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R Iwamoto

Publications and source records attributed to R Iwamoto.

31 records · Page 2Linked to original sources
PubMed1992

The 27-kD diphtheria toxin receptor-associated protein (DRAP27) from vero cells is the monkey homologue of human CD9 antigen: expression of DRAP27 elevates the number of diphtheria toxin receptors on toxin-sensitive cells.

Diphtheria toxin (DT) receptor associates with a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A human-mouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.

Amino Acid Sequence↗
PubMed1992

Intraoperative radiation therapy.

1. Local and regional recurrence of disease is a major problem in cancer management. Intraoperative radiation therapy (IORT) combines surgery with irradiation to treat any disease remaining after surgery and to decrease the local recurrence rates for certain cancers. 2. The development of an IORT treatment program is often undertaken by groups and institutions with expertise in multidisciplinary oncology, particularly surgical and radiation oncology. The program uses multiple resources, requiring cooperation and teamwork to achieve an efficient procedure with maximum patient outcome. 3. The process should be reviewed and modified each time it is performed. There is no "standard" way to use IORT, and every program will have variations that adapt to the circumstances existing in each specific facility.

Combined Modality Therapy↗
PubMed1991

Purification of diphtheria toxin receptor from Vero cells.

Diphtheria toxin receptor has been solubilized from Vero cell membranes with octyl beta-D-glucoside. CRM197, the product of a mutated diphtheria toxin gene, was used for the identification of the receptor. The binding activity of the solubilized receptor was assayed by precipitating the receptor with acetone in the presence of phospholipids and carrier proteins. The solubilized receptor was purified by the combination of several chromatographic steps in the presence of the detergent, resulting in about a 10(6)-fold purification of the receptor. The purified receptor showed essentially a single band of 14.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When partially purified receptor fractions were subjected to ligand blotting analysis using 125I-CRM197 as the probe, the 14.5-kDa protein and a few minor protein bands were identified as diphtheria toxin-binding molecules. These results show clearly that the 14.5-kDa protein is the diphtheria toxin receptor, or at least the major diphtheria toxin-binding molecule. When partially purified receptor was applied to a Sephacryl S-300 column in the presence of detergent, the receptor was eluted in the fractions corresponding to the 60-90-kDa size range. This suggests that the protein forms a complex with itself or with another protein.

Animals↗
PubMed1991

An antibody that inhibits the binding of diphtheria toxin to cells revealed the association of a 27-kDa membrane protein with the diphtheria toxin receptor.

A monoclonal antibody that blocks the binding of diphtheria toxin to Vero cells was isolated by immunizing mice with Vero cell membrane. The antibody inhibits the binding of diphtheria toxin and also CRM197, a mutant form of diphtheria toxin, to Vero cells, and consequently inhibits the cytotoxicity of diphtheria toxin. This antibody does not directly react with the receptor molecule of diphtheria toxin (DTR14.5). Immunoprecipitation and immunoblotting studies revealed that this antibody binds to a novel membrane protein of 27 kDa (DRAP27). When diphtheria toxin receptor was passed through an affinity column made with this antibody, the receptor was trapped only in the presence of DRAP27. These results indicate that DRAP27 and DTR14.5 closely associate in Vero cell membrane and that the inhibition of the binding of diphtheria toxin to the receptor is due to the binding of the antibody to the DRAP27 molecule. Binding studies using 125I-labeled antibody showed that there are many more molecules of DRAP27 on the cell surface than diphtheria toxin-binding sites. However, there is a correlation between the sensitivity of a cell line to diphtheria toxin and the number of DRAP27 molecules on the cell surface, suggesting that DRAP27 is involved in the entry of diphtheria toxin into the target cell.

Animals↗
PubMed1991

Direct evidence of the Entner-Doudoroff pathway operating in the metabolism of D-glucosamine in bacteria.

Pseudomonas fluorescens (Migula) (IFO 14808) has both a membrane-bound PQQ-dependent D-glucose (D-Glc) dehydrogenase [EC 1.1.99.17] [which also acts on D-glucosamine (D-GlcN)] and a PLP-dependent D-glucosaminate (D-GlcNA) dehydratase [EC 4.2.1.26]. Further, these two enzymes were induced when D-GlcN was added to the culture medium. However, D-glucosamine-6-phosphate (D-GlcN-6-P) isomerase [EC 5.3.1.10], another enzyme involved in the metabolism of D-GlcN, was only present at a low level in this bacterium. The bacterium was able to grow in a minimal medium containing D-GlcN or D-GlcNA as the sole source of carbon and nitrogen. Intact cells of P. fluorescens (Migula) converted D-GlcN to D-GlcNA and then to 2-keto-3-deoxy-D-gluconate (KDGA). These results demonstrate that D-GlcN is metabolized via D-GlcNA to KDGA in P. fluorescens (Migula) (Entner-Doudoroff pathway). In contrast, Enterobacter cloacae(IFO 13535) and Agrobacterium radiobacter (IAM 1526) have significant amounts of D-GlcN-6-P isomerase with low levels of the D-Glc dehydrogenase and D-GlcNA dehydratase. Further, only the isomerase activity was induced on the addition of D-GlcN to the culture medium. These results demonstrate that there is a new route (Entner-Doudoroff pathway), i.e., in addition to the known one (Embden-Meyerhof pathway), for the metabolism of D-GlcN in bacteria and one of the two routes is predominant in the each of bacteria examined.

Aldose-Ketose Isomerases↗
PubMed1988

Antibodies to Asp-Asp-Glu-Asp can inhibit transport of nuclear proteins into the nucleus.

The signal sequence of simian virus 40 (SV40) large T-antigen for translocation into the nucleus is composed of positively charged amino acids Lys-Lys-Lys-Arg-Lys. Rabbit antibodies to a synthetic peptide containing the negatively charged amino acid sequence Asp-Asp-Asp-Glu-Asp were obtained. Indirect immunofluorescence of the antigens recognized by the antibody was punctate at the nuclear rim or the nuclear surface, depending on the plane of focus. The antibody blocked transport of nuclear proteins into the nucleus. The antigens recognized by the antibody were predominantly localized to the nuclear pores.

Amino Acid Sequence↗
PubMed1988

ATP-dependent association of nuclear proteins with isolated rat liver nuclei.

In vitro association of Xenopus nucleoplasmin and mammalian nonhistone chromosomal high mobility group 1 (HMG1) protein with nuclei isolated from rat liver was examined. Efficient association of nuclear proteins with isolated nuclei requires ATP, HCO3-, and Ca2+. Association occurred at 33 degrees C but not at 4 degrees C. ATP could be replaced by adenosine 5'-[alpha,beta-methylene]triphosphate (pp[CH2]pA), a nonhydrolyzable ATP analog. pp[CH2]pA associated with nuclei at 33 degrees C and nucleoplasmin and HMG1 rapidly associated with the pp[CH2]pA-bound nuclei at 4 degrees C. Competition studies showed that these associations at both 33 degrees C and 4 degrees C were specific. More than 80% of the bindings of nuclear proteins to the nuclear surface were blocked by wheat germ agglutinin.

Adenosine Triphosphate↗
PubMed1986

Quantitative surface analysis of Cardiothane 51 by FT-IR-ATR spectroscopy.

Surface analysis has been carried out for Cardiothane 51 samples as commercially available balloon pumps and cast films prepared from the solution. Silicone contents on surfaces have been quantitatively determined by FT-IR-ATR spectroscopy coupled with a subtraction technique. The contents on the air-facing surface vary from 0.7 to 5.3% among six balloon pumps examined; three of them contain less silicone on the air-facing surface than on the substrate-facing surface, whereas just the opposite is true for one, and the other two have nearly and quite an equal content of it between the two surfaces. Silicone contents on surfaces of cast films vary also from a sample to another even when sample films were prepared from the same Cardiothane solution, indicating difficulty in quality control of Cardiothane surfaces. An appreciable fraction of silicone was found not to be covalently bonded to polyurethane in Cardiothane 51 for the cast film samples.

Biocompatible Materials↗
PubMed1984

D-glucosaminate dehydratase: spectrometric properties of the enzyme-bound pyridoxal 5'-phosphate.

The holoenzyme of D-glucosaminate dehydratase [EC 4.2.1.26] from Agrobacterium radiobacter showed absorption peaks at 280 and 415 nm with a shoulder in the region of 320 to 330 nm. The treatment of the enzyme with hydroxylamine followed by dialysis led to disappearance of both the absorption peak at 415 nm and the shoulder, giving the apoenzyme. The fluorescence excitation maximum of the holoenzyme was at 320 nm with a shoulder at 420 nm (emission at 510 nm), and the emission maxima were at 420 nm with a shoulder at 370 nm (excitation at 320 nm) and at 510 nm (excitation at 420 nm). The holoenzyme showed a negative circular dichroic band at 418 nm and a positive shoulder at around 320 nm. Reduction of the holoenzyme with sodium borohydride caused a loss of the absorption peak at 415 nm with a concomitant increase of 325 nm absorbance and an irreversible loss of the activity. The occurrence of epsilon-N-pyridoxyllysine in the acid hydrolysate of the reduced enzyme showed that D-glucosaminate dehydratase contains a catalytically essential lysine residue whose epsilon-amino group binds the 4-formyl group of pyridoxal 5'-phosphate to form a Schiff base. The plots of absorption of the apoenzyme against the amount of pyridoxal 5'-phosphate added showed that four and two molar equivalents of the cofactor bind to the apoenzyme and subunit, respectively. The biphasic nature of the spectrometric titration curve of the apoenzyme with pyridoxal 5'-phosphate and the two Km values obtained for the cofactor suggest the occurrence of two distinct types of binding sites for pyridoxal 5'-phosphate in the enzyme.

Circular Dichroism↗
PubMed1983

Stereochemistry of an alpha, beta-elimination reaction by D-glucosaminate dehydratase.

The proton NMR analysis of D-glucosaminate dehydratase reaction in D2O revealed the incorporation of a deuterium atom at C-3 carbon of the product, 2-keto-3-deoxy-D-gluconate. Based on the chemical shift of C-3 proton of the product and the coupling constant characteristic for the C-3 and C-4 axial-axial coupling in the 2C5 pyranose conformation, the deuterium is in the pro-S position. Thus, the dehydration of D-glucosaminate by the enzyme proceeds in a retention mode at C-3 carbon. Kinetic parameters show that the rate-determining step is the abstraction of alpha-proton from the substrate.

Deuterium↗
PubMed1982

D-glucosaminate dehydratase from Agrobacterium radiobacter. Physicochemical and enzymological properties.

The bacterial distribution of D-glucosaminate dehydratase [EC 4.2.1.26] was investigated and Agrobacterium radiobacter (IAM 1526) was found to have the highest enzyme activity. The enzyme was formed inducibly in a glycerol-urea medium by D-glucosamine, D-galactosamine, and D-glucosamine, but not by D-mannosamine. The enzyme purified from the cells grown in the glucosamine-glycerol-urea medium was shown to be homogeneous by ultracentrifugation. The molecular weight was determined to be about 66,000 by the sedimentation equilibrium method, and 72,800 by the gel permeation chromatography low-angle light scattering method. The pH optimum is 8.3-9.0. The enzyme catalyzed the dehydration of D-glucosaminate (relative activity: 100, Km: 2.8 mM), D-galactosaminate (31.5, 5.0 mM), D-mannosaminate (17.5, 29 mM), D-threonine (5.1, 4.8 mM), D-serine (3.2, 0.026 mM), and L-serine (1.1, ND), but not L-threonine. The reverse reaction does not occur. The enzyme is inhibited by typical inhibitors of pyridoxal 5'-phosphate enzymes, such as L'penicillamine, and also by carbonyl reagents, thiol reagents, divalent metals, and several D-amino acids and D-amino sugars.

Drug Stability↗