signals were observed in neurons but not in any other types of cells. Neurons expressing the mRNA were far more numerous in the diencephalon than in the telencephalon. In the thalamus, a number of neurons with high levels of signals were localized to all nuclei of the dorsal thalamus, habenular nuclei and subthalamic nucleus, but not the reticular nucleus and zona incerta. Moderate signal levels were seen in many neurons throughout the hypothalamus, particularly the ventromedial, paraventricular, supraoptic and arcuate nuclei, lateral hypothalamic area and mammillary complex. In contrast, expression of DNPI mRNA in the telencephalon was generally at a low level and occurred locally in some restricted regions within the neocortex, retrosplenial cortex, piriform cortex, olfactory regions, hippocampal formation and medial amygdaloid nucleus. The present results suggest that DNPI functions in heterogeneous neuron populations as a neuron-specific Na(+)-dependent inorganic phosphate cotransport system predominantly expressed in the diencephalon of the rat.

Animals↗
PubMed2000

Direct interaction of Rab4 with syntaxin 4.

In the present study, we examined the possible interaction between Rab4 and syntaxin 4, both having been implicated in insulin-induced GLUT4 translocation. Rab4 and syntaxin 4 were coimmunoprecipitated from the lysates of electrically permeabilized rat adipocytes. The interaction between the two proteins was reduced by insulin treatment and increased by the addition of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). An in vitro binding assay revealed that the bacterially expressed Rab4 was bound to a glutathione S-transferase fusion protein containing the cytoplasmic domain of syntaxin 4 (GST-syntaxin 4-(1-273)) but not to syntaxin 1A or vesicle-associated membrane protein-2. The interaction between Rab4 and syntaxin 4 seemed to be regulated by the guanine nucleotide status of Rab4, because 1) GTPgammaS treatment of the cells significantly increased, but guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) treatment decreased the amount of Rab4 pulled down with GST-syntaxin 4-(1-273) from the cell lysates; 2) GTPgammaS loading on Rab4 caused a marked increase in the affinity of Rab4 to syntaxin 4 whereas GDPbetaS loading had little effect; and 3) a GTPase-deficient mutant of Rab4 (Rab4(Q67L)), but not a GTP-binding-defective mutant (Rab4(S22N)), was bound to GST-syntaxin 4-(1-273). Although insulin stimulated [gamma-(32)P]GTP binding to Rab4 in a time-dependent fashion, its effect on the Rab4 interaction with syntaxin 4 was apparently biphasic; an initial increase in Rab4 associated with syntaxin 4 was followed by a gradual dissociation of the GTPase from syntaxin 4. Finally, the binding of Rab4(Q67L) to GST-syntaxin 4-(1-273) was inhibited by munc-18c in a dose-dependent manner, indicating that GTP-loaded Rab4 binds to syntaxin 4 in the open conformation. These results suggest that 1) Rab4 interacts with syntaxin 4 in a direct and specific manner, and 2) the interaction is regulated by the guanine nucleotide status of Rab4 as well as by the conformational status of syntaxin 4.

Adipocytes↗
PubMed2000

Actin filaments play a critical role in insulin-induced exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes.

Actin-based cytoskeletons have been implicated in insulin-stimulated glucose transport and translocation of the insulin-regulated glucose transporter, GLUT4, from the intracellular pool to the plasma membrane. However, most previous studies were done using adherent cell systems such as L6 myotubes and 3T3-L1 adipocytes, and very little information is available on the significance of the actin filaments to the insulin action in isolated adipocytes, a widely used experimental system. In the present study, we investigated the physiological role of actin filaments in the subcellular trafficking of GLUT4 in isolated rat adipocytes. We first compared the effects of two actin-disrupting reagents, latrunculin A and cytochalasin D, on the organization of the actin filaments as well as on the insulin action on glucose transport by laser confocal microscopy combined with biochemical analysis of the insulin action. Treatment of the cells with latrunculin A induced dose- and time-dependent disappearance of the filamentous actin, which correlated very well with inhibition of the insulin effect on glucose transport. Although cytochalasin D at 50 microM significantly inhibited insulin-stimulated glucose transport, it was not effective in disassembly of the actin filaments; rather, many intense punctate signals were observed in cytochalasin D-treated cells. In the actin-disrupted adipocytes treated with latrunculin A, insulin-induced GLUT4 translocation was inhibited completely. In addition, latrunculin A remarkably inhibited both insulin-induced glucose transport and GLUT4 translocation in the presense of D(k)-(62-85), a potent inhibitor of GLUT4 endocytosis, suggesting that intactness of the actin filaments was necessary for insulin-induced exocytosis of the GLUT4-containing vesicles. On the other hand, latrunculin A showed little inhibitory effect on either endocytosis of the trypsin-cleaved 35-kDa fragment of GLUT4 or decay of the glucose transport activity after addition of wortmannin in insulin-stimulated cells. The results of our experiment show clearly that, in rat adipocytes, (i) latrunculin A may be a more suitable tool than cytochalasin D for disruption of actin filaments, and (ii) actin filaments play a crucial role in exocytotic recruitment of GLUT4 to the plasma membrane from the intracellular pool, but not in its endocytosis.

Actins↗
PubMed2000

Number of glomeruli is increased in the kidney of transgenic mice expressing the truncated type II activin receptor.

Histological analyses of the kidney were performed in transgenic mice expressing the truncated type II activin receptor. In these mice, signaling through the activin receptor was attenuated. Size and wet weight of the kidneys were identical to those of normal mice. Histologically, the number of glomeruli was approximately 180% of that in normal mice. The sizes and shapes of the glomeruli were variable, but many of them were smaller than those in normal mice. Morphometrically, the total glomerular area was 130% of that of the normal mice. Abnormality of the epithelia in Bowman's capsule was observed and the number of tubular epithelial cells was increased in the transgenic mice. The serum levels of blood urea nitrogen, creatinine, and creatinine clearance were identical to those in normal mice. These results suggest that the action of activin or related ligands is critical for determination of the nephron number.

Activin Receptors, Type II↗
PubMed2000

Molecular cloning of a novel brain-type Na(+)-dependent inorganic phosphate cotransporter.

We have isolated a human cDNA encoding a protein, designated DNPI, that shows 82% amino acid identity and 92% similarity to the human brain-specific Na(+)-dependent inorganic phosphate (Na(+)/P(i)) cotransporter (BNPI), which is localized exclusively to neuron-rich regions. Expression of DNPI mRNA in Xenopus oocytes resulted in a significant increase in Na(+)-dependent P(i) transport, indicating that DNPI is a novel Na(+)/P(i) cotransporter. Northern blot analysis shows that DNPI mRNA is expressed predominantly in brain, where the highest levels are observed in medulla, substantia nigra, subthalamic nucleus, and thalamus, all of which express BNPI mRNA at low levels. In contrast, DNPI mRNA is expressed at low levels in cerebellum and hippocampus, where BNPI mRNA is expressed at high levels. No hybridizing signal for DNPI mRNA is observed in the glia-rich region of corpus callosum. In other regions examined, both mRNAs are moderately or highly expressed. These results indicate that BNPI and DNPI, which coordinate Na(+)-dependent P(i) transport in the neuron-rich regions of the brain, may form a new class within the Na(+)/P(i) cotransporter family.

Animals↗
PubMed2000

Hepatocyte growth factor induces branching tubulogenesis in MDCK cells by modulating the activin-follistatin system.

BACKGROUND: The activin-follistatin system is expressed in tubular cells of the kidney. The present study was conducted to examine the role of the activin-follistatin system in tubulogenesis using Madin-Darby canine kidney (MDCK) cells as a model system. METHODS: Tubulogenesis was assessed using MDCK cells cultured in collagen gel. The effect of recombinant human activin A on tubulogenesis was examined. Blockade of the action of endogenous activin was achieved by either adding follistatin or transfection of dominant-negative mutant of the type II activin receptor. The production of activin A was examined by Northern blotting, in situ hybridization, and Western blotting. RESULTS: MDCK cells expressed mRNA for the betaA subunit of activin. These cells formed spherical cysts when cultured in collagen gel. Hepatocyte growth factor (HGF) added to the spherical cysts induced branching tubulogenesis. When activin A was added together with HGF, activin A blocked the branching tubulogenesis induced by HGF, and the activin-treated cells were scattered. Conversely, follistatin, an antagonist of activin A, induced branching tubulogenesis qualitatively similar to that induced by HGF. Adenovirus vector-mediated transfer of the gene encoding truncated type II activin receptor, which acts as a dominant negative mutant, also induced branching tubulogenesis. Finally, HGF markedly inhibited the production of activin A in MDCK cells cultured in collagen gel. CONCLUSION: Activin A produced in MDCK cells tonically inhibits branching tubulogenesis, and HGF induced branching tubulogenesis mainly by blocking the production of activin A.

Activins↗
PubMed2000

The role of activin and transforming growth factor-beta in the regulation of organ mass in the rat liver.

The present study was conducted to assess the role of activin(s) in the regulatory mechanism to maintain constant liver mass. To this end, we infused follistatin, an activin antagonist, into the portal vein of the rat. Follistatin induced DNA synthesis, as assessed by bromodeoxy uridine labeling, in intact livers. Small peaks of bromodeoxy uridine labeling were observed after 3 and 18 hours of infusion, and a large peak was observed after 48 hours. In follistatin-treated rats, the DNA content of the liver was significantly elevated after 72 hours and returned to the basal value within 120 hours. Likewise, liver weight increased significantly after 60 and 72 hours, but returned to the control value within 120 hours. Apoptosis of hepatocytes, assessed by the Tdt-mediated, dUTP-biotin nick end labeling method was observed after 72 hours or later. Messenger RNA (mRNA) expression of hepatocyte growth factor, transforming growth factor-alpha, tumor necrosis factor-alpha, and interleukin-6 did not increase after the addition of follistatin. The mRNA expression and immunoreativity of transforming growth factor-beta increased after the administration of follistatin. These results suggest that the blockade of activin action leads to the initiation of DNA synthesis in the intact liver. Activins may tonically inhibit hepatocyte growth in the intact liver. Transforming growth factor-beta may also act to maintain constant liver mass when activin action is blocked.

Activins↗
PubMed2000

Purification and characterization of 2,6-beta-D-fructan 6-levanbiohydrolase from Streptomyces exfoliatus F3-2.

Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a beta-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60 degrees C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50 degrees C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only beta-2,6-linkage of levan, but also beta-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-beta-D-fructan 6-levanbiohydrolase (EC 3.2.1.64).

Disaccharides↗
PubMed1999

Molecular identification of a eukaryotic, stretch-activated nonselective cation channel.

Calcium-permeable, stretch-activated nonselective cation (SA Cat) channels mediate cellular responses to mechanical stimuli. However, genes encoding such channels have not been identified in eukaryotes. The yeast MID1 gene product (Mid1) is required for calcium influx in the yeast Saccharomyces cerevisiae. Functional expression of Mid1 in Chinese hamster ovary cells conferred sensitivity to mechanical stress that resulted in increases in both calcium conductance and the concentration of cytosolic free calcium. These increases were dependent on the presence of extracellular calcium and were reduced by gadolinium, a blocker of SA Cat channels. Single-channel analyses with cell-attached patches revealed that Mid1 acts as a calcium-permeable, cation-selective stretch-activated channel with a conductance of 32 picosiemens at 150 millimolar cesium chloride in the pipette. Thus, Mid1 appears to be a eukaryotic, SA Cat channel.

Amino Acid Sequence↗
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↗