Biomedical subjects

Takashi Sato

Publications and source records attributed to Takashi Sato.

At least 19 recordsLinked to original sources

PubMed2006

Chondroitin sulfate N-acetylgalactosaminyltransferase-1 plays a critical role in chondroitin sulfate synthesis in cartilage.

Cartilage destruction leads to severe joint diseases, such as osteoarthritis and spinal disorders with back pain, and cartilage regeneration is very inefficient. A major component of the cartilage extracellular matrix is the proteoglycan aggrecan that contains approximately 100 chondroitin sulfate (CS) chains, which impart water absorption and resistance to compression. Here, we demonstrate that chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) plays a critical role in CS biosynthesis in cartilage. By in situ hybridization and real time reverse transcription-PCR of developing cartilage, CSGalNAcT-1 exhibited the highest level of expression. Its expression in chondrogenic ATDC5 cells correlated well with that of aggrecan core protein. In heterozygote and homozygote aggrecan-null cartilage where aggrecan transcription is decreased, CSGalNAcT-1 transcription diminished accordingly. Overexpression of the enzyme in chondrocytic cells further enhanced CS biosynthesis but not that of the aggrecan core protein, indicating that the enzyme activity is not saturated in the cells and that aggrecan synthesized in the overexpressing cells is heavier than the native molecule. Analysis of the CS chains synthesized in the overexpressing cells by gel chromatography and that of disaccharide composition revealed that the CS chains had similar length and sulfation patterns. Furthermore, adenoviral gene delivery of the enzyme into intervertebral discs displayed a substantial increase in the level of CS biosynthesis. These observations indicate that CSGalNAcT-1 overexpression increases the number of CS chains attached to aggrecan core protein. Our studies may lead to a new therapeutic intervention, ameliorating the outcome of cartilage degenerative diseases.

Animals↗

PubMed2006

Blastic natural killer cell leukaemia in a dog--a case report.

A case of canine non-T, non-B lymphoid leukaemia was determined to be of natural killer (NK) cell lineage by detecting specific expression of canine CD56 mRNA by reverse transcriptase polymerase chain reaction analysis. Although NK cells are usually considered to be morphologically large granular lymphocytes, the malignant NK cells in this case were agranular and blast-like, resembling human blastic NK cell leukaemia. The prognosis of human NK cell leukaemia is usually poor. In this case, the dog died 10 days after initial presentation, despite chemotherapy.

Animals↗

PubMed2006

Prospective study of positron emission tomography for evaluation of the activity of lapatinib, a dual inhibitor of the ErbB1 and ErbB2 tyrosine kinases, in patients with advanced tumors.

BACKGROUND: To evaluate the role of FDG-PET in assessing anti-tumor efficacy of molecular targeted drugs, we prospectively performed FDG-PET and CT for response evaluation in patients treated with lapatinib, a dual inhibitor of ErbB1 and ErbB2 tyrosine kinases. METHODS: Lapatinib was given orally once a day at doses ranging from 1200 to 1800 mg in a phase I study. CT and FDG-PET were performed before treatment, and at 1, 2 and 3 months after the initiation of the treatment and every 2 months thereafter. RESULTS: A total of 29 FDG-PET examinations were performed in eight patients with various solid tumors and the metabolic activity in the tumor was evaluated as SUVmax. The best responses, as assessed by CT, were as follows; one partial response, four stable disease and three disease progression. The partial response was observed in a patient with trastuzumab-resistant breast cancer, whose SUVmax was decreased by 60% from baseline. In all of the four patients whose best response was stable disease, the SUVmax was decreased by 6-42% one month after the start of treatment. Prolonged stable disease (10 months) was observed in a patient with colon cancer, whose SUVmax was decreased by 42%. In the patient group with disease progression, SUVmax was increased in two out of three patients. CONCLUSIONS: FDG-PET detected decreases in the metabolic activity of the tumors in patients who experienced clinical benefits on treatment with lapatinib. Thus, FDG-PET may be useful for the evaluation of molecular targeted drugs, such as lapatinib.

Adult↗

PubMed2006

[Examination of PET image evaluation experimentation method aiming at improved accuracy of data acquisition].

Multiple data evaluation is desirable for data obtained by positron emission tomography (PET), as the data follow the Poisson distribution. Such evaluation, however, tends to be very complicated, since the count- rates change with nuclide decay. To solve this problem, we propose a new data scan protocol in this communication. With this method, the true+scatter coincidence counts were computed during the initial one-minute scanning, which was fixed as the standard. A dynamic scan was then performed with the fixed counts from the high count-rate region. Regions with +/-2.5% of the image noise of the standard image was chosen to provide the data for evaluation. These were found to be the regions of 16.5-25.5 kcps (2D) and 81.1-138.5 kcps. Image quality was found to be affected by noise (2D) and random coincidence. Using this method, multiple data could be obtained by a single experiment, and very reliable image evaluation could be done.

Positron-Emission Tomography↗

PubMed2006

The identification of catalytic pentad in the haloalkane dehalogenase DhmA from Mycobacterium avium N85: reaction mechanism and molecular evolution.

Haloalkane dehalogenase DhmA from Mycobacterium avium N85 showed poor expression and low stability when produced in Escherichia coli. Here, we present expression DhmA in newly constructed pK4RP rhodococcal expression system in a soluble and stable form. Site-directed mutagenesis was used for the identification of a catalytic pentad, which makes up the reaction machinery of all currently known haloalkane dehalogenases. The putative catalytic triad Asp123, His279, Asp250 and the first halide-stabilizing residue Trp124 were deduced from sequence comparisons. The second stabilizing residue Trp164 was predicted from a homology model. Five point mutants in the catalytic pentad were constructed, tested for activity and were found inactive. A two-step reaction mechanism was proposed for DhmA. Evolution of different types of catalytic pentads and molecular adaptation towards the synthetic substrate 1,2-dichloroethane within the protein family is discussed.

Catalytic Domain↗

PubMed2006

Involvement of Ca2+-independent phospholipase A2 in the translocation of hypoxia-inducible factor-1alpha to the nucleus under hypoxic conditions.

We investigated the role of Ca2+-independent phospholipase A2 (iPLA2) as well as cytosolic phospholipase A2 (cPLA2) in hypoxia-inducible factor-1 (HIF-1)-dependent gene expression. An inhibitor of both iPLA2 and cPLA2, methyl arachidonyl fluorophosphonate (MAFP), prevented hypoxia-induced erythropoietin mRNA expression without affecting HIF-1alpha accumulation in Hep3B cells. The DNA-binding of HIF-1alpha was suppressed by MAFP as confirmed by luciferase reporter gene assays with the hypoxia response element. Translocation of HIF-1alpha to the nucleus assessed by its presence in the nuclear extracts of cells exposed to hypoxia, was diminished by MAFP. However, hypoxia-dependent gene expression was not affected in mesangial cells obtained from cPLA2alpha null mice. Furthermore, a specific iPLA2 inhibitor, bromoenol lactone, suppressed erythropoietin mRNA expression and HIF-1alpha translocation to the nucleus under hypoxic conditions. Thus, iPLA2, but not cPLA2alpha, may play an important role in regulating the transport of HIF-1alpha to the nucleus.

Active Transport, Cell Nucleus↗

PubMed2006

Molecular cloning and characterization of a novel human beta1,3-glucosyltransferase, which is localized at the endoplasmic reticulum and glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain.

Protein O-linked fucosylation is an unusual glycosylation associated with many important biological functions such as Notch signaling. Two fucosylation pathways synthesizing O-fucosylglycans have been reported on cystein-knotted proteins, that is, on epidermal growth factor-like (EGF-like) domains and on thrombospondin Type 1 repeat (TSR) domains. We report here the molecular cloning and characterization of a novel beta1,3-glucosyltransferase (beta3Glc-T) that synthesizes a Glcbeta1,3Fucalpha- structure on the TSR domain. We found a novel glycosyltransferase gene with beta1,3-glycosyltransferase (beta3GT) motifs in databases. The recombinant enzyme expressed in human embryonic kidney 293T (HEK293T) cells exhibited glucosyltransferase activity toward fucose-alpha-para-nitrophenyl (Fucalpha-pNp). Thin-layer chromatography (TLC) analysis revealed that the product of the recombinant enzyme migrated to the same position as did the product of endogenous beta3Glc-T of Chinese hamster ovary (CHO) cells. The two products could be digested by beta-glucosidase from almond and by exo-1,3-beta-glucanase from Trichoderma sp. These results strongly suggested that the product has the structure of Glcbeta1-3Fuc. Therefore, we named this novel enzyme beta3Glc-T. Immunostaining revealed that FLAG-tagged beta3Glc-T is an enzyme residing in the endoplasmic reticulum (ER) via retention signal, "REEL," which is a KDEL-like sequence, at the C-terminus. The TSR domain expressed in Escherichia coli was first fucosylated by the recombinant protein O-fucosyltransferase 2 (POFUT2), after which it became an acceptor substrate for the recombinant beta3Glc-T, which could apparently transfer Glc to the fucosylated TSR domain. Our results suggest that a novel glycosyltransferase, beta3Glc-T, contributes to the elongation of O-fucosylglycan and that this occurs specifically on TSR domains.

Amino Acid Sequence↗

PubMed2006

Susceptibility of brain microvascular endothelial cells to advanced glycation end products-induced tissue factor upregulation is associated with intracellular reactive oxygen species.

There is accumulating evidence that advanced glycation end products (AGEs) are relevant to the formation of vascular complications in diabetes mellitus. The aim of this study was to investigate whether AGEs have a significant effect on tissue factor (TF) expression in brain microvascular endothelial cells compared with that in other arterial endothelial cells. Cultured bovine brain microvascular endothelial cells (BBMECs) and aortic endothelial cells (BAECs) were incubated in medium containing glyceraldehyde-derived AGE (glycer-AGE). TF mRNA expression, protein expression, and activity were measured at multiple time points after glycer-AGE incubation. Participation of reactive oxygen species (ROS) in the effect of glycer-AGE on TF expression was investigated by treatment with a free radical scavenger, edaravone, and intracellular ROS measurements with dihydroethidium (DHE). Basic TF mRNA expression was greater in BBMECs than in BAECs. Glycer-AGE significantly upregulated TF mRNA expression in both cells, and the upregulation was more prominent in BBMECs than in BAECs. TF protein expression and activity were also upregulated with a pattern of being greater in BBMECs than in BAECs. Edaravone significantly attenuated the AGE-induced upregulation of TF mRNA expression, protein expression, and activity. Intracellular ROS levels measured with DHE-stained fluorescent intensity were significantly upregulated by glycer-AGE with a pattern of being greater in BBMECs than in BAECs. AGE-induced ROS upregulation was attenuated by edaravone like AGE-induced TF upregulation. These results suggest that brain microvascular endothelial cells are more susceptible to AGE-induced TF upregulation than aortic endothelial cells, and that this susceptibility is associated with levels of intracellular ROS.

Animals↗

PubMed2006

Heme oxygenase-1, a potential biomarker of chronic silicosis, attenuates silica-induced lung injury.

RATIONALE: Heme oxygenase-1 (HO-1), a rate-limiting enzyme in heme catabolism, has antioxidative, antiapoptotic, and antiinflammatory activities. We examined whether HO-1 might be involved in silicosis. OBJECTIVES: To investigate whether HO-1 can reduce silicosis in mice and humans. METHODS AND MEASUREMENTS: Silicosis was studied using a murine model, and in 46 male patients. Serum HO-1 and 8-hydroxydeoxyguanosine (a marker of oxidative stress) were measured by enzyme-linked immunosorbent assay. Levels of HO-1 were measured by immunohistochemistry and immunoblotting. MAIN RESULTS: Serum HO-1 levels were significantly elevated in patients with silicosis compared with age-matched control subjects or patients with chronic obstructive pulmonary disease. Serum HO-1 levels also correlated inversely with serum 8-hydroxydeoxyguanosine levels and positively with vital capacity and forced expiratory volume in one second in patients with silicosis. HO-1 was present in the lungs of humans and mice with silicosis, especially at sites of silica particle deposition. In mice, silica exposure was associated with acute leukocyte infiltration, leading to development of silicotic lung lesions. The inflammation was suppressed by treatment with hemin, an inducer of HO-1, and enhanced by zinc protoporphyrin, an inhibitor of HO-1. CONCLUSIONS: Pulmonary HO-1 expression is increased in silicosis. HO-1 suppresses reactive oxygen species activity, and subsequent pathologic changes, thereby attenuating disease progression.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2006

Acceleration of matrix metalloproteinase-1 production and activation of platelet-derived growth factor receptor beta in human coronary smooth muscle cells by oxidized LDL and 4-hydroxynonenal.

Increases in matrix metalloproteinases (MMPs) at atherosclerotic lesions are involved in the migration of smooth muscle cells (SMCs) into the intima and to the rupture of plaques, being implicated in the progression of atherosclerosis. The present study examined the mechanisms underlying the production of MMP-1, interstitial collagenase-1, induced by oxidized low-density lipoprotein (oxLDL) and 4-hydroxynonenal (4-HNE), factors proposed to play a pivotal role in atherogenesis, in human coronary SMCs. oxLDL promoted the production of MMP-1 with the preceding phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Immunoprecipitation of platelet-derived growth factor receptor beta (PDGFR-beta) revealed that oxLDL induced tyrosine phosphorylation of the receptor. Inhibition of the activation of PDGFR-beta and ERK1/2 resulted in a suppression of the production of MMP-1. Consistently, 4-HNE also elicited the production of MMP-1 with the preceding phosphorylation of PDGFR-beta and ERK1/2. The 4-HNE-induced production of MMP-1 was prevented when the activation of PDGFR-beta and ERK1/2 was inhibited. The present results suggest that the activation of PDGFR-beta and ERK1/2 is involved in the production of MMP-1 in oxLDL- and 4-HNE-stimulated human coronary SMCs.

Aldehydes↗

PubMed2006

Apical Golgi localization of N,N'-diacetyllactosediamine synthase, beta4GalNAc-T3, is responsible for LacdiNAc expression on gastric mucosa.

beta1,4-N-acetylgalactosaminyltransferase III (beta4GalNAc-T3), which was recently cloned and identified, exhibits GalNAc transferase activity toward a GlcNAcbeta residue with beta1,4-linkage, forming the N,N'-diacetyllactosediamine, GalNAcbeta1,4GlcNAc (LacdiNAc or LDN). Though LacdiNAc has not been found in the gastric mucosa, a large amount of transcript was detected in our previous study. To increase our knowledge of beta4GalNAc-T3 expression and its product LacdiNAc, we examined the exact localization of beta4GalNAc-T3 in human gastric mucosa using a newly developed antibody, monoclonal antibody (mAb) K1356. This antibody specifically detected the enzyme that transfected the beta4GalNAc-T3 gene into MKN45 cells, and the terminal betaGalNAc epitope yielded on the cell surface was recognized by a lectin, Wisteria floribunda agglutinin (WFA). beta4GalNAc-T3 was localized in the supra-nuclear region of surface mucous cells in gastric mucosa, and WFA positively stained the mucins secreted by the cells. In contrast, in the cells of the glandular compartment in the fundic glands and a few cells in the pyloric glands, beta4GalNAc-T3 was observed in the basolateral position of the nucleus, where no WFA reactivity was detected. The anti-Tn (GalNAcalpha-O-Ser/Thr) antibody staining did not overlap with the WFA staining. By measuring the binding activity of WFA using automated frontal affinity chromatography (FAC), we found WFA to bind most strongly LacdiNAc among the sugar chains examined. Neither beta4GalNAc-T3 nor WFA-positive staining was detected in intestinal metaplastic cells. These results suggest that the supra-nuclear expression of beta4GalNAc-T3 is essential for the formation of LacdiNAc on the surface mucous cells and that LacdiNAc and beta4GalNAc-T3 are novel differentiation markers of surface mucous cells in the gastric mucosa.

Antigens, Differentiation↗

PubMed2006

Progressive hearing loss in mice carrying a mutation in the p75 gene.

The neurotrophin receptor p75 (p75NTR) is expressed in the developmental stage of the cochlea. However, the role of the p75NTR in the inner ear remains to be established. In this study, we conducted electrophysiological and morphological analyses of the auditory function of mice carrying a mutation in the p75 gene at different longitudinal stages. The mice carrying a mutation in the p75 gene showed an age-related progressive hearing loss. At 1 month, there was no obvious morphological change in the cochlea of the mice carrying a mutation in the p75 gene compared to wild-type mice, except for a slight loss of spiral ganglion neurons (SGNs). Auditory function was not significantly different between both genotypes from 1 to up to 4 months of age. The mice carrying a mutation in the p75 gene started to show progressive hearing loss at 4 months, when both SGN degeneration and hair cell (HC) loss were observed at the basal turn. These results suggest that the neurotrophin receptor p75 may play a significant role in the maintenance of cochlear function, and that mice carrying a mutation in the p75 gene could be a good animal model of early onset progressive hearing loss.

Acoustic Stimulation↗

PubMed2006

Usefulness of mitral annular velocity in predicting exercise tolerance in patients with impaired left ventricular systolic function.

Left ventricular (LV) diastolic function is 1 of the determinants of exercise tolerance. However, the relation between early diastolic velocity of the mitral annulus (Ea) obtained by tissue Doppler imaging and exercise tolerance is unknown in patients with impaired LV systolic function. To investigate the feasibility of evaluating exercise tolerance using tissue Doppler imaging, we studied 53 consecutive patients (mean age 58 +/- 14 years) with a LV ejection fraction of <50% (mean 37 +/- 9%). We measured the peak early diastolic velocity of transmitral flow (E) and Ea at the lateral border of the mitral annulus and then calculated the E/Ea ratio. After echocardiography, we measured the peak oxygen consumption and anaerobic threshold (AT) by cardiopulmonary exercise testing. Of all the echocardiographic parameters, the best correlation for AT was the E/Ea ratio (r = -0.74, p <0.001). Peak oxygen consumption correlated well with Ea and the E/Ea ratio (r = 0.64 and r = -0.68, respectively, p <0.001). The AT and peak oxygen consumption did not correlate with conventional Doppler indexes. Using an AT of 8 ml/min/kg as the cutoff to separate severe exercise intolerance from normal, mild, or moderate exercise intolerance, a receiver-operating characteristic curve showed that an E/Ea ratio of >11.3 had the best combination of sensitivity (88%) and specificity (86%). Exercise tolerance correlated with the E/Ea ratio in patients with impaired LV systolic function. In conclusion, the evaluation of LV diastolic function using tissue Doppler imaging is useful for predicting exercise tolerance in patients with heart failure.

Adult↗

PubMed2006

Infants with acute lymphoblastic leukemia and a germline MLL gene are highly curable with use of chemotherapy alone: results from the Japan Infant Leukemia Study Group.

Although infants with acute lymphoblastic leukemia (ALL) and a germline MLL gene have a better prognosis than comparable infants with a rearranged MLL gene, their optimal therapy is controversial. In 2 consecutive studies, conducted between 1996 and 2002, we treated 22 cases of infant ALL with germline MLL using chemotherapy alone. The 5-year event-free survival rate was 95.5% with a 95% confidence interval of 86.9 to 100%. All 21 infants with precursor B-cell ALL have been in first complete remission for 3.5 to 8.8 years. Most treatment-related toxicities were predictable and well tolerated, and neither secondary malignancies nor physical growth impairments have been observed. These results indicate that chemotherapy of the type described here is both safe and highly effective against infant precursor B-cell ALL with MLL in the germline configuration.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2006

Comprehensive enzymatic characterization of glycosyltransferases with a beta3GT or beta4GT motif.

Bioinformatics is a very powerful tool in the field of glycoproteomics, as well as genomics and proteomics. The bioinformatics technique accelerates the comprehensive identification and in silico cloning of human glycogenes containing glycosyltransferases, glycolytic enzymes, sugar-nucleotide synthetases, sugar-nucleotide transporters, and so forth. Glycosyltransferase genes play central roles in carbohydrate chain biosynthesis and have been analyzed for their biological functions. At present, over 180 human glycosyltransferases were identified, cloned, and expressed in various expression systems to detect the activity for carbohydrate synthesis. The recombinant proteins for glycosyltransferase were successfully identified for their enzyme activities and substrate specificities. Their substrate specificities were determined using various donor substrates and acceptors. This section reviews the functions, substrate specificities, and enzymatic reactions of glycosyltransferases such as beta1,3-glycosyltransferase family and beta1,4-glycosyltransferase family.

Amino Acid Motifs↗

PubMed2006

Application of lectin microarray to crude samples: differential glycan profiling of lec mutants.

We recently developed a novel system for lectin microarray based on the evanescent-field fluorescence-detection principle, by which even weak lectin-oligosaccharide interactions are detectable without a washing procedure. For its practical application, cell glycan analysis was performed for Chinese hamster ovary (CHO) cells and their glycan profile was compared with those of their glycosylation-defective Lec mutants. Each of the cell surface extracts gave a significantly different profile from that of the parental CHO cells in a manner reflecting denoted biosynthetic features. Hence, the developed lectin microarray system is considered to be fully applicable for differential glycan profiling of crude samples.

Animals↗

PubMed2006

Analysis of hemodynamics during blood purification therapy using a newly developed noninvasive continuous monitoring method.

Advancement in blood purification therapy extends not only to consoles and dialyzers, but also to patient management during blood purification therapy. However, no monitor has been devised for hemodynamics during blood purification therapy that is carried out continuously and non-invasively. By studying the laser Doppler flowmeter (LDF), we have developed a probe that can continuously measure changes in blood flow in tissues of the head and lower extremities during blood purification therapy. By applying the improved LDF, we have developed a non-invasive continuous monitoring method (NICOMM). Hemodynamics in various types of blood purification therapies were also studied by simultaneously measuring with an automatic oscillometric sphygmomanometer.

Blood Pressure↗

PubMed2006

Detection of norovirus antigens from recombinant virus-like particles and stool samples by a commercial norovirus enzyme-linked immunosorbent assay kit.

The commercial norovirus enzyme-linked immunosorbent assay kit was evaluated for its reactivity to recombinant virus-like particles and the detection of natural viruses from stool samples of Japanese infants and children with sporadic acute gastroenteritis compared to reverse transcription-PCR. The kit had a sensitivity of 76.3% and a specificity of 94.9%. Our results clearly indicated that the kit allows the detection of the most prevalent genotype, GII/4. In order to increase the sensitivity of the kit, the reactivity with norovirus of GII/3 and GII/6 genotypes needs to be improved.

Antigens, Viral↗