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K Yoshizato

Publications and source records attributed to K Yoshizato.

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

Thyroid hormone response element-like sequence in anuran matrix metalloproteinase 1 gene is responsive to in vivo thyroid hormone administration.

Gene (Rmmp1) of matrix metalloproteinase 1 (MMP1) of the bullfrog, Rana catesbeiana, has been shown previously to contain a thyroid hormone response element (TRE)-like sequence in its 5'-upstream region. The present study aimed to determine whether this TRE-like sequence is functional in vivo as a true TRE, and to characterize the sequences of the 5'-upstream region with respect to the regulation of the activity of the TRE when the TRE-like sequence was proved to be a true TRE. With this aim, various sequences of TRE-like sequence-containing 5'-upstream region were constructed and fused to the enhanced green fluorescent protein gene as a reporter gene. The fusion constructs were bombarded to the skin of bullfrog tadpoles and the activity of the TRE was quantitatively determined by measuring increased intensities of fluorescence when the animals were exposed to thyroid hormone. The present study clearly demonstrated that the sequence of Rmmp1 is a biologically active TRE in vivo. In addition, a unique 36 bp long sequence directly flanked to the 3'-end of the TRE was identified which worked co-operatively with TRE to regulate the transcriptional promoter activity. It should be emphasized that the presence of TRE in the Rmmp1 gene is unique, because its presence has not been reported in the known promoter region of vertebrate MMPs.

Animals↗
PubMed2001

[Characterization of a highly proliferative population of adult hepatocytes and its use for tissue engineering].

It is well known among cell biologists that normal hepatocytes of adult mammals are difficult to replicate repeatedly in vitro, irrespective of the fact that these cells can grow well clonally in vivo. We developed a culture medium (HCGM) wherein the normal hepatocytes of adult Fischer rats replicate repeatedly and form clonal colonies. This growth requires the presence of hepatic stellate cells (HSCs). Fractionation and separation of hepatocytes by a combination of centrifugation and cell sorting revealed the presence of a highly proliferative population of hepatocytes in the adult liver, called small-sized hepatocytes (SHs-R3). SHs-R3 showed a 3- to 4-fold higher growth potential than large-sized hepatocytes (SHs-R2) both in vitro and in vivo. The in vivo growth potential was estimated using the retrorsine-dipeptidylpeptidase IV (DPPIV)--rat model in which DPPIV-positive SHs-R3 were transplanted into the liver of retrorsine-treated DPPIV-negative mutant rats which were then subjected to two-thirds partial hepatectomy. The results of our studies suggest a close relationship between SHs-R3 and small hepatocyte-like progenitor cells, as reported by Gordon et al. We showed that the liver of adult humans also contains a highly proliferative population of hepatocytes, which possibly corresponds to SHs-R3. Research is now being undertaken to utilize this population of human hepatocytes to develop an artificial liver.

Animals↗
PubMed2000

Kdap, a novel gene associated with the stratification of the epithelium.

The skin develops and differentiates during embryogenesis, which is concertedly regulated by a variety of genes. The present study isolated from the rat embryonic skin a novel differentiation-associated gene named Kdap (keratinocyte differentiation-associated protein) by suppression subtractive hybridization between the skin of 14day postcoitus (dpc) embryo (the prehair-germ stage) and that of 17dpc embryo (the hair-germ stage). Its mRNA contained four spliced forms in these tissues. The gene encoded a protein of total 98 amino acids with a calculated molecular mass of 11kDa and an isoelectric point of 6.1 as an unspliced form. The two splicing zones were well conserved among rat, mouse, and human. This protein had a high hydrophobic N-terminal region, a possible signal sequence, and contained two putative N-myristoylation sites and two casein kinase II phosphorylation sites. In situ hybridization experiments detected Kdap transcripts exclusively in the suprabasal cell layers of the embryonic epidermis. Intense expression was also seen in suprabasal cells in regions of infundibulum of the hair follicle. These results indicated that Kdap provides a new insight into the mechanism of differentiation and the maintenance of stratified epithelia.

Amino Acid Sequence↗
PubMed2000

Highly efficient electro-gene therapy of solid tumor by using an expression plasmid for the herpes simplex virus thymidine kinase gene.

We report successful electro-gene therapy (EGT) by using plasmid DNA for tumor-bearing mice. Subcutaneously inoculated CT26 tumor was subjected to EGT, which consists of intratumoral injection of a naked plasmid encoding a marker gene or a therapeutic gene, followed by in vivo electroporation (EP). When this treatment modality is carried out with the plasmid DNA for the green fluorescent protein gene, followed by in vivo EP with the optimized pulse parameters, numerous intensely bright green fluorescent signals appeared within the tumor. EGT, by using the "A" fragment of the diphtheria toxin gene significantly inhibited the growth of tumors, by about 30%, on the flank of mice. With the herpes simplex virus thymidine kinase gene, followed by systemic injection of ganciclovir, EGT was far more effective in retarding tumor growth, varying between 50% and 90%, compared with the other controls. Based on these results, it appears that EGT can be used successfully for treating murine solid tumors.

Animals↗
PubMed2000

Mass spectrometric approaches for the characterization of proteins on a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer.

This study demonstrates structural and conformational characterization of proteins by nanoflow electrospray ionization (nanoESI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) utilizing a quadrupole time-of-flight (Q-TOF) mass spectrometer (Micromass, Manchester, England). Model peptides were successfully sequenced at the 35 attomole (amol) level, and peptides derived from a tryptic in-gel digest of 25 femtomole (fmol) bovine serum albumin (BSA) were successfully sequenced. The results demonstrated that the MS/MS sensitivity of the Q-TOF clearly surpassed the detection limit of the silver stain. A silver destaining step greatly improved the mass analysis of peptides derived from in-gel digests. Interestingly, sequence analysis revealed BSA residue 424 (tyrosine) as a potential chlorination site. In addition, a modified procedure was successfully used to extract and measure the masses of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE)-resolved proteins in the 10-68.5 kDa range. The Q-TOF was also used to monitor conformational changes of proteins. These experiments demonstrated an acid-induced denaturation of BSA in the pH 3-4 range, and heat-induced unfolding of cytochrome c between 50 and 60 degrees C. Finally, Zn2+ binding was demonstrated for the carbonic anhydrase apoprotein. In summary, the wide range of applications and the high quality of the experimental data made the Q-TOF mass spectrometer a powerful analytical tool for protein characterization.

Animals↗
PubMed2000

Heterogeneity of growth potential of adult rat hepatocytes in vitro.

Nearly pure populations of small hepatocytes (SHs), parenchymal hepatocytes (PHs), and nonparenchymal cells (NPCs) were prepared from the adult rat, and cocultures of hepatocytes and NPCs were reconstituted from them first to obtain the direct evidence that NPCs promote the growth of hepatocytes and second to compare the growth potential between SHs and PHs. SHs and PHs underwent multiple divisions when cocultured with NPCs, whereas neither SHs nor PHs formed colonies at 10 days when cultured alone. Stellate cells in the NPCs were shown to be responsible for this growth promotion. SHs showed a higher growth capacity than PHs. To clearly show the relationship between the growth potential and the size of hepatocytes, SHs and PHs were further fractionated by a fluorescence-activated cell sorter, because the size distribution of SHs and PHs was half overlapped. SHs produced 2 cell populations, SH-R2 and SH-R3. The former showed a greater extent of granularity and autofluorescence than the latter. In contrast, PHs produced only 1 population (PH-R2), which corresponded to the SH-R2. The size of hepatocytes of SH-R3 was smaller (17.1 +/- 0.2 microm) than those of SH-R2 (22.6 +/- 0.5 microm) and PH-R2 (24.1 +/- 0.1 microm) and there was not a significant overlap in the size distribution between the 2 groups. The hepatocytes of SH-R3 were highly replicative and 4 or 5 times higher in their growth potential than those of SH-R2 and PH-R2. We concluded that the growth potential of hepatocytes is heterogeneous and is correlated with their size and the extent of their granularity and autofluorescence.

Animals↗
PubMed2000

Bradykinin enhances insulin receptor tyrosine kinase in 32D cells reconstituted with bradykinin and insulin signaling pathways.

We have previously shown that bradykinin potentiated insulin-induced glucose uptake through GLUT4 translocation in canine adipocytes and skeletal muscles. The aim of this study was to determine the molecular mechanism of bradykinin enhancement of the insulin signal. For this purpose, 32D cells, which express a limited number of insulin receptors and lack endogenous bradykinin B2 receptor (BK2R) or insulin receptor substrate (IRS)-1 were transfected with BK2R cDNA and/or insulin receptor cDNA and/or IRS-1 cDNA, and analyzed. In 32D cells that expressed BK2R and insulin receptor (32D-BKR/IR), bradykinin alone had no effect on the phosphorylation of the insulin receptor, but it enhanced insulin-stimulated tyrosine phosphorylation of the insulin receptor. In 32D cells that expressed BK2R, insulin receptor and IRS-1 (32D-BKR/IR/IRS1), bradykinin also enhanced insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1. An increase in insulin-stimulated phosphorylation of IRS-1 by treatment with bradykinin in 32D-BKR/IR/IRS1 cell was associated with increased binding of 85 kD subunit of phosphatidylinositol 3 (PI 3)-kinase and increased IRS-1 associated PI 3-kinase activity. These effects of bradykinin were not observed in 32D cells which lack the expression of BK2R (32D-IR/IRS1) or insulin receptor (32D-BKR/IRS1). Furthermore, tyrosine phosphatase activity against insulin receptor beta-subunit in plasma membrane fraction of 32D-BKR/IR cells was significantly reduced by bradykinin, suggesting that the effect of bradykinin was in part mediated by inhibition of protein tyrosine phosphatase(s). Our results clearly demonstrated that bradykinin enhanced insulin-stimulated tyrosine kinase activity of the insulin receptor and downstream insulin signal cascade through the BK2R mediated signal pathway.

Animals↗
PubMed2000

Developmentally and regionally regulated participation of epidermal cells in the formation of collagen lamella of anuran tadpole skin.

We investigated the cellular mechanism of formation of subepidermal thick bundles of collagen (collagen lamella) during larval development of the bullfrog, Rana catesbeiana, using cDNA of alpha1(I) collagen as a probe. The originally bilayered larval epidermis contains basal skein cells and apical cells, and the collagen lamella is directly attached to the basement membrane. The basal skein cells above the collagen lamella and fibroblasts beneath it intensively expressed the alpha1(I) gene. As the skin developed, suprabasal skein cells ceased expression of the gene. Concomitantly, the fibroblasts started to outwardly migrate, penetrated into the lamella and formed connective tissue between the epidermis and the lamella. These fibroblasts intensively expressed the gene. As the connective tissue developed, the basal skein cells ceased to express the gene and were replaced by larval basal cells that did not express the gene. These dynamic changes took place first in a lateral region of the body skin and proceeded to all other regions except the tail. Isolated cultured skein cells expressed the gene and extracellularly deposited its protein as the type I collagen fibrils. Thus, it is concluded that anuran larval epidermal cells can autonomously and intrinsically synthesize type I collagen.

Animals↗
PubMed2000

Proteome analysis of rat hepatic stellate cells.

Proteome analysis was performed on cellular and secreted proteins of normal (quiescent) and activated rat hepatic stellate cells. The stellate cells were activated either in vitro by cultivating quiescent stellate cells for 9 days or in vivo by injecting rats with carbon tetrachloride for 8 weeks. A total of 43 proteins/polypeptides were identified, which altered their expression levels when the cells were activated in vivo and/or in vitro. Twenty-seven of them showed similar changes in vivo and in vitro, including up-regulated proteins such as calcyclin, calgizzarin, and galectin-1 as well as down-regulated proteins such as liver carboxylesterase 10 and serine protease inhibitor 3. Sixteen of them showed different expression levels between in vivo and in vitro activated stellate cells. These results were reproducibly obtained in 3 independent experiments. The up-regulation of calcyclin, calgizzarin, and galectin-1, as well as the down-regulation of liver carboxylesterase 10 were directly confirmed in fibrotic liver tissues. Northern blots confirmed up-regulation of the messenger RNAs (mRNAs) of calcyclin, calgizzarin, and galectin-1 in activated stellate cells, indicating that these changes were controlled at the mRNA level. In addition a list compiling over 150 stellate cell proteins is presented. The data presented here thus provide a significant new protein-level insight into the activation of hepatic stellate cells, a key event in liver fibrogenesis.

Animals↗
PubMed2000

Involvement of bradykinin in acute exercise-induced increase of glucose uptake and GLUT-4 translocation in skeletal muscle: studies in normal and diabetic humans and rats.

Acute exercise induces glucose uptake in skeletal muscle in vivo, but the molecular mechanism of this phenomenon remains to be identified. In this study, we evaluated the involvement of bradykinin in exercise-induced glucose uptake in humans and rats. In human studies, plasma bradykinin concentrations increased significantly during an ergometer exercise (20 minutes) in 8 healthy normoglycemic subjects and 6 well-controlled type 2 diabetic patients (mean hemoglobin A1c [HbA1c], 6.4% +/- 0.6%), but not in 6 poorly controlled type 2 diabetics (mean HbA1c, 11.6% +/- 2.6%). In rat studies, plasma bradykinin concentrations also significantly increased after 1 hour of swimming in nondiabetic and mildly diabetic (streptozotocin [STZ] 45 mg/kg intravenously [IV]) rats, but not in rats with severe diabetes (STZ 65 mg/kg IV). Glucose influx (maximum velocity [Vmax]) and GLUT-4 translocation in skeletal muscle of nondiabetic rats significantly increased after 1 hour of swimming, but these increases were abrogated by subcutaneous infusion of bradykinin B2 receptor antagonist HOE-140 (400 microg x kg(-1) x d(-1)). Insulin-stimulated tyrosine phosphorylation and phosphatidylinositol (PI) 3-kinase activity in response to insulin injection (20 U/kg IV) in the portal vein were significantly attenuated in exercised rats pretreated with HOE-140 compared with saline-treated exercised rats. Our results suggest that plasma bradykinin concentrations increase in response to acute exercise and this increase is affected by blood glucose status in diabetic patients. Moreover, the exercise-induced increase in bradykinin may be involved in modulating exercise-induced glucose transport through an increase of GLUT-4 translocation, as well as enhancement of the insulin signal pathway, during the postexercise period in skeletal muscle, resulting in a decrease of blood glucose.

Adult↗
PubMed2000

Gene delivery with optimized electroporation parameters shows potential for treatment of gliomas.

Electroporation, a standard laboratory method of introducing exogenous molecules into cells, has been gaining importance as a very effective non-viral physical technique of gene delivery. In this study, we have used subcutaneous model of the C6 rat glioma cells and established an optimal condition to obtain very high gene expression in tumor tissues using both reporter and functional genes. Tumors grown on the flanks of Wistar rats are exposed and directly injected with plasmid DNA having the constructs of luciferase, green fluorescent protein and, the fragment of the diphtheria toxin, DT-A. The tumors are then subjected to square wave pulses from an electroporator. Gene expression is found to be several orders of magnitude higher when the tumors are pulsed with the optimized electrical parameters compared to the controls. For luciferase, the enhancement is approximately 135-fold, for the green fluorescent protein, gene expression is seen over a wide area within the sections examined, as contrast to a few punctate dots in the control specimens, and finally, DT-A shows massive death in the tumor tissue. A special circular array of six needles through which pulses are delivered with rotating electric field is found to be highly efficient in transferring genes inside the tumor. Direct injection of plasmid DNA followed by electroporation allows very high in vivo gene transfer and its subsequent expression into tumor tissues. This method may be applicable to any solid tumor.

Animals↗
PubMed2000

Cerebral function after aortic surgery using retrograde cerebral perfusion: report of three cases.

INTRODUCTION: The neurological analysis undertaken on three cases of aortic aneurysm treated surgically using retrograde cerebral perfusion (RCP) are reported herein. This is the first detailed analysis of postoperative neurological studies in such cases. METHODS: The oxygenation state of cerebral Hb and cytochrome aa3 levels were monitored by near-infrared spectroscopy (NIR) during RCP. Postoperative neurological scoring, computed tomography (CT), magnetic resonance imaging (MRI), and monitoring of local cerebral blood flow (LCBF) quantatively by N-isopropyl-p-[123I] iodoamphetamine (IMP) single-photon emission computed tomography (SPECT) were performed for an extended follow-up period after surgery. RESULTS: The T2WI of MRI findings which consisted of many spotty high intensity areas in the striatum, cerebellum and hippocampus, may reflect the postoperative severe consciousness and mental disturbances (2-4 weeks). However, in all cases the neurological deficit gradually improved until they returned to their normal preoperative states. The improvement of LCBF correlated well with neurological recovery. CONCLUSION: The conditions of the conventional method of RCP must be improved to prevent transient severe consciousness and mental disturbances which occur after the operation in relationship with the appearance of the high intensity area on the MRI T2WI.

Adult↗
PubMed1999

A long-term culture of human hepatocytes which show a high growth potential and express their differentiated phenotypes.

The present study succeeded for the first time in cultivating for more than 2 months human normal hepatocytes which showed a high growth potential and expressed their differentiated phenotypes. Constituents of culture medium were critical for this culture, and the medium optimized for their growth contained fresh human serum, fetal bovine serum, Swiss 3T3-cell conditioned medium, L-ascorbic acid 2-phosphate, epidermal growth factor, nicotinamide, and dimethyl sulfoxide. Hepatocytes steadily replicated and formed colonies which continued to increase in size up to around 35 days. The number of hepatocytes in the most replicative colonies increased 17-fold during 31 days. Cells in colonies expressed normal differentiated hepatocytic phenotypes for as long as 35 days. These hepatocytes retained normal liver functions at least for 70 days such as to secrete albumin, and to metabolize lidocaine and D-galactose.

3T3 Cells↗
PubMed1999

Pleiotrophin as a Swiss 3T3 cell-derived potent mitogen for adult rat hepatocytes.

Rat parenchymal liver cells were cultured in the presence of lethally treated Swiss 3T3 cells. This co-culture allowed hepatocytes to produce colonies containing more than 300 cells in 30 days. Hepatocytes in colonies appeared morphologically normal and some of them were suggested to have bipotental differentiation capacity. The initial growth stimulatory activity of the feeder cells was replaceable with their conditioned medium (CM). Biochemical analysis of an active principle in the 3T3 cell-CM identified pleiotrophin. Pleiotrophin purified from the 3T3 cell-CM, recombinant human pleiotrophin, chemically synthesized human pleiotrophin, and midkine promoted the growth of hepatocytes as well. Reverse transcription-polymerase chain reaction clearly showed that the synthesis of mRNA of pleiotrophin was stimulated in the regenerating liver induced by either partial hepatectomy or the treatment with d-galactosamine, strongly suggesting a biological significance of pleiotrophin in the proliferation of hepatocytes in vivo. From these results we concluded that pleiotrophin is a new potent growth factor for adult parenchymal hepatocytes. This study indicates the importance of mesenchymal stimulation for the growth of adult rat hepatocytes.

3T3 Cells↗
PubMed1999

Expression of genes of type I and type II collagen in the formation and development of the blastema of regenerating newt limb.

We cloned cDNAs of alpha1(I) and alpha1(II) collagen, and studied their expression profiles in regenerating limbs of newts, Cynops pyrrhogaster. The expression of the alpha1(I) gene was markedly up-regulated at the early bud stage of the blastema. In situ hybridization experiments revealed that the alpha1(I) gene was expressed in not only mesenchymal cells of the blastema, but also the basal cells of the wound epidermis at the wound healing stage when the epidermal basement membrane was absent. This unique expression continued until 21 days (late bud stage), while the basement membrane began to form at 14 days. These results indicate biochemical differences between the wound and normal epidermis, and suggest the direct involvement of the former in the synthesis of blastemal matrices of type I collagen. Actually, immunohistochemistry revealed that type I collagen began to be deposited beneath the wound epidermis at 8 days, and accumulated there and around blastemal mesenchymal cells at 14 to 21 days. Undifferentiated mesenchymal cells associated with the amputated muscle fibers actively expressed the alpha1(I) gene. Mesenchymal cells in the central region of blastemas deposited type I collagen fibers around them. Concomitantly with the appearance of prechondrocytes, the alpha1(II) collagen gene became activated. The present study clearly shows that the expression of the genes of both type I and type II collagen in blastemal cells is temporally and regionally well-regulated in a cooperative manner. Dev Dyn 1999;216:59-71.

Amino Acid Sequence↗
PubMed1999

Insulin inhibits glucagon secretion by the activation of PI3-kinase in In-R1-G9 cells.

Intracellular mechanisms through which insulin inhibits glucagon secretion remain to be elucidated in glucagon secreting cells. In this study, we confirmed that, in In-R1-G9 cells, a pancreatic alpha cell line, insulin stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) and activated phosphatidylinositol 3-kinase (PI3-kinase). We further studied, using wortmannin, an inhibitor of PI3-kinase, whether the inhibitory effect of insulin on glucagon secretion was mediated through PI3-kinase pathway in these cells. In static incubation studies, insulin significantly inhibited glucagon secretion at 2, 6 and 12 h, which was completely abolished by pretreatment with wortmannin. In perifusion studies, insulin significantly suppressed glucagon secretion after 10 min, which was also blocked by wortmannin. Insulin also reduced glucagon mRNA at 6 and 12 h but not at 2 h. Wortmannin also abolished insulin-induced reduction of glucagon mRNA. Insulin increased the amount of 85 kDa subunit of PI3-kinase in plasma membrane fraction (PM), with a reciprocal decrease of the kinase in cytosol fraction (CY). Insulin also increased PI3-kinase activity in PM, but not in CY. Our results suggest that insulin suppressed glucagon secretion by inhibiting glucagon release and gene expression. Both actions were mediated by activation of PI3-kinase. Recruitment and activation of PI3-kinase in plasma membrane might be relevant at least in part to insulin-induced inhibition of glucagon release.

Androstadienes↗