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Biomedical subjects

K Takata

Publications and source records attributed to K Takata.

At least 19 recordsLinked to original sources
PubMed1997

Insulin receptor substrate (IRS)-2 is dephosphorylated more rapidly than IRS-1 via its association with phosphatidylinositol 3-kinase in skeletal muscle cells.

Insulin receptor substrate (IRS)-2 is structurally and functionally similar to IRS-1. Indeed, stimulation with insulin or insulin-like growth factor I led to the rapid tyrosine phosphorylation of both IRS-1 and IRS-2, which in turn activated phosphatidylinositol (PI) 3-kinase in L6 cells and rat skeletal muscle. However, IRS-2 was rapidly dephosphorylated (3-10 min after the addition of insulin/insulin-like growth factor I), whereas IRS-1 phosphorylation continued for at least 60 min. The time courses of the PI 3-kinase activity associated with IRS-1 and IRS-2 paralleled the tyrosine phosphorylation of these proteins. Preincubation with sodium orthovanadate, an inhibitor of protein tyrosine phosphatase, blocked the rapid dephosphorylation of IRS-2, suggesting the involvement of tyrosine phosphatase. The activation of PI 3-kinase apparently plays an important role in the rapid dephosphorylation of IRS-2, as IRS-2 dephosphorylation was inhibited markedly by suppressing PI 3-kinase activity with wortmannin or overexpression of the dominant negative p85 subunit of PI 3-kinase, which cannot bind the p110 catalytic subunit. In addition, platelet-derived growth factor stimulation prior to insulin stimulation decreased IRS-associated PI 3-kinase and significantly inhibited the dephosphorylation of IRS-2. Taken together, these observations suggest that IRS-2 plays a unique role in mediating the signals from the insulin receptor to downstream molecules and that this effect is more transient than that of IRS-1. Tyrosine phosphatase and IRS-associated PI 3-kinase activity thus contribute to the rapid dephosphorylation of IRS-2.

Androstadienes↗
PubMed1997

Proprotein-processing endoprotease furin controls the growth and differentiation of gastric surface mucous cells.

Gastric surface mucous cells originate from progenitor cells at the isthmus of the gastric gland, from where the cells migrate to the luminal surface. With migration they form secretory granules and express TGF alpha. We found that proprotein-processing endoprotease furin-positive cells were layered around the upper one fourth of the gastric glands of adult rats, whereas they were distributed along an outer epithelial layer in fetal rats. Because the furin-positive cell layer was localized from the upper cell proliferating zone to the less proliferating pit-cell region in the gastric gland unit, we examined the role of furin in the growth and differentiation of surface mucous cells by using the cell line, GSM06. This cell line is derived from the gastric surface mucous cells of transgenic mice harboring the temperature-sensitive simian virus 40 T antigen. At T antigen-active temperature (33 degrees C), the cells grew to confluency, whereas at T antigen-inactive temperature (39 degrees C), the cells ceased growing. At 33 degrees C, the cells exhibited a high level of furin expression with a negligible level of periodic acid Schiff (PAS)-positive materials and a low level of TGF alpha. In contrast, at 39 degrees C the cells produced a high level of PAS-positive materials, TGF alpha, and secretory granules, with a negligible level of furin expression. To further examine the role of furin, we established a GSM06 cell line introduced with either a sense or an antisense furin cDNA. The cells with sense furin expression produced fewer PAS-positive materials and a low level of TGF alpha even at 39 degrees C, whereas the cells with antisense furin expression exhibited more PAS-positive materials and TGF alpha even at 33 degrees C. When furin expression was suppressed by its antisense oligonucleotide, the cell growth was retarded with enhanced expression of the differentiated characteristics. Thus, we conclude that furin is instrumental in controlling the growth of the surface mucous cells.

Animals↗
PubMed1997

Neural activity affects distribution of glutamate receptors during neuromuscular junction formation in Drosophila embryos.

Changes in the distribution and density of transmitter receptors in the postsynaptic cell are required steps for functional synapse formation. We raised antibodies against Drosophila glutamate receptors (DGluR-II) and visualized the distribution of receptors during neuromuscular junction formation in embryos. In wild-type embryos, embryonic development is complete within 22 hr after egg lying (AEL) and neuromuscular junction (NMJ) formation begins at 13 hr AEL. At the time of initial synapse formation, DGluR-IIs appeared as clusters closely associated with some muscle nuclei. Subsequently, these nonjunctional clusters dispersed while DGluR-IIs accumulated at the junctional region. In a paralytic temperature-sensitive mutant, para(ts1), neural activity decreases drastically at restrictive temperatures. When neural activity was blocked throughout synaptogenesis by rearing embryos at a restrictive temperature prior to the beginning of synaptogenesis, 12 hr AEL, the dispersal of extrajunctional clusters was significantly suppressed and no accumulation of receptors at the junction was observed at 22 hr AEL. However, when neural activity was blocked later, by rearing embryos at a restrictive temperature from 13 hr AEL, DGluR-IIs did not accumulate at the NMJ, although extrajunctional clusters dispersed normally. These findings suggest that the neural activity differentially regulates dissipation of receptor clusters in the nonjunctional region and accumulation of receptors at the junctional region.

Amino Acid Sequence↗
PubMed1997

p85alpha gene generates three isoforms of regulatory subunit for phosphatidylinositol 3-kinase (PI 3-Kinase), p50alpha, p55alpha, and p85alpha, with different PI 3-kinase activity elevating responses to insulin.

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by association with a variety of tyrosine kinase receptors and intracellular tyrosine-phosphorylated substrates. We isolated a cDNA that encodes a 50-kDa regulatory subunit of PI 3-kinase with an expression cloning method using 32P-labeled insulin receptor substrate-1 (IRS-1). This 50-kDa protein contains two SH2 domains and an inter-SH2 domain of p85alpha, but the SH3 and bcr homology domains of p85alpha were replaced by a unique 6-amino acid sequence. Thus, this protein appears to be generated by alternative splicing of the p85alpha gene product. We suggest that this protein be called p50alpha. Northern blotting using a specific DNA probe corresponding to p50alpha revealed 6.0- and 2.8-kb bands in hepatic, brain, and renal tissues. The expression of p50alpha protein and its associated PI 3-kinase were detected in lysates prepared from the liver, brain, and muscle using a specific antibody against p50alpha. Taken together, these observations indicate that the p85alpha gene actually generates three protein products of 85, 55, and 50 kDa. The distributions of the three proteins (p85alpha, p55alpha, and p50alpha), in various rat tissues and also in various brain compartments, were found to be different. Interestingly, p50alpha forms a heterodimer with p110 that can as well as cannot be labeled with wortmannin, whereas p85alpha and p55alpha associate only with p110 that can be wortmannin-labeled. Furthermore, p50alpha exhibits a markedly higher capacity for activation of associated PI 3-kinase via insulin stimulation and has a higher affinity for tyrosine-phosphorylated IRS-1 than the other isoforms. Considering the high level of p50alpha expression in the liver and its marked responsiveness to insulin, p50alpha appears to play an important role in the activation of hepatic PI 3-kinase. Each of the three alpha isoforms has a different function and may have specific roles in various tissues.

Amino Acid Sequence↗
PubMed1997

Two isoforms of orthodenticle-related proteins (HpOtx) bind to the enhancer element of sea urchin arylsulfatase gene.

The sea urchin (Hemicentrotus pulcherrimus) arylsulfatase (HpArs) gene, expressed specifically in aboral ectoderm, contains a 229-bp enhancer in its first intron that is required for the activation of HpArs gene expression. Deletion analysis shows that a tandem repeat of orthodenticle-related protein (Otx) binding sites are responsible for the activity of this enhancer. Gel mobility shift analysis reveals that three types of Otx-proteins, which show different mobilities in gel shift assays, form complexes with the enhancer. Band I appears before hatching and gradually decreases by the gastrula stage. Band III appears at the blastula stage and Band II appears at the mesenchyme blastula stage; the levels of Band II and III remain constant until the gastrula stage. Two distinct types of HpOtx cDNA clones have been isolated from cDNA libraries of unfertilized eggs and gastrulae. Nucleotide sequences of the homeobox and downstream regions are well conserved in the two types of HpOtx cDNAs, while the region upstream from the homeobox has different nucleotide sequences. By genomic Southern blot analysis, only a single copy of HpOtx gene is detectable in the Hp genome, making it likely that two HpOtx isoforms are generated from the same gene. Results from Northern blot analysis confirm the presence of two types of HpOtx transcripts. Transcriptional regulation of the HpArs gene may, in part, be carried out through switching of Otx isoforms.

Amino Acid Sequence↗
PubMed1997

Serum concentrations of cortisone and cortisol in premature infants.

To determine the relationship between biological active cortisol and its inert metabolite cortisone accurately in premature infants, serum cortisone and cortisol concentrations were measured by reversed-phase high-performance liquid chromatography (HPLC) in a group of 232 premature infants and in a control group of 127 children and 88 adults. In the control group, serum cortisone concentrations were greater than serum cortisol levels during the first 2 months after birth; cortisol levels were higher than cortisone levels after 2 months of age. However, in premature infants, serum cortisone concentrations were greater than serum cortisol levels even after the first 2 months, and total concentrations of cortisone and cortisol were equal to those in controls. Results were then analyzed according to the equivalent gestational age of premature infants. Cortisone was predominant in premature infants older than 32 weeks of equivalent gestational age, but cortisol was higher than cortisone from equivalent gestational age 24 to 31 weeks. These findings suggest that the ability of premature infants to secrete glucocorticoids resembled that of normal controls. Also, the fetal zone of the cortex, which is associated with a predominance of cortisone, remained functional in premature infants for a longer time than in control infants. Our findings that in premature infants cortisone was predominant compared with cortisol and the sum of cortisone and cortisol was equal to that in the controls indicate that cortisone cannot be disregarded whenever the cortisol level is estimated, although cortisone itself is recognized to be biologically inactive. Simultaneous measurement of serum cortisone and cortisol concentrations is important when adrenocortical function is being determined, especially in premature infants.

Adolescent↗
PubMed1997

Transport of glucose across the blood-tissue barriers.

In specialized parts of the body, free exchange of substances between blood and tissue cells is hindered by the presence of a barrier cell layer(s). Specialized milieu of the compartments provided by these "blood-tissue barriers" seems to be important for specific functions of the tissue cells guarded by the barriers. In blood-tissue barriers, such as the blood-brain barrier, blood-cerebrospinal fluid barrier, blood-nerve barrier, blood-retinal barrier, blood-aqueous barrier, blood-perilymph barrier, and placental barrier, endothelial or epithelial cells sealed by tight junctions, or a syncytial cell layer(s), serve as a structural basis of the barrier. A selective transport system localized in the cells of the barrier provides substances needed by the cells inside the barrier. GLUT1, an isoform of facilitated-diffusion glucose transporters, is abundant in cells of the barrier. GLUT1 is concentrated at the critical plasma membranes of cells of the barriers and thereby constitutes the major machinery for the transport of glucose across these barriers where transport occurs by a transcellular mechanism. In the barrier composed of double-epithelial layers, such as the epithelium of the ciliary body in the case of the blood-aqueous barrier, gap junctions appear to play an important role in addition to GLUT1 for the transfer of glucose across the barrier.

Animals↗
PubMed1997

Immunohistochemical expression of glucose transporter-1 in human penile proliferative lesions.

Glucose transporters (GLUTs) are a family of membrane proteins responsible for the transport of glucose across cellular membranes. In terms of their mRNA levels, they have been reported to be expressed in some human tumours. However, the immunohistochemical localization of GLUTs in human urogenital lesions has rarely been studied. This study was performed to evaluate the expression of GLUT1 in penile proliferative lesions (18 cases of penile carcinoma and 13 cases of condyloma acuminatum). Using an isoform-specific anti-GLUT1 antibody, formalin-fixed paraffin-embedded sections were stained by the avidin-biotin complex method. In all cases of penile carcinoma, GLUT1 staining was diffusely recognized on the cell membrane of the carcinoma cells in the mainly infiltrating areas. However, the inner areas of the tumour were more weakly and focally stained. The intensity of staining for the penile carcinoma (staining score = 2.8 +/- 0.6) was stronger than that for condyloma acuminatum and that for adjacent non-proliferative areas. All cases of condyloma acuminatum showed a diffuse staining on the cell membrane in the basal and intermediate layers (staining score = 2.4 +/- 0.5). Non-proliferative (histologically normal) glans areas adjacent to the above lesions expressed the weakest GLUT1 staining only in the stratum basale (staining score = 1.8 +/- 0.5). These three areas showed significantly different staining scores from each other (p < 0.01). In conclusion, GLUT1 is expressed dominantly in penile proliferative lesions, especially in infiltrating areas of penile carcinoma.

Adult↗
PubMed1997

Mennen plate fixation for fractures of the femoral shaft after ipsilateral hip arthroplasty.

We performed osteosynthesis with the use of a Mennen plate for six patients with femoral fractures in the vicinity of the stem, which occurred after ipsilateral hip arthroplasty. The fixation was so favorable that postoperative deformity was slight. At 4 months after surgery, bone fusion was obtained in all patients, and their hip joint functions recovered to the preinjury level. For femoral fractures in the vicinity of the stem after ipsilateral arthroplasty, there are no suitable fixation methods other than treatment with the Mennen plate. Therefore, this method is recommended.

Adult↗
PubMed1997

DNA staining for fluorescence and laser confocal microscopy.

We examined five nucleic acid binding fluorescent dyes, propidium iodide, SYBR Green I, YO-PRO-1, TOTO-3, and TO-PRO-3, for nuclear DNA staining, visualized by fluorescence and laser confocal microscopy. The optimal concentration, co-staining of RNA, and bleaching speeds were examined. SYBR Green I and TO-PRO-3 almost preferentially stained the nuclear DNA, and the other dyes co-stained the cytoplasmic RNA. RNAse treatment completely prevented the cytoplasmic RNA staining. In conventional fluorescence microscopy, these dyes can be used in combination with fluorescence-labeled antibodies. Among the dyes tested, TOTO-3 and TO-PRO-3 stained the DNAs with far-red fluorescence under red excitation. Under Kr/Ar-laser illumination, TOTO-3 and TO-PRO-3 were best suited as the nuclear staining dyes in the specimens immunolabeled with fluorescein and rhodamine (or Texas red).

Animals↗
PubMed1997

Expression of thyrotropin-releasing hormone (TRH) receptor mRNA in somatotrophs in the rat anterior pituitary.

A response of growth hormone (GH) to thyrotropin-releasing hormone (TRH) is observed in lower mammals and patients with diseases such as a cromegaly, but not in normal subjects. We have previously demonstrated the existence of intact TRH receptor mRNA in GH-secreting adenoma. To examine whether intact somatotrophs in the anterior pituitary also express TRH receptor, we attempted to localize both TRHR mRNA and GH immunoreactivity simultaneously. In situ hybridization analysis revealed TRHR mRNAs specifically in the anterior pituitary, and 61.1% of the anterior pituitary cells expressed this transcript. Staining for GH and PRL on the same samples showed that the somatotrophs apparently expressed TRHR mRNA and approximately 62.3% and 30.9% of hybridization-positive cells were somatotorophs and mammotrophs, respectively. Moreover, TRHR mRNA level in the somatotrophs expressed as the number of silver grains per cell was equivalent to that in the mammotrophs. These findings demonstrated expression of the TRHR mRNA in somatotrophs in the rat anterior pituitary, and also showed that more than 50% of the TRHR mRNA detected in the anterior pituitary was derived from these cells.

Animals↗
PubMed1997

Targeting of GLUT1-GLUT5 chimeric proteins in the polarized cell line Caco-2.

Caco-2, a human differentiated intestinal epithelial cell line, is a promising model for investigating the mechanism of polarized targeting of apical and basolateral membrane proteins. We stably transfected rat GLUT5 cDNA and rabbit GLUT1 cDNA into Caco-2 cells with an expression vector. Immunohistochemical study revealed that the GLUT5 protein expressed was localized at apical membranes and that the GLUT1 expressed was present primarily in the basolateral membranes of cells grown on permeable support. Next, to investigate the domain responsible for determining apical vs. basolateral sorting in glucose transporters, we prepared several GLUT1-GLUT5 chimeric cDNAs and transfected them into Caco-2 cells. A GLUT1 [N terminus approximately sixth transmembrane domain (TM6)]-GLUT5 [intracellular loop (IL) approximately C terminus] chimera was observed exclusively at the apical membrane, while GLUT1 (N terminus approximately IL)-GLUT5 (TM7 approximately C terminus) and GLUT1 (N terminus approximately TM12)-GLUT5 (C-terminal domain) chimeras were observed mainly at the basolateral membrane, a localization similar to that of GLUT1. Moreover, using a recombinant adenovirus expression system, we expressed a GLUT5 (N terminus approximately TM6)-GLUT1(IL)-GLUT5(TM7 approximately C-terminus) chimera, which was observed at the basolateral membrane. Based on these results, the C-terminal domain does not determine isoform-specific targeting of GLUT1 and GLUT5. Rather, it is the intracellular loop in glucose transporters that appears to play a pivotal role in apical-basolateral sorting signals in Caco-2 cells.

Animals↗
PubMed1996

Modulatory role of drebrin on the cytoskeleton within dendritic spines in the rat cerebral cortex.

Morphological changes in the dendritic spines have been postulated to participate in the expression of synaptic plasticity. The cytoskeleton is likely to play a key role in regulating spine structure. Here we examine the molecular mechanisms responsible for the changes in spine morphology, focusing on drebrin, an actin-binding protein that is known to change the properties of actin filaments. We found that adult-type drebrin is localized in the dendritic spines of rat forebrain neurons, where it binds to the cytoskeleton. To identify the cytoskeletal proteins that associated with drebrin, we isolated drebrin-containing cytoskeletons using immunoprecipitation with a drebrin antibody. Drebrin, actin, myosin, and gelsolin were co-precipitated. We next examined the effect of drebrin on actomyosin interaction. In vitro, drebrin reduced the sliding velocity of actin filaments on immobilized myosin and inhibited the actin-activated ATPase activity of myosin. These results suggest that drebrin may modulate the actomyosin interaction within spines and may play a role in the structure-based plasticity of synapses.

Actins↗
PubMed1996

Vitamin D3 elicits calcium response and activates blood monocyte-derived macrophages from patients with vitamin D dependent rickets type II.

We studied the effects of vitamin D3 metabolites on intracellular free Ca2+ concentration ([Ca2+]i) and the respiratory burst of monocyte-derived macrophages (MDM) from patients with vitamin D dependent rickets type II. Treatment of MDM from the patients and healthy donors with 1 nM 1,25(OH)2D3 produced a rapid elevation of [Ca2+]i and similarly primed both types of cells for enhanced capacity for O2- release with phorbol diester. These results suggest that macrophages may have distinct non-genomic pathways of vitamin D3, which partly explain the absence of immunodeficiency and the disappearance of rickets after treatment with vitamin D3 in the patients.

24,25-Dihydroxyvitamin D 3↗
PubMed1996

Immuno-localization of H+/peptide cotransporter in rat digestive tract.

In the mammalian digestive tract, small peptides are absorbed by a H+-coupled peptide transport system. Using an antibody against the rat H+/peptide cotransporter (PepT1), we examined the localization of PepT1 immunohistochemically along the rat digestive tract. PepT1 was detected in the small intestine (duodenum, jejunum, and ileum), but not in the esophagus, stomach, colon, or rectum. PepT1 was especially enriched in the villi, where it was localized in the brush border of the absorptive epithelial cells. PepT1 was not detected in the mucus-secreting goblet cells or less-differentiated epithelial cells in the crypts. These observations show that PepT1 is specific to the brush border of the differentiated absorptive epithelial cells and suggest that H+-coupled uptake of small peptides and peptide-like drugs occurs at the apical membrane of these cells in the small intestine.

Animals↗
PubMed1996

Na(+)-dependent glucose transporter SGLT1 is localized in the apical plasma membrane upon completion of tight junction formation in MDCK cells.

SGLT1, an isoform of Na(+)-dependent glucose transporters, is localized at the apical plasma membrane in the epithelial cells of the small intestine and the kidney. In the present study we examined its location in SGLT1 cDNA-transfected MDCK cells, which form an epithelial sheet connected by tight junctions in culture. Formation of tight junctions was monitored by staining for occludin, an integral tight junction protein. In the cells demarcated by an uninterrupted occludin meshwork, SGLT1 was specifically localized at the apical plasma membrane, showing that SGLT1 has a signal to accomplish this restricted localization. In the cells with little or no occludin accumulation in the tight junction, however, SGLT1 was present along the entire aspect of the plasma membrane. Similar distribution of SGLT1 was observed in the cells as long as the occludin meshwork remained incomplete. These observations suggest that apical localization of SGLT1 occurs upon the completion of the uninterrupted meshwork of tight junctions.

Actins↗
PubMed1996

Connexin 43 and the glucose transporter, GLUT1, in the ciliary body of the rat.

To investigate the relationship between the gap junction protein connexin 43 and the glucose transporter GLUT1, their localization was visualized by double-immunofluorescence microscopy using frozen sections as well as immunogold staining of ultrathin frozen sections. In pigmented epithelial cells, most of the GLUT1 was localized along the plasma membrane facing the blood vessels, whereas in non-pigmented epithelial cells, it was present along the plasma membrane facing the aqueous humor. Connexin 43 was abundant in the ciliary body and localized mainly in the gap junctions connecting the pigmented and non-pigmented epithelial cells. Localization of GLUT1 and connexin 43 in the blood-aqueous barrier suggests that GLUT1, connexin 43, and GLUT1 disposed in this order could be a machinery responsible for the transport of glucose across the blood-aqueous barrier.

Animals↗