Hypermethylation of p15(INK4B) gene in a patient with acute myelogenous leukemia evolved from paroxysmal nocturnal hemoglobinuria.
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Acute Disease↗
Biomedical subjects
R Taguchi
Publications and source records attributed to R Taguchi.
Characterization of aminopeptidase N from the brush border membrane of the larvae midgut of silkworm, Bombyx mori as a zinc enzyme.
Three GPI-anchored proteins, aminopeptidase N, alkaline phosphatase and alkaline phosphodiesterase I were released from the midgut brush border membrane of Bombyx mori by phosphatidylinositol-specific phopholipase C and the aminopeptidase N was purified to a homogeneous state. N-terminus and 6 internal sequences, one of which possessed part of zinc-binding motif, showed homology with those from other species. The zinc content in purified aminopeptidase N was estimated as approximately 0.72 mol/mol of the protein and 1,10-phenanthroline completely inhibited the enzyme activity, suggesting zinc requirement for the activity. The aminopeptidase N activity was inhibited not only by probestin and actinonin, but also strongly depressed by amastatin, while leuhistin and bestatin were less inhibitory. These suggest that the active site of aminopeptidase N might be structurally different from those of mammals. Calcium and magnesium ions stimulated the aminopeptidase N activity, but copper ion was rather inhibitory. Zinc ion showed bi-modal effect on the activity, i.e., stimulatory at low concentration, but inhibitory at higher than 100 microM. This inhibition was completely restored by EDTA. These results suggest that the aminopeptidase N possesses two zinc ion-binding sites with high and low affinity as essential and inhibitory one, as well as some regulatory metal-binding sites.
Amino Acid Sequence↗
A hypertensive father, but not hypertensive mother, determines blood pressure in normotensive male offspring through body mass index.
This investigation was to assess the role of genetic loading of hypertensive parents in the determination of blood pressure (BP) in their normotensive offspring. The medical check-up data from 7279 Japanese university students aged 19.22 +/- 0.01 years were analysed of which 641 students had only one hypertensive parent with or without hypertensive grandparents, and from this number 609 cases were available for the present analysis. The BP in the students having only one hypertensive parent were in the normotensive range, but was significantly higher than in those students without hypertensive relatives. Analyses of the data from the students having only one hypertensive parent revealed that systolic BP (SBP) and body mass index (BMI) were higher in the male than in the female students. In addition, there were no differences in BP and BMI between the male students with a hypertensive father and the male students having a normotensive father. However, multivariate analyses revealed that BMI was an independent predictor of SBP solely in the male students having a hypertensive father, but not in the male students having a normotensive father. Such a relationship between BMI and BP determination was not observed in the female students with one hypertensive parent. It is suggested that there are different mechanisms for the determination of BP in normotensive offspring of hypertensive parents, and genetic loading of a hypertensive father plays a critical role in the determination of BP through BMI.
Adolescent↗
Identification of chiro-inositol and its formation by isomerization of myo-inositol during hydrolysis of glycosylphosphatidylinositol-anchored proteins.
myo-Inositol has been believed to be a sole inositol isomer existing in phosphatidylinositol (PI) and related derivatives. In this experiment, chiro-inositol, an inositol isomer other than myo-inositol, was identified in hydrolytic products from several GPI-anchored proteins. The chiro-inositol contents in several different GPI-anchored proteins including 5'-nucleotidase of bovine liver and alkaline phosphatase of mouse NS-1 varied with hydrolytic conditions of these GPI anchor. Isomerization of 20-60% of myo-inositol occurred on the hydrolysis in 6 N HCl solution. Under the hydrolytic conditions of a HCl gas stream in place of solution, however, isomerization was very low (less than 0.1%). Even in the hydrolysis under HCl gas stream, existence of CNBr accelerated the isomerization of inositol in GPI up to 70-95%. In the hydrolysis of phosphatidylinositol or myo-inositol 1-phosphate, however, a significant amount of chiro-inositol was not detected in 6 N HCl solution or in the existence of CNBr under the HCl stream. These facts indicated that isomerization occurred during the hydrolysis of the GPI anchor, when myo-inositol is substituted by glucosamine at 6-OH and is substituted by phosphate at 1-OH. It also suggested that the former identification of chiro-inositol in GPI structure in the various reports might be due to isomerization.
5'-Nucleotidase↗
Release of ectoenzymes from small intestine brush border membranes of mice by phospholipases.
This study investigated ectoenzyme release from small intestine brush border membranes (duodenum and jejunum, Preparation A; ileum, Preparation B) of mice by the action of phosphatidylinositol-specific phospholipase C or glycosyl-phosphatidylinositol-specific phospholipase D. Most of the alkaline phosphatase was solubilized from Preparation A, but about 60% was released from Preparation B. As for alkaline phosphodiesterase I activity, 15 and 10% were released from Preparations A and B, respectively. With Preparation B, octylglucoside treatment followed by phosphatidylinositol-specific phospholipase C or glycosyl-phosphatidylinositol-specific phospholipase D completely solubilized the alkaline phosphatase activity. However, this treatment did not change the ratio of release of alkaline phosphodiesterase I from Preparation A or B. These results indicate that the resistance to alkaline phosphatase found in Preparation B is due to hindered accessibility of the bonding splitting by phosphatidylinositol-specific phospholipase C and not to a modified glycosyl-phosphatidylinositolanchor.
Alkaline Phosphatase↗
Growth inhibition, morphological change, and ectoenzyme release of LLC-PK1 cells by phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis.
Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis added to a culture of LLC-PK1 cells inhibited cell growth by 40%. In contrast with normal cells, the cells cultured in the presence of PI-PLC showed needle-like appendages which seemed to have been formed due to portions of the cell remaining adhered to the culture dish as the cell shrank. When LLC-PK1 cells were treated with PI-PLC, significant amounts of alkaline phosphatase and alkaline phosphodiesterase I were released specifically from the apical surface of the LLC-PK1 cells. Furthermore, PI-PLC treatment caused a delay of enzyme production and dome formation. These data indicate that glycosyl-phosphatidylinositol (GPI)-anchored proteins on the surface of LLC-PK1 cells are important in cell growth and differentiation. Also, the combined use of LLC-PK1 cells and PI-PLC of B. thuringiensis is effective for investigating the function of GPI-anchor proteins.
Alkaline Phosphatase↗
High-level expression of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C by the Bacillus brevis host-vector system.
We succeeded in hyperproduction of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC), using a Bacillus brevis 47 expression system. The recombinant B. thuringiensis PIPLC was expressed under the control of the middle wall protein gene promoter in B. brevis expression vector pNU211. A large amount of recombinant PIPLC (0.4 g per liter culture) was secreted into the medium as a mature enzyme, and the enzymatic properties of purified recombinant PIPLC were similar to those of the enzyme from wild-type B. thuringiensis. This system provides a useful approach to the three-dimensional structure-function relationship of PIPLC.
Bacillus↗
Direct evidence of involvement of glycosylphosphatidylinositol-anchored proteins in the heavy metal-mediated signal delivery into T lymphocytes.
The biological significance of the action of glycosylphosphatidylinositol (GPI)-anchored proteins in cell physiology and pathology when stimulated with their natural agonists is not known. Here we provide evidence that GPI-anchored proteins play a crucial role in the recently defined heavy metal (HgCl2)-triggered signal delivery to T lymphocytes. Thiol-reactive HgCl2, a multi-potent crosslinker of cell membrane proteins, induced heavy aggregation of Thy-1, a representative GPI-anchored protein, on murine thymocytes, and delivered a signal to induce heavy tyrosine phosphorylation of cellular proteins. This rather unusual signal delivery by HgCl2 is diminished by the pre-treatment of cells with phosphatidylinositol-specific phospholipase C, which partially cleaved GPI-anchored proteins from the cell surface. Direct evidence for the involvement of GPI or GPI-anchored proteins in the HgCl2-mediated signaling is provided by the loss of signaling in a mutant thymoma cell line defective in the phosphatidylinositol glycan-class A gene (PIG-A), and its restoration in a transfectant with PIG-A.
Animals↗
Promotion of cytotoxic T-cell generation in mixed leukocyte culture by phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis.
Phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus thuringiensis, which cleaves phosphatidylinositol or glycosylphosphatidylinositol on the external cell surface to generate a second messenger for intracellular signal transduction (S. Rahman et al., FEBS Lett. 303:193-196, 1992), was found to preferentially promote the generation of alloantigen-specific cytotoxic T lymphocytes in mixed leukocyte culture. PIPLC affected an early stage of cytotoxic T-lymphocyte activation in culture, and there was no evidence of any soluble cellular mediators of this PIPLC action. PIPLC neither enhanced overall cell proliferation nor noticeably promoted interleukin-2 and -4 production in mixed leukocyte culture. The relative population size of Ly-2+ T cells was increased, however, in a late mixed leukocyte culture with PIPLC. In addition, PIPLC enhanced an anti-CD3 monoclonal antibody-induced early increase in [Ca2+]i. These results suggest a new parasite (bacterium)-oriented mechanism for enhancing antigen-driven host cytotoxic T-lymphocyte immunity which does not include promotion of interleukin-2 production.
Animals↗
Microheterogeneity in glycosylphosphatidylinositol anchor structures of bovine liver 5'-nucleotidase.
In our study, 5'-nucleotidase was released from bovine liver by the treatment with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and purified to a homogeneous state by concanavalin A-Sepharose and (diethylaminoethyl)-Toyopearl column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified 5'-nucleotidase were then cleaved by cyanogen bromide (CNBr), and then inositol phosphoglycan-containing C-terminal peptides (IPG peptides) were separated by C18 reverse-phase liquid chromatography and analyzed by peptide sequencer, amino acid analyzer, gas chromatography (GC), and GC-mass spectrometry (MS). Ser523 of the amino acid sequence deduced from 5'-nucleotidase cDNA [Suzuki et al. (1993) J. Biochem. (Tokyo) 113, 607-613] is revealed to be the C-terminal amino acid to which a glycosylphosphatidylinositol is anchored. Separated peaks of CNBr-cleaved IPG peptides were then analyzed by electron spray ionization (ESI)-MS. Eight different molecular weight (MW) species of CNBr-cleaved IPG peptides were detected. Three fractions of CNBr-cleaved IPG peptides were separately treated by trypsin, and trypsinized IPG peptides were purified by C18 reverse-phase liquid chromatography. Finally, five different MW species of trypsinized IPG peptides (1629.4, 1752.7, 1791.8, 1832.8, and 1994.5) were detected by ESI-MS. Together with sequential exoglycosidase treatment and quantitative analysis of sugar moieties by GC and GC-MS, microheterogeneity in the structures of these five glycosylphosphatidylinositol (GPI) anchor species was determined. The common core structure was ethanolamine phosphate-mannose-mannose-mannose(-ethanolamine phosphate)-glucosamine-myoinositol phosphate. Variations observed in additional mannose, N-acetylhexosamine, and ethanolamine phosphate moieties form this heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)
5'-Nucleotidase↗
Peripheral blood cells are predominantly chimeric of affected and normal cells in patients with paroxysmal nocturnal hemoglobinuria: simultaneous investigation on clonality and expression of glycophosphatidylinositol-anchored proteins.
To investigate clonal compositions of hematologic cells in paroxysmal nocturnal hemoglobinuria (PNH), we analyzed peripheral blood (PB) cells of 12 female patients with PNH, by clonality analysis using X-chromosome inactivation and assessment of expression of glycophosphatidylinositol-anchored proteins (GPI-APs) by flow cytometry. Southern hybridization showed that granulocytes were monoclonal in three and polyclonal in eight patients, respectively, whereas lymphocytes were polyclonal in all nine patients examined. Expressions of CD16 and CD59 on granulocytes varied greatly in seven patients examined. Clonality analysis of granulocytes by the polymerase chain reaction showed that CD59- and CD59low+ cells were monoclonal, whereas CD59+ cells were polyclonal. It was shown that PB cells are predominantly chimeric of clonal (GPI-AP- or GPI-APlow+) and nonclonal (GPI-AP+) cells in PNH, and that degrees of chimerism differ greatly from patient to patient.
Adolescent↗
Studies on the GPI-anchored enzyme, hepatic 5'-nucleotidase. Microheterogeneity of the anchor, processing and localization.
Hepatic 5'-nucleotidases of vertebrates were investigated for localization in the lysosomes and the plasma membrane, microheterogeneity of the glycosylphosphatidylinositol (GPI)-anchor moiety and minimal requirement of the C-terminal signal peptide for GPI attachment. Using PIPLC of Bacillus thuringiensis and subcellular fractionation by Percoll gradient centrifugation, we found that chicken liver 5'-nucleotidase can be transferred from plasma membrane to lysosomes in the GPI-anchored or soluble form. Bovine liver ecto 5'-nucleotidase was solubilized by PIPLC, purified to a homogeneous state, and analyzed for the structures of GPI-anchor isoforms by HPLC and ESI-MS in combination with glycosidase treatments, after peptide-bond cleavage by CNBr or trypsin. Several isomers of the GPI anchor were thus characterized; major components contained two phosphorylethanolamine residues, whereas the component containing three phosphorylethanolamine residues was present only as a small percentage of the total. The cleavage/attachment site of the GPI anchor in the C-terminal of 5'-nucleotidase was shown to be Ser523. The peptide region cleaved off at the posttranslational processing has a length of 25 amino acid residues which contains a hydrophobic stretch of 17 amino acids. By site-directed mutagenesis, we determined the minimal length of the hydrophobic peptide to be 13 amino acids for expression of 5'-nucleotidase as a GPI-anchored form on the COS cell surface. When peptide length was shortened to less than 13 amino acids, the expressed enzyme was not sorted to the cell surface but present within, or secreted out of the cells.
5'-Nucleotidase↗
The role of acidic amino-acid residues in catalytic and adsorptive sites of Bacillus cereus sphingomyelinase.
By the modification of acidic amino-acid residues with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate), the activity of sphingomyelinase of Bacillus cereus was decreased by 80-90%. Also, the reduction of Cys residues in the sphingomyelinase molecule by dithiothreitol caused a drastic decrease in enzymatic activity, whereas the sphingomyelinase activity was not affected by treatment with p-chloromercuribenzenesulfonic acid. Actually, no inactivation of sphingomyelinase activity was observed after selective modification of basic amino-acid residues such as Lys, His and Arg, and of the uncharged amino-acid residues Ser and Thr. The treatment of the sphingomyelinase molecule with Woodward's reagent K or dithiothreitol also brought about the inhibition of the specific adsorption of sphingomyelinase toward intact erythrocyte membranes. However, the extent of inhibition in the enzyme adsorption, 20-50%, was less than that observed in the sphingomyelinase activity. These results suggest that acidic amino-acid residues, such as Asp and Glu, in the sphingomyelinase molecule are involved in the catalytic sites and the adsorptive sites. Apparently, the disruption of disulfide linkage in the sphingomyelinase molecule by dithiothreitol destabilized its structure, resulting in a drastic decrease in sphingomyelin-hydrolyzing activity and specific adsorption of sphingomyelinase towards erythrocyte membranes.
Amino Acid Sequence↗
Proof of alkaline phosphodiesterase I as a phosphatidylinositol-anchor enzyme.
1. Ectoenzyme release from kidney brush border membranes of Rattus norvegicus and Sus scrofa domesticus by phosphatidylinositol-specific phospholipase C (PIPLC) of Bacillus thuringiensis was studied. 2. The levels of specific activities of ectoenzymes in R. norvegicus kidney brush border membranes were higher than those in S. scrofa domesticus. About 10-fold higher values were found for specific activities of alkaline phosphatase and gamma-glutamyl transpeptidase in R. norvegicus. 3. Alkaline phosphodiesterase I, alkaline phosphatase and 5'-nucleotidase were released from both R. norvegicus and S. scrofa domesticus brush border membranes, while gamma-glutamyl transpeptidase and dipeptidyl peptidase IV were not solubilized. The enzyme release by the action of PIPLC was suppressed when purified anti-PIPLC antibody was added to the reaction mixture. This suggests that enzyme release must be due to the direct action of PIPLC on kidney brush border membranes. 4. The released alkaline phosphodiesterase I from kidney of S. scrofa domesticus had a molecular weight of 240,000 and was activated by Mg2+ and Ca2+, but strongly inhibited by EDTA.
5'-Nucleotidase↗
Alkaline phosphodiesterase I release from eucaryotic plasma membranes by phosphatidylinositol-specific phospholipase C. III. The release from tumor cells.
1. Alkaline phosphodiesterase I release from two tumor cell lines, KB III or AH-130 cells, by the action of phosphatidylinositol-specific phospholipase C (PIPLC) of Bacillus thuringiensis was studied. 2. A significant amount of alkaline phosphodiesterase I was released from both the cell suspension and homogenate of KB III cells, but not from AH-130 cells. 3. The release of the enzyme from KB III cells was dependent on, or proportional to, the reaction time and the PIPLC or cell concentrations. 4. Alkaline phosphatase and 5'-nucleotidase were also released from KB III cells, while gamma-glutamyl transpeptidase and dipeptidyl peptidase IV were not solubilized. The enzyme release by the action of PIPLC was suppressed when purified anti-PIPLC antibody was added to the reaction mixture. This suggests that the enzyme release must be due to the direct action of PIPLC on KB III cells. 5. The alkaline phosphodiesterase I released from KB III cells had a mol. wt of 240,000 and was activated by Mg2+, but strongly inhibited by EDTA and thiol reagents and by 5'-nucleotide-containing compounds. Although KB III cells were derived from Homo sapiens tumor, the released alkaline phosphodiesterase I appeared to be very similar to enzymes obtained from normal tissues of Rattus norvegicus.
5'-Nucleotidase↗
Purification and cDNA cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells.
A glycosylphosphatidylinositol (GPI)-anchored protein, 5'-nucleotidase [EC 3.1.3.5], was released from the membrane of bovine liver by use of phosphatidylinositol-specific phospholipase C (PI-PLC) of Bacillus thuringiensis and purified by several column chromatographies to a homogeneous state. The purified protein has an apparent molecular mass of 61 kDa, as estimated by SDS-polyacrylamide gel electrophoresis. From the partial amino acid sequence of a tryptic peptide, mixed oligonucleotides were synthesized and used to screen a lambda gt11 liver cDNA library, and one positive clone, pE1, was isolated. Since the insert of the clone lacked the NH2-terminal coding region, another lambda gt11 liver cDNA library was screened by using a synthetic probe corresponding to the 5' region of the insert of pE1. Three additional cDNA clones were obtained. Sequencing of these cDNAs revealed an open reading frame that encodes a 574-residue polypeptide with a calculated mass of 63,084 Da. The predicted structure showed two highly hydrophobic stretches at both ends of the protein, like those of rat and human 5'-nucleotidases. The NH2-terminal 26 residues comprise a signal peptide and the COOH-terminal hydrophobic stretch may serve as a signal for the posttranslational GPI modification. An expression vector of the cDNA, pSVNT, was constructed in a mammalian expression vector pSVL and the 5'-nucleotidase activity was transiently expressed in COS-1 cells. The expressed activity was about 8 times higher than the pSVL-transfected control activity. PI-PLC released 45% of the transiently expressed 5'-nucleotidase activity, indicating that the cDNA isolated here encodes this enzyme expressed as a GPI-anchored protein.
5'-Nucleotidase↗
Alteration in reactivity of sphingomyelinase from Streptomyces sp. modified with a polyethylene glycol derivative.
Purified sphingomyelinase of Streptomyces A9107 (NRRL 15100) was modified with ss-PEG, methoxypolyethyleneglycol succinimidyl succinate, without loss of activity toward sphingomyelin in the mixed micelles with detergents such as sodium deoxycholate and Triton X-100 or toward a water-soluble, synthetic substrate, HNP, 2-(N-hexadecanoylamino)-4-nitrophenyl phosphocholine. The hemolytic and sphingomyelin-hydrolyzing activities of the enzyme toward intact bovine erythrocyte membrane were completely abolished by modification with ss-PEG, whereas the activity toward membranous sphingomyelin in the erythrocyte ghosts was fully retained. The enzyme activity toward liposomal sphingomyelin (large and small unilamellar vesicles) was significantly decreased by PEG-modification. The thermostability of sphingomyelinase was greatly increased by modification with ss-PEG, whereas the sensitivity of the enzyme to proteolysis was not influenced by that modification.
Animals↗
Delivery of an accessory signal for cell activation by exogenous phosphatidylinositol-specific phospholipase C.
Digestion of phosphatidylinositol (PI) or glycosylphosphatidylinositol (GPI) anchors of membrane proteins on the external cell surface with exogenous PI-specific phospholipase C (PIPLC) from Bacillus thuringiensis was shown to transmit a signal into the thymocyte to modulate the TCR/CD3 complex-induced signal delivery for cell activation. This was demonstrated for very early protein tyrosine phosphorylation, early c-fos transcription and late DNA synthesis. For this effect preincubation of the cells with PIPLC was required, but there was no evidence of involvement of any soluble products released from the cell surface by PIPLC in the signaling, suggesting a crucial role of the membrane-bound counterpart (diacylglycerol or diradylglycerol) of the PI/GPI hydrolysate. A possible role for this accessory signal in the microorganism-linked control of the (diacylglycerol or diradylglycerol) of the PI/GPI hydrolysate. A possible role for this accessory signal in the microorganism-linked control of the T cell receptor function is discussed.
Animals↗