Biomedical subjects

S Taketani

Publications and source records attributed to S Taketani.

At least 37 records · Page 2Linked to original sources

PubMed2000

ELISA for urinary trehalase with monoclonal antibodies: a technique for assessment of renal tubular damage.

BACKGROUND: alpha,alpha-Trehalase, located on renal proximal tubules, is a glycoprotein that hydrolyses alpha,alpha-trehalose to two glucose molecules. Urinary trehalase reflects damage to renal proximal tubules, but its activity has not been measured routinely because measurement of catalytic activity is rather complicated and because conventional assays for enzyme activity might not reflect all of the trehalase protein because of enzyme inactivation in urinary samples. METHODS: We established novel monoclonal antibodies for human trehalase and a sandwich ELISA for quantification of urinary trehalase. We determined the urinary trehalase protein concentration with this ELISA and trehalase catalytic activity, and the results of these two methods were compared. RESULTS: The ELISA system was more sensitive than the detection of enzyme activity and could detect a subtle difference in the amount of trehalase present in renal diseases. The within- and between-assay CVs in the ELISA were 6.7-7.6% and 6.2-8.2%, respectively. Highly significant increases in both the quantity and activity were seen in patients with nephrotic syndrome (acute phase), Lowe syndrome, and Dent disease. The quantities were 70- to 200-fold greater, whereas enzyme activities were, at most, 10-fold higher than those of control subjects. In the detection of small amounts of trehalase in patients with chronic glomerulonephritis and renal anomalies, quantities were better than enzyme activities. CONCLUSIONS: We have established an ELISA system for quantification of urinary trehalase that uses novel monoclonal antibodies. Our ELISA system is simpler and more sensitive than a conventional activity assay and reflects trehalase protein. This ELISA can be a useful as a common tool for clinical assessment of renal proximal tubular damage.

Acetylglucosaminidase↗

PubMed2000

Analysis of the candidate genes responsible for non-syndromic cleft lip and palate in Japanese people.

In order to assess the association of alleles for candidate genes with non-syndromic cleft lip and palate, DNA samples from 43 Japanese patients were compared with those from 73 control subjects with respect to the genes encoding transforming growth factor alpha (TGFalpha), TGFbeta and gamma-aminobutyric acid type A receptor beta3 (GABRB3). The restriction fragment length polymorphisms of the 3'-non-coding region of the TGFalpha gene K-primer region were observed after digestion with NcoI and HinfI. Allele 4 was the most common among cases of cleft lip with or without cleft palate, whereas allele 2 was the most common among controls. A significant difference was found in this region between groups with cleft lip (with or without cleft palate) and controls (chi2=10.190; P=0.017). Three alleles of the TGFbeta2 gene were tested, and allele 2 was the most common in both cases and controls. The proportion of allele 2 in the case group was greater than that in the control group, showing a significant difference between cases of cleft lip (with or without cleft palate) and controls (chi(2)=19.208; P<0.0001). No significant differences in variants of TGFbeta3 or GABRB3 between case and control populations were observed. Thus it is concluded that TGF genes play a role in craniofacial development, and that alleles of TGFalpha or/and TGFbeta2 are associated with cleft lip and cleft palate in Japanese populations.

Adolescent↗

PubMed1999

Activated Rac1 selectively up-regulates the expression of integrin alpha6beta4 and induces cell adhesion and membrane ruffles of nonadherent colon cancer Colo201 cells.

Functions of small GTPases in integrin expression were investigated when the interaction of nonadherent human colon carcinoma 201 cells with the extracellular matrix (ECM) was examined. By transfection of the constitutively active form of a small GTPase Rac1, Rac V12, adhesion of cells to the ECM increased with concomitant cell spreading and formation of membrane ruffles. Activated Cdc42 and Cdc42 V12, but not wild-type Rac1, Cdc42, or RhoA, also induced the adhesion and spreading of Colo201 cells. This adhesion is integrin beta4 dependent since an antibody for integrin beta4 inhibited the RacV12-dependent cell adhesion and numbers of adhesive cells on laminin-coated plates exceeded those on collagen- and fibronectin-coated plates. By immunofluorescence, in addition to clustering of integrin molecules, expression of integrin alpha6beta4 on the cell surface of Rac V12- and Cdc42 V12-expressing cells was selectively up-regulated without an increase in biosynthesis of alpha6beta4 integrin. Treatment of Rac V12-expressing cells with wortmannin or LY294002, specific inhibitors of phosphoinositide 3-OH kinase, decreased the up-regulated alpha6beta4 and cell adhesion. In light of this evidence, we propose that the regulation of integrin alpha6beta4 expression induced by Rac1 and Cdc42 may play an important role in cell adhesion and tumorigenesis of colon carcinoma cells.

Antibodies, Monoclonal↗

PubMed1999

MAP kinase-independent induction of proto-oncogene c-fos mRNA by hemin in human cells.

Treatment of HeLa cells or human skin fibroblast cells with hemin led to a time- and dose-dependent rapid induction of c-fos mRNA. This induction was absent in the cells treated with actinomycin D, indicating that the c-fos induction by hemin occurs at the level of transcription. Metalloporphyrins, including zinc-, cobalt-, and tin-protoporphyrin, ferric ion, and protoporphyrin also induced c-fos mRNA. Transient reporter assay with the reporter constructs of the human c-fos gene promoter up to -404 bp connected to the luciferase gene showed high activity but no induction by hemin, suggesting that cis-acting elements, including the serum response element located about -310 bp upstream of the human c-fos gene promoter, may not contribute to the heme-dependent induction. With in-gel assay of protein kinases, the activity of the mitogen-activated protein (MAP) kinases such as extracellular signal-regulated kinase 12 or p38 MAP kinase in hemin-treated HeLa cells was not stimulated. Stimulation of c-Jun N-terminal kinase by hemin was nil. Furthermore, PD58059 and SB203580, inhibitors for MAP kinases, did not affect the hemin-dependent c-fos induction. Of the inhibitors for protein kinases so far tested, KN-62, a specific inhibitor for calmodulin-dependent protein kinase II (CaMK II), inhibited the induction of c-fos mRNA by hemin. Phosphorylation of CaMK II in hemin-treated cells increased. With gel mobility assay, the DNA AP-1 binding activity transiently increased when treating HeLa cells with hemin. Therefore, induction of c-fos led to an activation of AP-1 in the presence of hemin. We suggest that calmodulin-dependent protein kinase II rather than the MAP kinase family regulates the induction of the human c-fos gene expression by hemin.

Arsenites↗

PubMed1999

Structure and transcriptional regulation of the mouse ferrochelatase gene.

Ferrochelatase (EC.4.99.1.1), the final step in the biosynthesis of heme, is widely expressed in various tissues and is induced in erythroid cells. We determined the structure of the mouse ferrochelatase gene after isolation and characterization of lambda phage clones mapping discrete regions of the cDNA. The gene spans about 25 kb and consists of 11 exons. The exon/intron boundary sequences conform to consensus acceptor (GTn)/donor (nAG) sequences, and exons in the gene encode functional protein domains. The promoter region contains multiple Sp1 sites, a CACCC box and GATA-1 binding sites. Function analysis of the promoter by transient transfection assay demonstrated that one Sp1 binding site located at -37/-32 is essential for basic expression of the ferrochelatase gene in both mouse erythroleukemia (MEL) and non-erythroid EL4 cells. In addition, the region (-66/-51) containing a CACCC box and the neighboring GC box partly contributes to the inducible activity of the reporter in MEL cells upon induction with dimethylsulfoxide. It appears that at least two promoter regions of the mouse ferrochelatase gene function in basic and inducible expression.

Animals↗

PubMed1999

HGF activates signal transduction from EPO receptor on human cord blood CD34+/CD45+ cells.

Hepatocyte growth factor (HGF) is a multifunctional cytokine with early hematopoiesis-stimulatory activity. Here, we focus on its erythropoiesis-stimulatory effect on highly purified human hematopoietic progenitor cells (CD34+/CD45+ cells) derived from the cord blood. In immunoblot analyses, c-met protein (a receptor of HGF) was detected in the CD34+/CD45+ cells, although the expression levels were different among samples. The c-met expression was facilitated by incubation of the cells with stem cell factor (SCF) or interleukin 3 (IL-3), even if the expression level had been low. IL-6, G-CSF, or erythropoietin (EPO) did not show such a stimulatory effect on the c-met expression of the cells. When HGF was added to the CD34+/CD45+ cells in the presence of SCF, the numbers of CD36+/CD11b- cells (very early erythroid lineage cells) and BFU-E increased. EPO-dependent tyrosine phosphorylation of Stat 5 also increased, but the EPO receptor (EPO-R) expression remained unchanged in the CD34+/CD45+ cells treated with SCF + HGF. Our present study suggests that stimulation of the HGF/c-met signal is concomitant with induction of c-met protein by SCF. The subsequent enhancement of signal transduction via the activation of Stat 5 from the EPO-R plays a crucial role in the commitment of hematopoietic stem cells into erythroid lineage cells.

Antigens, CD34↗

PubMed1999

Age-dependent abnormalities of hematopoietic stem cells in (NZW x BXSB)F1 mice.

The (NZW x BXSB)F1 (W/BF1) mouse is known as an autoimmune-prone strain which develops lupus nephritis, thrombocytopenia due to platelet-specific autoantibodies, leukocytosis, and myocardial infarction. In this experiment, we investigated the age-dependent abnormalities of the hematopoietic stem cells (HSCs) and hematopoiesis in this mouse. White blood cell counts (especially Mac-1- or Gr-1-positive cells) in the peripheral blood of 12-week-old W/BF1 mice increased in comparison with those of four-week-old W/BF1 or normal mice. To investigate whether the abnormal hematopoiesis can be attributed to the HSCs of W/BF1 mice, colony-forming unit in spleen (CFU-S) and colony-forming unit in culture (CFU-C) assays were performed. Day 12 CFU-S counts of 12-week-old W/BF1 mice significantly increased in comparison with those of four-week-old W/BF1 mice or normal mice. In the CFU-C assay, CFU-GEMM and CFU-GM counts in 12-week-old W/BF1 mice increased in comparison with those of four-week-old W/BF1 or control mice. The bone marrow cells (BMCs) from 12-week-old W/BF1 mice showed a high level of G-CSF and a low level of GM-CSF in mRNA expression. To examine the effect of HSCs from 12-week-old W/BF1 mice on the onset of autoimmune diseases and the abnormal hematopoiesis, T- and B-cell-depleted BMCs of four-week-old or 12-week-old W/BF1 mice were transplanted to C3H mice. Recipient C3H mice that had received the BMCs from 12-week-old W/BF1 mice showed an earlier onset of autoimmune diseases and a shorter survival rate than those that had received the BMCs from four-week-old W/BF1 mice. These data suggest that the HSCs from 12-week-old W/BF1 mice showing the symptoms of autoimmune diseases have the capacity to induce autoimmune diseases earlier than the HSCs from four-week-old W/BF1 mice.

Age of Onset↗

PubMed1999

A crucial role of caspase 3 and caspase 8 in paclitaxel-induced apoptosis.

The anticancer drug paclitaxel is well known as an inhibitor of microtubule depolymerization, resulting in mitosis arrest. We investigated the mechanism underlying antitumor effects of paclitaxel on the lung adenocarcinoma cell line LC-2-AD. Less than 10 microg/ml paclitaxel induced mitosis arrest upon LC-2-AD, followed by apoptosis, but more than 30 microg/ml paclitaxel induced apoptosis without mitosis arrest. LC-2-AD with less than 1 microg/ml paclitaxel showed a loss of mitochondrial transmembrane potential (deltapsim), which correlated with antitumor effects. However, LC-2-AD with more than 10 microg/ml paclitaxel showed slight changes in the loss of deltapsim in spite of its ability to induce apoptosis significantly. The cleavage of caspase 3, caspase 8, and DFF45/ICAD was also observed in paclitaxel-induced apoptosis, and the inhibitor of caspase 3 and caspase 8 inhibited both antitumor effects and apoptosis induced by paclitaxel. These results suggest that activation of caspase 3 and caspase 8 plays a crucial role in paclitaxel-induced apoptosis under any concentrations of paclitaxel.

Antineoplastic Agents, Phytogenic↗

PubMed1999

Oxidation of protoporphyrinogen IX in Escherichia coli is mediated by the aerobic coproporphyrinogen oxidase.

Protoporphyrinogen oxidase, the penultimate enzyme involved in the biosynthetic pathway for heme, catalyzes the removal of six electrons from protoporphyrinogen IX to generate protoporphyrin IX. In Escherichia coli, this enzyme is encoded by the hemG gene. In this study we examined possible alternate pathways for the oxidation of protoporphyrinogen IX to protoporphyrin IX, by isolating and investigating E. coli mutants that can still grow normally when the hemG gene is disrupted. One of these mutants was characterized in detail and had a mutation in the promoter region of the hemF gene, which encodes aerobic coproporphyrinogen oxidase, the enzyme involved in the step immediately before protoporphyrinogen oxidase. Measurement of the promoter activity of the hemF gene showed that the level of transcription was elevated by the mutation. Overexpression of a wild-type hemF gene cloned in a multicopy plasmid also restored the growth of deltahemG strain. Extracts from cells that overexpress hemF exhibited an increased ability to oxidize protoporphyrinogen IX to protoporphyrin IX. These findings suggest that the E. coli aerobic coproporphyrinogen oxidase has an intrinsic capacity to oxidize not only coproporphyrinogen III but also protoporphyrinogen IX.

Base Sequence↗

PubMed1999

Ischemia induces metallothionein III expression in neurons of rat brain.

Metallothionein III (MT-III) is a brain-specific member of the metallothionein family and binds zinc in vivo. In order to confirm the precise localization of MT-III in normal rat brain and the change of MT-III expression after transient whole brain ischemia, we raised a high affinity phagemid-antibody specific for rat MT-III. Immunohistochemical analysis revealed that MT-III in normal brain is localized abundantly in neuronal cell bodies in CA1-3 regions of hippocampus, dentate gyrus, cerebral cortex, olfactory bulb and Purkinje cells in cerebellum. This expression pattern of MT-III was similar to that of MT-III mRNA observed by in situ hybridization studies. ELISA and Northern blot analysis revealed that MT-III protein as well as mRNA levels were up-regulated in cerebrum soon after ischemic stress. Immunohistochemical analysis also demonstrated intense staining in neurons in injured brain after ischemia, which distributed in the same regions as in normal brain. These results suggest that MT-III plays an important role in protecting neurons from ischemic insult by reducing neurotoxic zinc levels and inhibits uncontrolled growth of neurites after ischemia.

Animals↗

PubMed1999

Facile degradation of apolipoprotein B by radical reactions and the presence of cleaved proteins in serum.

A facile cleavage of peptide bonds of apolipoprotein B (apoB) by radical reaction is reported. When human LDL was subjected to oxidative damage using Cu2+, extensive degradation of apoB was observed based on immunoblotting. The degradation of apoB was inhibited by radical scavengers (beta-mercaptoethanol, butylated hydroxytoluene, and probucol) and promoted by a radical initiator [2, 2'-azobis(2-amidinopropane)dihydrochloride]. When human serum was treated with Cu2+, a similar cleavage pattern of apoB was observed. The cleaved apoB proteins were also detected in normal serum on the basis of immunoblots. These results suggest that apoB is highly reactive toward radicals in vitro and in vivo, with reaction resulting in the cleavage of peptide bonds.

Adult↗

PubMed1999

Detection of a variety of Ser/Thr protein kinases using a synthetic peptide with multiple phosphorylation sites.

A novel peptide with multiple phosphorylation sites, which we designated as multide, was developed to detect a wide variety of protein kinases in crude cell extracts. Multide, KKRKSSLRRWSPLTPRQMSFDC, has been designed to contain consensus sequences for various Ser/Thr protein kinases including cAMP-dependent protein kinase, protein kinase C, MAP kinases, and Ca(2+)/calmodulin-dependent protein kinases in a single peptide. In-gel protein kinase assay using multide was found to be very useful for analyzing the activities of protein kinases that are altered in response to various extracellular stimuli. The substrate specificities of the protein kinases thus detected were further determined by using five multide analogs with different phosphorylation sites.

Amino Acid Sequence↗

PubMed1999

Apoptosis of colorectal adenocarcinoma induced by 5-FU and/or IFN-gamma through caspase 3 and caspase 8.

The cytotoxic effect of 5-FU has been shown by the induction of apoptosis in cancer cells, and reported to be enhanced by IFN-gamma. We examined the role of caspases on the apoptosis induction of 5-FU and IFN-gamma using a colorectal adenocarcinoma cell line. The activities of caspase 3 and caspase 8 increased when apoptosis was induced by 5-FU and/or IFN-gamma. Moreover, all apoptotic cells showed high caspase 3 activity in these conditions. Although the inhibitors of caspase 3 and caspase 8 inhibit apoptosis, anti-Fas ligand antibody does not affect the apoptosis induced by 5-FU. Thus, caspase 3 and caspase 8 play crucial roles in apoptosis induced by 5-FU and/or IFN-gamma, regardless of the Fas-Fas ligand system.

Adenocarcinoma↗

PubMed1998

Molecular characterization of a newly identified heme-binding protein induced during differentiation of urine erythroleukemia cells.

A heme-binding protein with a molecular mass of 22 kDa, termed p22 HBP, was purified from mouse liver cytosol, using blue Sepharose CL-6B. We identified a cDNA encoding p22 HBP, and sequence analysis revealed that p22 HBP comprises 190 amino acid residues (Mr 21,063) and has no homology to any other known heme-binding protein. The p22 HBP mRNA (approximately 1.0 kilobases) is ubiquitously expressed in various tissues and is extremely abundant in the liver. cDNA allows for expression of active p22 HBP, with a high affinity for 55Fe-hemin, with a Kd of 26 +/-1.8 nM. The Bmax of hemin binding to p22 HBP was 0.55 +/- 0.021 mol/mol of protein, a value consistent with one heme molecule binding per molecule of protein. The order of potency of different ligands to compete against 55Fe-hemin binding to p22 HBP was hemin = protoporphyrin IX > coproporphyrin III > bilirubin > palmitic acid > all-trans-retinoic acid. Treatment of mouse erythroleukemia (MEL) cells with dimethyl sulfoxide or hemin resulted in an increase in p22 HBP mRNA. The immunoblot analysis showed that p22 HBP increased with time in dimethyl sulfoxide- and hemin-induced MEL cells. Conversely, transfer of antisense oligonucleotides to p22 HBP cDNA resulted in a decrease of p22 HBP in dimethyl sulfoxide-treated MEL cells, and the heme content in these cells decreased to 66-71% of sense oligonucleotides-transferred cells. Thus, this newly identified heme-binding protein, p22 HBP, may be involved in heme utilization for hemoprotein synthesis and even be coupled to hemoglobin synthesis during erythroid differentiation.

Amino Acid Sequence↗

PubMed1998

Differential regulation of coproporphyrinogen oxidase gene between erythroid and nonerythroid cells.

Coproporphyrinogen oxidase (CPO) catalyzes the sixth step of the heme biosynthetic pathway. To assess the tissue-specific regulation of the CPO gene promoter, mouse genomic DNA clones for CPO were isolated. Structural analysis demonstrated that the mouse CPO gene spans approximately 11 kb and consists of seven exons, just like its human counterpart. Functional analysis of the promoter by transient transfection assays indicated that synergistic action between an SP-1-like element at -21/-12, a GATA site at -59/-54, and a novel regulatory element, CPRE (-GGACTACAG-) at -49/-41, is essential for the promoter activity in murine erythroleukemia (MEL) cells. In nonerythroid NIH3T3 cells, however, the GATA site is not required. Gel mobility shift assays demonstrated that specific DNA-protein complexes can be formed with each element, and that there are cell-specific differences in factors, which bind to the SP-1-like element between MEL and NIH3T3 cells. These results provide evidence for differential regulation of the promoter function of CPO gene between erythroid and nonerythroid cells.

3T3 Cells↗

PubMed1998

Molecular properties and chromosomal location of cadherin-8.

Cloning of rat cadherin-8 cDNA demonstrated two types of cDNAs. The overall structure of the protein defined by one type of the cDNA is essentially the same as that of classic cadherins, whereas the protein defined by the other type of cDNA ends near the N-terminus of the fifth repeat of the extracellular domain (EC5) and contains a short unique sequence at the C-terminus. The same truncated type of cDNA was also obtained from a human cDNA library. In Northern blot analysis of rat brain mRNA, a probe for EC5 detected multiple bands of about 3.5-4.3 knt, whereas a probe for the alternative form hybridized with a band of about 3.5 knt. Western blot experiments showed that an antibody against the extracellular domain of rat cadherin-8 stained a band of about 95 kDa and a faint band of about 130 kDa in rat brain extract. These results suggest that cadherin-8 is expressed in two forms, a complete form and a truncated form without a transmembrane domain or cytoplasmic domain, in brain. The complete form of cadherin-8 expressed in L cells was about 130 kDa in molecular mass and was located at the cell periphery, mainly at the cell-cell contact sites. However, we failed to express the truncated form in L cells. The transfectants of the complete form showed weak cell adhesion activity. The complete form of cadherin-8 was sensitive to trypsin digestion, and Ca2+ did not protect cadherin-8 from digestion, in contrast to the classic cadherins. The complete form of cadherin-8 coprecipitated with beta-catenin, but did not immunoprecipitate well with alpha-catenin or gamma-catenin. Cadherin-8, as well as cadherin-11, was mapped to a specific region of chromosome 8 that also includes cadherins-1, -3, and -5.

Amino Acid Sequence↗

PubMed1998

Hemopexin from four species inhibits the association of heme with cultured hepatoma cells or primary rat hepatocytes exhibiting a small number of species specific hemopexin receptors.

Hemopexin (Hx) binds heme with a very high affinity (Kd<0.1 pmol/L). It has been implicated as a major vehicle for the transport of heme into liver cells, involving a receptor-mediated recycling mechanism. However, previous studies indicated that heme is not taken up by cultured embryonic chick or adult rat hepatocytes by such a mechanism, because heme added as heme hemopexin failed to affect heme-responsive activities of 5-aminolevulinic acid synthase and heme oxygenase. Here, we investigated the importance of hemopexin in hepatic heme uptake in cultured rat hepatocytes and human HepG2 hepatoma cells, and determined the number and species specificity of hemopexin receptors on the rat hepatocytes. We also tested whether there is a difference between heterologous and homologous hemopexins. We found the following: 1) heme is inhibited from associating with hepatocytes by apo hemopexins from rat, human, rabbit, and chicken; 2) heme readily associates with hepatocytes when heme hemopexin preparations are added in which the ratio of heme to hemopexin exceeds 1.0; 3) heme induces heme oxygenase mRNA in rat hepatocytes and this induction is prevented by excess hemopexin; and 4) rat hepatocytes exhibit only about 2,000 hemopexin receptors per cell when using rat hemopexin, and none when using hemopexin of rabbit and human. We conclude that hemopexin plays a limited role in heme uptake by cultured hepatocytes and hepatoma cells, and that heme which exceeds the hemopexin binding capacity is taken up directly from heme-albumin.

Animals↗

PubMed1998

[Evaluation of cerebral circulation during cardiopulmonary bypass using near-infrared spectroscopy].

Cerebral oxygenation level during cardiopulmonary bypass (CPB) was measured using near-infrared spectroscopy as a monitor of cerebral circulation in 30 patients. Six adult cases with thoracic aortic aneurysm were operated on using selective cerebral perfusion (SCP). CPB was established under moderate hypothermic temperature in 9 adult cases (hypothermic group, lowest blood temperature during CPB; 25 degrees C) and under normothermic temperature in 9 adult cases (normothermic group, lowest blood temperature during CPB; 34 degrees C). In congenital cases (n = 6), CPB was established under moderate hypothermic temperature (congenital group, lowest blood temperature; 25 degrees C). The oxyhemoglobin (HbO2) level showed a significant positive correlation with cerebral blood flow during SCP (r = 0.715). There was no significant correlation between SjO2 and HbO2 in the SCP group. The HbO2 levels in the hypothermic group after 30 and 60 min, from the initiation of CPB and 30 min. before the weaning of CPB were significant lower than the control level (p < 0.05). HbO2 levels in the congenital group after 0, 30 and 60 min. from the initiation of CPB and 30 min. before the weaning of CPB were significantly lower than the control level (p < 0.01). The deoxyhemoglobin (HbR) level in the hypothermic group after 30 and 60 from the initiation of CPB and 30 min. before the wearing of CPB were significantly higher than the pre level (p < 0.05). The mixed venous saturation (SvO2) in the normothermic group showed significant lower levels than those in the hypothermic group (p < 0.01). However, there was no significant difference in HbO2 levels between the two groups. In conclusion, these results suggest that near-infrared spectroscopy may be a noninvasive and useful technique for the cerebral circulation monitoring during CPB.

Adolescent↗