[Molecular mechanism of stop codon recognition by eRF1: a wobble hypothesis for peptide anticodons].
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Amino Acid Sequence↗
Biomedical subjects
T Muramatsu
Publications and source records attributed to T Muramatsu.
Mechanism of superoxide anion generation in the toxic red tide phytoplankton Chattonella marina: possible involvement of NAD(P)H oxidase.
Red tide phytoplankton Chattonella marina is known to produce reactive oxygen species (ROS), such as superoxide anion (O(2)(-)), hydrogen peroxide (H(2)O(2)) and hydroxyl radical (&z.rad;OH), under normal physiological conditions. Although several lines of evidence suggest that ROS are involved in the mortality of fish exposed to C. marina, the mechanism of ROS generation in C. marina remains to be clarified. In this study, we found that the cell-free supernatant prepared from C. marina cells showed NAD(P)H-dependent O(2)(-) generation, and this response was inhibited by diphenyleneiodonium, an inhibitor of mammalian NADPH oxidase. When the cell-free supernatant of C. marina was analyzed by immunoblotting using antibody raised against the human neutrophil cytochrome b558 large subunit (gp91phox), a main band of approximately 110 kDa was detected. The cell surface localization of the epitope recognized with this antibody was also demonstrated in C. marina by indirect immunofluorescence. Furthermore, Southern blot analysis performed on genomic DNA of C. marina with a probe covering the C-terminal region of gp91phox suggested the presence of a single-copy gene coding for gp91phox homologous protein in C. marina. These results provide evidence for the involvement of an enzymatic system analogous to the neutrophil NADPH oxidase as a source of O(2)(-) production in C. marina.
Amino Acid Sequence↗
Randomized comparison of primary stenting and provisional balloon angioplasty guided by flow velocity measurement. Doppler Endpoints Balloon Angioplasty Trial Europe (DEBATE) II Study Group.
BACKGROUND: Coronary stenting improves outcomes compared with balloon angioplasty, but it is costly and may have other disadvantages. Limiting stent use to patients with a suboptimal result after angioplasty (provisional angioplasty) may be as effective and less expensive. METHODS AND RESULTS: To analyze the cost-effectiveness of provisional angioplasty, patients scheduled for single-vessel angioplasty were first randomized to receive primary stenting (97 patients) or balloon angioplasty guided by Doppler flow velocity and angiography (523 patients). Patients in the latter group were further randomized after optimization to either additional stenting or termination of the procedure to further investigate what is "optimal." An optimal result was defined as a flow reserve >2.5 and a diameter stenosis <36%. Bailout stenting was needed in 129 patients (25%) who were randomized to balloon angioplasty, and an optimal result was obtained in 184 of the 523 patients (35%). There was no significant difference in event-free survival at 1 year between primary stenting (86.6%) and provisional angioplasty (85.6%). Costs after 1 year were significantly higher for provisional angioplasty (EUR 6573 versus EUR 5885; P:=0.014). Results after the second randomization showed that stenting was also more effective after optimal balloon angioplasty (1-year event free survival, 93.5% versus 84.1%; P:=0. 066). CONCLUSIONS: After 1 year of follow-up, provisional angioplasty was more expensive and without clinical benefit. The beneficial value of stenting is not limited to patients with a suboptimal result after balloon angioplasty.
Analysis of Variance↗
Randomized comparison of elective stent implantation and coronary balloon angioplasty guided by online quantitative angiography and intracoronary Doppler. DESTINI Study Group (Doppler Endpoint STenting INternational Investigation).
BACKGROUND: The purpose of this study was to compare long-term outcomes of coronary stenting in all lesions (elective stenting) or only in lesions with inadequate morphological and functional results after balloon angioplasty (guided PTCA). METHODS AND RESULTS: Treatment of multivessel disease, with any lesion length and vessel size, was allowed provided that all lesions were suitable for stent implantation. Patients were randomized to elective stent implantation (n=370) or guided PTCA (n=365). An optimal PTCA result (residual diameter stenosis </=35%, coronary flow reserve measured with a Doppler guidewire >2.0, absence of threatening dissections) was achieved in 166 lesions (43%). The remaining 218 lesions underwent stent implantation (provisional stenting). Final residual diameter stenosis was lower in the elective and provisional stent groups (9.3% and 10.2%) than in the optimal PTCA group (24.8%, P:<0. 00001). On an intention-to-treat analysis, the probability of >/=1 major adverse cardiac event at 12 months was 17.8% in the elective stenting group and 18.9% in the guided PTCA group (20.1% for optimal PTCA and 18.0% for the provisional stenting subgroup, P:=NS). The incidence of repeat target lesion revascularization at 1 year was 14. 9% in the elective stent group and 15.6% in the guided PTCA group (17.6% for optimal PTCA and 14.1% for the provisional stenting subgroup, P:=NS). CONCLUSIONS: When balloon angioplasty is guided by online quantitative angiography and Doppler-derived coronary flow reserve, with provisional stenting reserved for suboptimal results, early and late clinical outcomes are comparable to those achieved by elective stenting of all patients.
Angiography↗
Neuronal cell adhesion, mediated by the heparin-binding neuroregulatory factor midkine, is specifically inhibited by chondroitin sulfate E. Structural ans functional implications of the over-sulfated chondroitin sulfate.
The heparin-binding neurotrophic factor midkine (MK) has been proposed to mediate neuronal cell adhesion and neurite outgrowth promotion by interacting with cell-surface heparan sulfate. We have observed that over-sulfated chondroitin sulfate (CS) D and CS-E show neurite outgrowth-promoting activity in embryonic day (E) 18 rat hippocampal neurons (Nadanaka, S., Clement, A., Masayama, K., Faissner, A., and Sugahara, K. (1998) J. Biol. Chem. 273, 3296-3307). In the present study, various CS isoforms were examined for their ability to inhibit the MK-mediated cell adhesion of cortical neuronal cells in comparison with heparin from porcine intestine and heparan sulfate from bovine kidney. E17-18 rat cortical neuronal cells were cultured on plates coated with recombinant MK in a grid pattern. The cells attached to and extended their neurites along the MK substratum. Cell adhesion was inhibited by squid cartilage over-sulfated CS-E as well as by heparin, but not by heparan sulfate or other CS isoforms. Direct interactions of MK with various glycosaminoglycans were then evaluated using surface plasmon resonance, showing that CS-E bound MK as strongly as heparin, followed by other over-sulfated CS isoforms, CS-H and CS-K. Furthermore, E18 rat brain extracts showed an E disaccharide unit, GlcUAbeta1-3GalNAc(4,6-O-disulfate). These findings indicate that CS chains containing the E unit as well as heparin-like glycosaminoglycans may be involved in the expression and/or modulation of the multiple neuroregulatory functions of MK such as neuronal adhesion and migration and promotion of neurite outgrowth.
Animals↗
Midkine inhibits bradykinin-stimulated Ca(2+) signaling and nitric oxide production in endothelial cells.
Effects of the heparin-binding growth factor midkine (MK) were investigated on endothelial Ca(2+) signaling and nitric oxide (NO) production. Bradykinin (10 nM) and thapsigargin (1 microM) provoked large Ca(2+) influxes under fura-2/AM fluoroscopy. Pretreatment with human MK dose-dependently (1-500 ng/ml) inhibited the Ca(2+) response to bradykinin but not that to thapsigargin. Anti-MK antibody prevented this effect. In Ca(2+)-free medium, MK greatly inhibited intracellular Ca(2+) store release by bradykinin and not that by thapsigargin, which effect was prevented by the antibody. Bradykinin increased NO production by 6.7-fold, which was inhibited 6, 44, 79, and 90% by MK at 1, 10, 100, and 500 ng/ml, respectively. MK did not affect thapsigargin-induced NO production. Our data clearly indicate that MK inhibits bradykinin-induced Ca(2+) response and NO production from endothelial cells.
Animals↗
Midkine promoter-based adenoviral vector gene delivery for pediatric solid tumors.
It is important to develop a system to express therapeutic genes in tumor cells with sufficient selectivity for cancer gene therapy. Midkine (MK) is a newly identified heparin-binding growth factor that is transiently expressed in the early stages of retinoic acid-induced differentiation of embryonal carcinoma cells. It has been reported that many human malignant tumors express high levels of MK mRNA or protein. However, no MK expression is detected in human or mouse liver. These interesting features of MK led us to examine the MK promoter as a candidate for tumor-specific gene expression. We thus developed new recombinant adenoviral (Ad) vectors containing either luciferase reporter gene (AdMKLuc) or herpes simplex thymidine kinase gene (AdMKTK) under the control of the human MK promoter. AdMKLuc achieved relatively high activity in Wilms' tumor (G-401) and neuroblastoma (SK-N-SH) cell lines. In addition, AdMKTK induced marked cell death in response to ganciclovir (GCV) in these same lines. Conversely, very low activity of the MK promoter was observed in mouse liver in vivo compared with the cytomegalovirus promoter. Importantly, AdMKTK + GCV did not induce liver toxicity, whereas substantial toxicity was seen with AdCMVTK + GCV treatment. On the basis of these findings, we conclude that the MK promoter is a candidate tumor-specific promoter for Wilms' tumor or neuroblastoma.
Adenoviridae↗
Diversity of N-acetylglucosamine-6-O-sulfotransferases: molecular cloning of a novel enzyme with different distribution and specificities.
N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) transfers sulfate to the C-6 position of non-reducing N-acetylglucosamine (GlcNAc) residues. We cloned human and mouse cDNAs encoding a novel GlcNAc6ST, designated as GlcNAc6ST-4, which showed sequence identities of 26 to 41% to other GlcNAc6STs. Human organs with strong expression of the enzyme mRNA were the heart, spleen, and ovary, while in the mouse strong expression was detected in the kidney. The enzyme expressed in CHO cells preferentially acted on mannose-linked GlcNAc, while a core 2 mucin-type oligosaccharide and an N-acetyllactosamine oligomer also served as acceptors. The distribution and the specificity of GlcNAc6ST are different from those of GlcNAc6STs identified previously.
Animals↗
Molecular cloning of endo-beta -galactosidase C and its application in removing alpha -galactosyl xenoantigen from blood vessels in the pig kidney.
Galalpha1-3Gal is the major xenoantigenic epitope responsible for hyperacute rejection upon pig to human xenotransplantation. Endo-beta-galactosidase C from Clostridium perfringens destroys the antigenic epitope by cleaving the beta-galactosidic linkage in the Galalpha1-3Galbeta1-4GlcNAc structure. Based on partial peptide sequences of the enzyme, we molecularly cloned the enzyme gene, which encodes a protein with a predicted molecular mass of about 93 kDa. The deduced protein sequence of the enzyme has limited homology in the C-terminal half with endo-beta-galactosidase from Flavobacterium keratolyticus and beta-1,3-glucanases. The enzyme expressed in Escherichia coli removed the alpha-galactosyl epitope nearly completely from pig erythrocytes and from pig aortic endothelial cells. The enzyme-treated endothelial cells in culture were greatly reduced in cell surface antigens, which were recognized by IgM, IgG, or IgA in human sera, and became much less susceptible to complement-mediated cytotoxicity caused by human sera. When the pig kidney was perfused with the enzyme, the vascular endothelial cells became virtually devoid of the alpha-galactosyl epitope, with concomitant decrease in binding to IgM in human plasma. These results demonstrated that the recombinant endo-beta-galactosidase C is a valuable aid in xenotransplantation.
Amino Acid Sequence↗
LDL receptor-related protein as a component of the midkine receptor.
Midkine (MK) is a heparin-binding growth factor with migration-promoting and survival-promoting activities. To identify signaling receptor(s) of MK, membrane glycoproteins with MK-binding activity were isolated from day 13 mouse embryos by lectin- and MK-affinity chromatography. SDS-PAGE followed by protein sequence analysis revealed the presence of LDL receptor-related protein (LRP) and NCAM in the fraction. The dissociation constant of binding between LRP and MK was 3.5 nM. Receptor-associated protein (RAP), which interfered with the binding, inhibited MK-dependent survival of embryonic neurons. Brushin/megalin, which is also a high molecular weight protein belonging to the LDL receptor family, bound to MK less strongly than LRP. These findings suggest that LRP is a component of the receptor complex for MK.
Amino Acid Sequence↗
Secretory leukocyte protease inhibitor is a novel inhibitor of fibroblast-mediated collagen gel contraction.
Cultured epithelial cells, including those from the oral epithelium, have been successfully applied in the promotion of scarless wound healing. Factors released from the epithelial cells are thought to contribute significantly to the beneficial effects. In the conditioned medium of human oral epithelial cells, we found a factor that inhibited fibroblast-mediated collagen gel contraction, an in vitro model of wound healing and scar formation. Biochemical analysis identified the factor to be human secretory leukocyte protease inhibitor (SLPI). Fibroblasts transfected with SLPI cDNA showed reduced gel-contracting activity. SLPI purified from the conditioned medium inhibited gel contraction in a dose-dependent manner, and anti-SLPI antibody counteracted this activity. Upon SLPI treatment, human skin fibroblasts in collagen gel became shorter in length and were inhibited in pseudopodia extension. Furthermore, after SLPI treatment, alpha(1)-integrin immunoreactivity decreased, and cyclic AMP levels increased. Excessive gel contraction was observed when fibroblasts treated with TGF-beta1 and fibroblasts from hypertrophic and from keloid scar tissue were cultured in collagen gel. SLPI was also effective in inhibiting gel contraction in the above three models of scar formation. These results suggest that SLPI may be useful in promoting scarless wound healing.
Adolescent↗
Syndecan-4 deficiency impairs focal adhesion formation only under restricted conditions.
Two domains of fibronectin deliver two different but cooperative signals required for focal adhesion formation. The signal from the cell-binding domain is mediated by integrins, whereas the signal from the heparin-binding domain is recognized by heparan sulfate proteoglycans, of which syndecan-4 has been hypothesized to be involved in focal adhesion formation. We generated mice deficient in syndecan-4 to study its role directly. Even in fibroblasts from syndecan-4-deficient mice, focal adhesions were formed, and actin fibers terminated normally at focal adhesions when they were cultured on coverslips coated with fibronectin or with a mixture of its cell-binding and heparin-binding fragments. However, when the cells were cultured on the cell-binding fragment and the heparin-binding fragment was added to the medium, focal adhesion formation was impaired in the syndecan-4 null fibroblasts as compared with that in wild-type cells. Therefore, syndecan-4 is essential for promoting focal adhesion formation only when the signal of the heparin-binding domain of fibronectin is delivered as a soluble form, most probably from the apical surface. When the signal is delivered as a substratum-bound form, other molecule(s) also participate(s) in the signal reception.
Actins↗
Syndecan-4 deficiency impairs the fetal vessels in the placental labyrinth.
Syndecan-4 is a transmembrane protein bearing heparan sulfate chains, involved in anticoagulation and focal adhesion formation. Here, we revealed that syndecan-4 was expressed in the fetal vessels in the placental labyrinth by in situ hybridization and immunohistochemical staining. At 17.5 gestational days, the area of degenerated fetal vessels in the placental labyrinth was more diffuse and larger in Synd4(-/-) embryos than wild-type controls. Calcium and fibrin(ogen) depositions in the degenerated vessels were also more extensive and more severe in the placentas of Synd4(-/-) embryos. These findings suggest that syndecan-4 deficiency impairs the fetal vessels in the placenta, probably due to a deficit in the anticoagulation mechanism. This article is the first report demonstrating that among a large number of core proteins of heparan sulfate proteoglycans, a defect of a single core protein caused impaired anticoagulation in a specific site.
Animals↗
cDNA cloning of bovine midkine and production of the recombinant protein, which affects in vitro maturation of bovine oocytes.
In the present study, we cloned bovine midkine (bMK) cDNA by RT- and RACE-PCR, and determined its nucleotide sequence. The nucleotide and deduced amino acid sequences of bMK showed a high degree of homology to those of mouse and human MK. Moreover, a large amount of recombinant bMK (rbMK) was produced using a baculovirus expression system and the protein was purified to homogeneity by heparin affinity chromatography. To examine whether treatment with rbMK during in vitro maturation (IVM) of bovine cumulus-enclosed oocytes affects their nuclear maturation and postfertilization development to the blastocyst stage, bovine cumulus-enclosed oocytes obtained from slaughterhouse-derived ovaries were cultured for 24 hr in IVM medium without (control) or with various concentrations (50-400 ng/ml) of rbMK, followed by in vitro fertilization (IVF) and culture. Although rbMK treatment during IVM did not affect the rates of nuclear maturation and postfertilization cleavage of oocytes, rbMK at all experimental concentrations significantly (P < 0.05) increased the blastocyst yields per tested and per cleaved oocyte compared to the control. Next, it was examined whether heparitinase (HTN) treatment of cumulus-enclosed oocytes would affect the enhancing activity of rbMK during IVM on the developmental competence of oocyte after IVF. The enhancing activity of rbMK was completely suppressed by HTN (1.0 mU/ml) treatment. These results indicate that the presence of rbMK during IVM of bovine cumulus-enclosed oocytes can enhance their developmental competence to the blastocyst stage after IVF and suggest that heparan sulfate chains on the cell surface of cumulus cells and/or oocytes are required for bMK to exert its effect.
Amino Acid Sequence↗
Mutation analysis of two Japanese patients with Fanconi-Bickel syndrome.
Fanconi-Bickel syndrome (FBS), or glycogen storage disease type XI, is a rare autosomal recessive disorder characterized by hepatorenal glycogen accumulation, Fanconi nephropathy, and impaired utilization of glucose and galactose. Recently, this disease was elucidated to link mutations in the glucose transporter 2 (GLUT2) gene. Only three mutations in three FBS families have been reported. Therefore, it is important to elucidate mutations in the GLUT2 gene in FBS by answering the question of whether the syndrome is a single gene disease. In this report, we describe two patients in two unrelated families clinically diagnosed with FBS. No mutation in the entire protein coding region of the GLUT2 gene was detected in patient 1, which suggested that no mutation existed in the GLUT2 gene, or that some mutations had affected the expression of the GLUT2 gene. In patient 2, a novel homozygous nonsense mutation (W420X, Trp at codon 420 to stop codon) was detected. These results support the correlation between GLUT2 gene mutation and FBS syndrome. However, many patients must be analyzed to determine whether other genes are involved in FBS.
Adult↗
Expression of connexin 32 and 43 in developing rat submandibular salivary glands.
The expression of the gap-junction proteins connexin 32 and 43 in the developing rat submandibular gland was determined using reverse transcription-polymerase chain reaction and immunohistochemistry. Sprague-Dawley rats from the 17th gestational day through to the 28th postnatal day were used. Connexin 43 gene expression was detected on the 17th gestational day. Connexin 32 gene expression was also detected on the 17th gestational day but less intensely. Immunolabelling for connexin 43 was also found in the developing submandibular gland from the 17th gestational day. Prenatally the most immunoreactive areas for connexin 43 were located in the periphery of terminal tubules, but some staining was discernible between the cells in terminal buds. In the postnatal period, reactive areas were located at both the periphery of acinus-like structures and around the intercalated duct. This is consistent with the known association of connexin 43 with myoepithelial cells. Connexin 32 immunostaining was first detected in the developing submandibular glands on the 18th day of prenatal development. Positive staining was present on the lateral side of the proacinar and mature acinar cells. The number of immunoreactive areas per cell increased during early development followed by a significant decrease perinatally. During postnatal development the density of areas again showed a pattern of increase. These results suggest that connexin 43 is associated with growth and differentiation in the pre- and perinatal periods and also with the contractile function of myoepithelial cells in the postnatal period of the developing submandibular gland. It is also implied that the number of connexin 32-positive areas may correspond to an increase or decrease in the number of proacinar and mature acinar cells.
Animals↗
Removal of terminal alpha-galactosyl residues by endo-beta-galactosidase C, a potent endoglycosidase which acts under physiological pH conditions.
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Animals↗
Suppression of antigen-antibody reaction in xenotransplantation.
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ABO Blood-Group System↗