Biomedical subjects

K Yoshizato

Publications and source records attributed to K Yoshizato.

At least 19 recordsLinked to original sources

PubMed2006

Gene expression during chemically induced liver fibrosis: effect of halofuginone on TGF-beta signaling.

Hepatic fibrosis is associated with the activation of stellate cells (HSCs), the major source of extracellular matrix (ECM) proteins. Transforming growth factor-beta (TGF-beta), signaling via Smad3, is the most profibrogenic cytokine and the major promoter of ECM synthesis. Halofuginone, an inhibitor of liver fibrosis, inhibits TGF-beta-dependent Smad3 phosphorylation in human HSCs in culture. We have used transcriptional profiling to evaluate the effect of halofuginone on gene expression during the progression of thioacetamide (TAA)-induced liver fibrosis in the rat and have focused on genes that are associated with TGF-beta. TAA treatment causes alterations in the expression of 7% of liver genes. Halofuginone treatment prevents the changes in the expression of 41% of these genes and results in the inhibition of HSC activation and collagen synthesis. During the early stages of the disease, halofuginone affects genes involved in alcohol, lipid, protein, and phosphate metabolism and cell adhesion and, at later stages, in the cell cycle (cell development, differentiation, cell proliferation, and apoptosis). The activation of TGF-beta-dependent genes, such as tartrate-resistant acid phosphatase, its putative substrate osteopontin, stellate cell activation-association protein, and fibrillin-1, during chemically induced fibrosis is prevented by halofuginone. This study thus highlights the role of TGF-beta signaling in liver fibrosis and especially its potential for pharmacological intervention. Halofuginone, which has demonstrated efficacy and tolerance in animals and humans, could become an effective and novel therapy for liver fibrosis.

Acid Phosphatase↗

PubMed2006

Detection of COL1A1-PDGFB fusion transcripts and platelet-derived growth factor alpha and beta receptors in giant cell fibroblastoma of the postsacrococcygeal region.

We describe a 2-year-old girl with recurrent giant cell fibroblastoma (GCF) of the postsacrococcygeal region. Both the initial and recurrent tumours contained solid and angiectoid areas. The former was composed of loosely arranged wavy spindle cells and giant cells with a well-vascularized myxoid to collagenous stroma. The angiectoid spaces were often lined by multinucleated giant cells. Immunohistochemically, the tumour cells and small vessels in the tumour tissue were positive for platelet-derived growth factor (PDGF) alpha and beta receptors. Molecular analysis revealed fusion of collagen type Ialpha1 exon 26 with PDGF-B chain exon 2 that induced unscheduled production of PDGF-BB. These findings suggest that PDGF and its receptors significantly contribute to the development of GCF in both an autocrine and a paracrine manner.

Base Sequence↗

PubMed2005

In vivo induction of human cytochrome P450 3A4 by rifabutin in chimeric mice with humanized liver.

The induction of human cytochrome P450 enzymes (CYPs) often poses a serious problem in clinical practice. The induction of CYP3A leads to a decrease in the pharmacological potency of drugs, since many drugs are substrates of CYP3A. The present study examined the in vivo induction potency of human CYP3A in chimeric mice with humanized liver, recently established in Japan, by a specific inducer of human CYP3A enzyme activity in this experimental condition, rifabutin, which is an analogue of rifampicin. The chimeric mice were treated intraperitoneally daily for 4 days with rifabutin (50 mg kg(-1) day(-1)). The mRNA, protein and enzyme activity in liver of the chimeric mice were measured by reverse-transcriptase polymerase chain reaction, Western blot analysis and high-performance liquid chromatography, respectively. In the chimeric mice, the human CYP3A4 mRNA expression, CYP3A4 protein content, testosterone 6ss-hydroxylase activity and dexamethasone 6-hydroxylase activity were increased 7.4-, 3.0-, 2.4- and 1.9-fold, respectively, by treatment with rifabutin. The mRNA expression of other human CYPs, transporters and nuclear receptors was not significantly changed by rifabutin. On the other hand, rifabutin was demonstrated not to increase the murine Cyp3a enzyme activities in the control mice. It was demonstrated that human CYP3A4 expressed in the chimeric mice with humanized liver was induced by rifabutin, suggesting that human CYP3A4 in the chimeric mice had induction potency. This chimeric mouse model may be a useful animal model to estimate and predict the in vivo induction of CYPs in human.

Animals↗

PubMed2005

Evaluation of mRNA expression of human drug-metabolizing enzymes and transporters in chimeric mouse with humanized liver.

The hepatic mRNA expression of human drug-metabolizing enzymes and transporters in chimeric mise with almost-completely humanized liver (replacement index: 71-89%) was investigated. The mRNAs of 58 human phase I enzymes, 26 human phase II enzymes, 23 human transporters, and five mouse Cyps were measured in the chimeric mice with humanized liver generated using hepatocytes from a Japanese donor. The mRNA expression of 52 human phase I enzymes, which includes 20 human CYPs, 26 human phase II enzymes and 21 human transporters was ascertained in the chimeric mouse liver. Among them, the expression of the target mRNAs vital for liver function such as the metabolism and secretion of endogenous compounds appeared to be maintained. The central value for the expression ratio in all target genes in chimeric mouse liver to the donor liver was 0.46, which was lower than the substitution rate of chimeric mouse liver by donor liver. The ratio of mouse Cyp mRNA expression of chimeric mouse liver to that of control mouse liver was 0.19 or less, except for that of Cyp2b10. There were good correlations between the mRNA expression levels of human hepatic albumin gene, the values of the rate of replacement of mouse liver by human liver, and the human blood albumin concentration in the chimeric mice. The chimeric mice with humanized liver may be a useful tool for the evaluation of drug-drug interactions such as the inhibition and induction of drug-metabolizing enzymes and transporters.

Animals↗

PubMed2002

Proteomic profiling and neurodegeneration in Alzheimer's disease.

Quantitative proteome analysis of Alzheimer's disease (AD) brains was performed using 2-D gels to identify disease specific changes in protein expression. The task of characterizing the proteome and its components is now practically achievable because of the development and integration of four important tools: protein, EST, and complete genome sequence databases, mass spectrometry, matching software for protein sequences and protein separation technology. Mass spectrometry (MS) instrumentation has undergone a tremendous change over the past decade, culminating in the development of highly sensitive, robust instruments that can reliably analyze biomolecules, particularly proteins and peptides; we identified 35 proteins from over 100 protein spots on a 2-D gel. Using this current technology, protein-expression profiling, which is actually a specialized form of mining, is an important principal application of proteomics. The information obtained has tremendous potential as a means of determining the pathogenesis, and detecting disease markers and potential targets for drug therapy in AD.

Aged↗

PubMed2002

Expression of pleiotrophin in hepatic nonparenchymal cells and preneoplastic nodules in carbon tetrachloride-induced fibrotic rat liver.

Pleiotrophin (PTN) is a heparin-binding protein, which induces growth, angiogenesis, differentiation, and transformation of cells. The aim of this study was to examine the role of PTN in liver fibrogenesis. Rats were treated with carbon tetrachloride (CCl4) for 3-9 weeks to induce liver fibrosis. The sirius-red staining of these liver tissue sections clearly showed the development of fibrosis and glutathione S-transferase placental type-positive preneoplastic nodules emerged at 7 weeks of the treatment. PTN expression was investigated in fibrotic liver tissues at the mRNA level using a real-time reverse transcription polymerase chain reaction and at the protein level by immunohistochemistry. Quantity of PTN mRNA increased 5-fold in fibrotic liver tissues at 7 weeks of CCl4-treatment over the control values. Immunohistochemistry localized PTN protein on hepatic nonparenchymal cells, mostly stellate cells and some of Kupffer cells, and the preneoplastic nodules in fibrotic liver tissues. PTN mRNA expression is significantly upregulated in the CCl4-induced chronic rat fibrotic liver tissues. We suggest that PTN might be involved in fibrogenesis and preneoplastic changes of liver.

Animals↗

PubMed2002

Heterozygous knockout of the IRS-1 gene in mice enhances obesity-linked insulin resistance: a possible model for the development of type 2 diabetes.

Insulin receptor substrate 1 (IRS-1) gene polymorphisms have been identified in type 2 diabetic patients; however, it is unclear how such polymorphisms contribute to the development of diabetes. Here we introduced obesity in heterozygous IRS-1 knockout (IRS-1(+/-)) mice by gold-thioglucose (GTG) injection and studied the impact of reduced IRS-1 expression on obesity-linked insulin resistance. GTG injection resulted in approximately 30% weight gain in IRS-1(+/-) and wild type (WT) mice, compared with saline-injected controls. There was no difference in insulin sensitivity between lean IRS-1(+/-) and lean WT. Elevated fasting insulin levels but no change in fasting glucose were noted in obese IRS-1(+/-) and WT compared with the respective lean controls. Importantly, fasting insulin in obese IRS-1(+/-) was 1.5-fold higher (P<0.05) than in obese WT, and an insulin tolerance test showed a profound insulin resistance in obese IRS-1(+/-) compared with obese WT. The islets of obese IRS-1(+/-) were 1.4-fold larger than those of obese WT. The expression of insulin receptor and IRS-1 and IRS-2 was decreased in obese IRS-1(+/-), which could in part explain the profound insulin resistance in these mice. Our results suggest that IRS-1 is the suspected gene for type 2 diabetes and its polymorphisms could worsen insulin resistance in the presence of other additional factors, such as obesity.

Animals↗

PubMed2001

Production of EGF-collagen chimeric protein which shows the mitogenic activity.

Collagen has been utilized as a natural biomaterial because of its high biocompatibility, adhesiveness to cells and tissues, and biodegradability. The present study developed a recombinant technology to confer a mitogenic activity on type III collagen by fusing it to epidermal growth factor (EGF) at the collagen's N-terminus. The chimeric protein of EGF-collagen was synthesized in insect cells by the baculovirus-insect cell expression system. The fusion protein was shown to hold the triple helical conformation of collagen and the mitogenic activity of EGF. It was also demonstrated that the chimeric protein can be immobilized on tissue culture dishes as a fibrous form and in collagen fibrils without abolishing the original mitogenic activity of EGF. This fusion protein can be utilized as a biocompatible, biodegradable, and adhesive fibrous mitogen for a variety of purposes in the area of tissue engineering.

Animals↗

PubMed2001

Expression of thyroid hormone receptor betaA gene assayed by transgenic Xenopus laevis carrying its promoter sequences.

The responsiveness of thyroid hormone responsive element (TRE)-containing promoter sequence to thyroid hormone (TH) was studied utilizing Xenopus laevis carrying a transgene containing 5'-upstream region of TRbetaA1 gene and green fluorescent protein (EGFP) gene. EGFP-expression was seen first in neurulae, which continued to stage 45, then became weak, and again started to increase at the prometamorphic stage, culminating at the metamorphic climax stage. Immunohistochemistry identified eyes, viscera, and muscles as the EGFP-expressing larval tissues. The treatment of premetamorphic tadpoles with TH induced the precocious EGFP-expression. We also showed that the transgenic Xenopus adults were responsive to exogenous TH, a high responsiveness being seen in brain, small intestine, kidney, and bone. TRbeta-expression in the embryo, larva, and adult was verified by Western blotting. Thus, TH not only regulates the metamorphosis, but also might play some biological role(s) in embryos and adults.

Animals↗

PubMed2001

Regulation of cell growth by redox-mediated extracellular proteolysis of platelet-derived growth factor receptor beta.

Redox-regulated processes are important elements in various cellular functions. Reducing agents, such as N-acetyl-l-cysteine (NAC), are known to regulate signal transduction and cell growth through their radical scavenging action. However, recent studies have shown that reactive oxygen species are not always involved in ligand-stimulated intracellular signaling. Here, we report a novel mechanism by which NAC blocks platelet-derived growth factor (PDGF)-induced signaling pathways in hepatic stellate cells, a fibrogenic player in the liver. Unlike in vascular smooth muscle cells, we found that reducing agents, including NAC, triggered extracellular proteolysis of PDGF receptor-beta, leading to desensitization of hepatic stellate cells toward PDGF-BB. This effect was mediated by secreted mature cathepsin B. In addition, type II transforming growth factor-beta receptor was also down-regulated. Furthermore, these events seemed to cause a dramatic improvement of rat liver fibrosis. These results indicated that redox processes impact the cell's response to growth factors by regulating the turnover of growth factor receptors and that "redox therapy" is promising for fibrosis-related disease.

Animals↗

PubMed2001

Characterization of a stellate cell activation-associated protein (STAP) with peroxidase activity found in rat hepatic stellate cells.

A proteome approach for the molecular analysis of the activation of rat stellate cell, a liver-specific pericyte, led to the discovery of a novel protein named STAP (stellate cell activation-associated protein). We cloned STAP cDNA. STAP is a cytoplasmic protein with molecular weight of 21,496 and shows about 40% amino acid sequence homology with myoglobin. STAP was dramatically induced in in vivo activated stellate cells isolated from fibrotic liver and in stellate cells undergoing in vitro activation during primary culture. This induction was seen together with that of other activation-associated molecules, such as smooth muscle alpha-actin, PDGF receptor-beta, and neural cell adhesion molecule. The expression of STAP protein and mRNA was augmented time dependently in thioacetamide-induced fibrotic liver. Immunoelectron microscopy and proteome analysis detected STAP in stellate cells but not in other hepatic constituent cells. Biochemical characterization of recombinant rat STAP revealed that STAP is a heme protein exhibiting peroxidase activity toward hydrogen peroxide and linoleic acid hydroperoxide. These results indicate that STAP is a novel endogenous peroxidase catabolizing hydrogen peroxide and lipid hydroperoxides, both of which have been reported to trigger stellate cell activation and consequently promote progression of liver fibrosis. STAP could thus play a role as an antifibrotic scavenger of peroxides in the liver.

Amino Acid Sequence↗

PubMed2001

New epidermal keratin genes from Xenopus laevis: hormonal and regional regulation of their expression during anuran skin metamorphosis.

Xenopus larval keratin (XLK) was isolated by gel electrophoresis of proteins of tadpole skin. Screening of an expression cDNA library of tail tissues by specific polyclonal antibodies against XLK produced XLK cDNA (xlk). Its complete nucleotide and predicted amino acid sequences revealed that XLK was a new member of type II keratin. Screening of a cDNA library of adult Xenopus skin using an oligonucleotide probe which had been designed from well-conserved N-terminal amino acid sequences of the rod domain of type I keratin produced two cDNAs, xak-a and xak-b, which were found to be new members of type I keratin gene. Northern blot analysis showed that xlk was expressed exclusively in the larval skin whereas xak-a and xak-b were expressed exclusively in the adult skin. Their expression level was regulated in a region- and metamorphic stage- dependent manner during larval skin development. mRNA in situ hybridization experiments identified the cells that expressed xlk, and xak-a and xak-b as larva- specific epidermal cells (skein cells and basal cells), and adult suprabasal epidermal cells, respectively. These three genes were found to be late responsive to thyroid hormone. Phylogenetic relationships of these keratins with known ones are discussed.

Amino Acid Sequence↗

PubMed2001

Identification of a cis-acting element and a novel trans-acting factor of the human insulin receptor gene in HepG2 and rat liver cells.

The liver is a major target organ of insulin and is important for glucose homeostasis. We analyzed the tissue specific regulation of the insulin receptor gene in the liver by studying the cis-acting element and trans-acting factor of the human insulin receptor gene in human hepatoma cell line, HepG2 cells. In the chloramphenicol acetyl transferase (CAT) assay with chimeric plasmids containing various deletions and insertions of the human insulin receptor promoter/CAT gene, a HepG2 cell specific cis-acting element was identified between nt -592 to -577 of the promoter. In electrophoretic mobility shift assay and UV cross-link analysis, a 35-kDa nuclear protein that bound to 5'-TCCCTCCC-3' (nt -588 to -581) sequence was identified in HepG2 cells as well as in rat hepatocytes. This nuclear protein, designated as hepatocyte-specific transcription factor of the insulin receptor gene (HTFIR), might play an important role in tissue-specific expression of the insulin receptor gene in the liver.

Animals↗

PubMed2001

Identification and characterization of newt rad (ras associated with diabetes), a gene specifically expressed in regenerating limb muscle.

Formation of the blastema is a key event for limb regeneration in urodele amphibians, and skeletal muscle has been thought to be a major origin of the multipotent blastemal mesenchyme. In the present study, we used differential display to identify the genes expressed differentially in the muscle at the amputation site. We have isolated a cDNA clone that was upregulated during limb regeneration of the Japanese newt, Cynops pyrrhogaster. Deduced amino acid sequence revealed that the cloned cDNA was a newt homolog of rad (ras associated with diabetes), a gene overexpressed in skeletal muscle of Type II diabetic patients. Expression of newt rad (nrad) was not observed in unamputated normal limb muscle, increased within 4 hr after amputation, and then decreased to the level of normal muscle between 11 and 21 days after amputation. In situ hybridization showed that the transcripts of nrad were localized around most of the nuclei of skeletal muscle near the amputation site, indicating the expression of nrad in the multinucleate myotubes. This expression gradually decreased along the distal to proximal axis. No signals were observed in apical epidermal cap or blastemal mesenchyme. However, reverse transcription-PCR analysis detected a very low level of nrad expression in blastema, suggesting the carry-over of nrad expression in blastema from muscle. Administration of retinoic acid, which has been shown to cause an enhanced dedifferentiation in the regenerating limbs, increased nrad expression in more proximally located limb muscle tissues and prolonged the expression period. Thus, it was strongly suggested that the nrad expression is correlated with the dedifferentiation of myotubes of regenerating limbs. We also analyzed the expression of nrad during development. Transcripts were observed in immature oocytes, seen faintly or not seen thereafter until stage 57 when its expression increased again. These results indicated that nrad may play a role(s) in the developmental process as well as limb regeneration.

Amino Acid Sequence↗

PubMed2001

Thyroid-hormone-dependent and fibroblast-specific expression of BMP-4 correlates with adult epithelial development during amphibian intestinal remodeling.

We have identified one of the genes that are up-regulated by thyroid hormone (TH) in Xenopus laevis small intestine as the Xenopus homolog of bone morphogenetic protein-4 (BMP-4). To clarify possible roles of BMP-4 in intestinal remodeling during metamorphosis, we have examined its expression in X. laevis intestine by using in situ hybridization and organ culture techniques. At the beginning of metamorphic climax, BMP-4 mRNA first becomes detectable in the connective tissue, concurrently with the appearance of adult epithelial primordia. Subsequently, when the adult epithelial primordia are actively proliferating, BMP-4 mRNA becomes more abundant only in the connective tissue with a gradient toward the epithelium. Thereafter, as the adult primordia differentiate, the level of BMP-4 mRNA gradually decreases. Thus, BMP-4 expression correlates well with cell proliferation and/or initial differentiation of the adult epithelium, but not with apoptosis of the larval epithelium. Furthermore, the present culture study indicates that (1) TH-induced expression of BMP-4 mRNA is higher in the anterior part of the intestine than in the posterior part, which agrees with the better development of the adult epithelium in the more anterior part, and that (2) the expression of BMP-4 mRNA is up-regulated by TH in the presence of epithelium, but not in its absence. Therefore, BMP-4, which is indirectly induced by TH through some epithelial factor(s), probably plays important roles in adult epithelial development during amphibian intestinal remodeling.

Age Factors↗

PubMed2001

Size-dependent in vivo growth potential of adult rat hepatocytes.

The present study was performed to determine whether hepatocytes show a size-dependent growth in vivo using as a growth assay system, a retrorsine/partial hepatectomy model of dipeptidyl dipeptidase IV-deficient (DPPIV(-)) mutant Fischer rats. Nearly pure populations of small hepatocytes (SHs) and parenchymal hepatocytes (PHs) were prepared from DPPIV(+) rats. The same number of these SHs and PHs was transplanted into the liver of retrorsine-treated and two-thirds partial hepatectomized DPPIV(-) rats. At 21 days after transplantation, colonies derived from donor hepatocytes were detected as DPPIV(+) cells by enzyme histochemistry. SHs were approximately three times more proliferative than PHs (673 +/- 25 cells/colony versus 226 +/- 10 cells/colony, mean +/- SE). SHs were subfractionated by a fluorescence-activated cell sorter into SH-R2s and SH-R3s. SH-R3s showed a lower extent of granularity and autofluorescence, and a smaller size than SH-R2s that showed characteristics similar to PHs. The growth potential of SH-R3s assayed as above was approximately three times higher than that of SH-R2s (1,101 +/- 46 cells/colony versus 341 +/- 13 cells). These results indicate that the in vivo growth potential of hepatocytes is heterogeneous and is correlated with their size, and the extent of their granularity and autofluorescence.

Albumins↗

PubMed2001

Novel Rana keratin genes and their expression during larval to adult epidermal conversion in bullfrog tadpoles.

The conversion of the larval to adult epidermis during metamorphosis of tadpoles of bullfrog, Rana catesbeiana, was investigated utilizing newly cloned Rana keratin cDNAs as probes. Rana larval keratin (RLK) cDNA (rlk) was cloned using highly specific antisera against Xenopus larval keratin (XLK). Tail skin proteins of bullfrog tadpoles were separated by 2-dimensional gel electrophoresis and subjected to Western blot analysis with anti-XLK antisera. The Rana antigen detected by this method was sequenced and identified as a type II keratin. We cloned rlk from tadpole skin by PCR utilizing primers designed from these peptide sequences of RLK. RLK predicted by nucleotide sequences of rlk was a 549 amino acid -long type II keratin. Subtractive cloning between the body and the tail skin of bullfrog tadpole yielded a cDNA (rak) of Rana adult keratin (RAK). RAK was a 433 amino acid-long type I keratin. We also cloned a Rana keratin 8 (RK8) cDNA (rk8) from bullfrog tadpole epidermis. RK8 was 502 amino acid-long and homologous to cytokeratin 8. Northern blot analyses and in situ hybridization experiments showed that rlk was actively expressed through prometamorphosis in larva-specific epidermal cells called skein cells and became completely inactive at the climax stage of metamorphosis and in the adult skin. RAK mRNA was expressed in basal cells of the tadpole epidermis and germinative cells in the adult epidermis. The expression of rlk and rak was down- and up-regulated by thyroid hormone (TH), respectively. In contrast, there was no change in the expression of RK8 during spontaneous and TH-induced metamorphosis. RK8 mRNA was exclusively expressed in apical cells of the larval epidermis. These patterns of keratin gene expression indicated that the expression of keratin genes is differently regulated by TH depending on the type of larval epidermal cells. The present study demonstrated the usefulness of these genes for the study of molecular mechanism of postembryonic epidermal development and differentiation.

Amino Acid Sequence↗

PubMed2001

Membrane-type 1 matrix metalloproteinase is induced in decidual stroma without direct invasion by trophoblasts.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) in endometrium and decidua may greatly affect attachment of the embryo to the epithelium, invasion of the trophoblast into the stroma, and extracellular matrix remodelling in the endometrium and decidua. We investigated the expression of this enzyme in normally cycling endometrium and in decidua associated with normal and tubal pregnancies at both the gene and protein level. Localization of expression (but not the overall level of expression), differed between endometrium and decidua parietalis and tubal pregnancy decidua. MT1-MMP mRNA was expressed mainly in epithelium and only faintly in stroma throughout the menstrual cycle, while in decidua parietalis and tubal pregnancy decidua, this mRNA was expressed predominantly in stromal cells. MT1-MMP protein was detected in the epithelium alone throughout the menstrual cycle, while in decidua parietalis and tubal pregnancy decidua, it was detected in stromal cells as well as the epithelium. Since decidua showed altered expression in the absence of trophoblastic contact, trophoblast invasion may not directly affect MT1-MMP gene expression.

Adult↗