A restriction enzyme, BpuI is an isoschizomer of BanII.
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Bacillus↗
Biomedical subjects
T Shibata
Publications and source records attributed to T Shibata.
Effect of verapamil on the class I major histocompatibility complex antigen expression in K562 chronic myelogenous leukemia cells treated with recombinant human interferon-gamma.
The effects of various compounds which modulated the intracellular signal transduction on the induction of class I major histocompatibility complex (MHC) antigens by recombinant human interferon-gamma (rIFN-gamma) were investigated using K562, chronic myelogenous leukemia cells. Class I or class II MHC antigens were not expressed in untreated K562 cells and rIFN-gamma (600 units/ml) weakly induced class I antigens on the cells. Among the compounds tested, verapamil but not the calcium ionophore A23187 enhanced the rIFN-gamma-induced class I antigen expression at both the surface molecule and mRNA levels and enhancement by verapamil occurred in a dose-dependent manner at non-toxic concentrations examined (approximately 50 microM). Verapamil alone had no inducible effect on MHC antigen expression. Deprivation of Ca2+ in culture medium by ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) could not cause an enhancement of class I antigen induction by rIFN-gamma. Simultaneous exposure of K562 cells to rIFN-gamma (600 units/ml) and recombinant human tumor necrosis factor (rTNF; 1000 units/ml) in combination with verapamil (50 microM) resulted in a further increase of class I antigens in the cells. The expressions of c-myc oncogene in K562 cells were not changed when the cells were treated with rIFN-gamma (600 units/ml) or verapamil (50 microM), either alone or in combination. These results indicate that verapamil synergistically interacts with rIFN-gamma on the class I antigen induction in K562 cells irrespective of c-myc gene expression and that class I antigen induction in this cell line may not be relevant to calcium influx triggered by IFN-gamma.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
The rat angiotensin II AT1A receptor couples with three different signal transduction pathways.
To examine whether the subpopulation of the rat type 1 angiotensin II (AII) receptor (AT1A) couples with a single or multiple signal transduction pathways, we constructed Chinese hamster ovary (CHO) cell lines producing the recombinant receptor. The expressed AT1A receptor exhibits typical pharmacological characteristics of the AT1 receptor, known to mediate the main physiological function of AII. Addition of AII to the CHO cells induced a rapid, transient increase in intracellular free Ca2+ concentrations ([Ca2+]i) followed by a lower, sustained phase. Nicardipine, a blocker of voltage-dependent L-type Ca2+ channels, attenuated the transient [Ca2+]i response and abolished the sustained phase. The transient phase was also reduced dose-dependently by the phospholipase C inhibitor neomycin. Furthermore, AII inhibited forskolin-evoked cAMP accumulation. These data suggest, although another subpopulation named AT1B is present, that the rat AT1A receptor can independently couple with all three signal transduction pathways known to be induced by AII: i.e., i) activation of phospholipase C resulting in InsP3 generation with a subsequent release of intracellularly stored Ca2+, ii) activation of dihydropyridine-sensitive voltage-dependent Ca2+ channels, and iii) inhibition of adenylate cyclase activity.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Glycosylation of lipoprotein lipase in human subcutaneous and omental adipose tissues.
Human adipose tissues from the abdomen (subcutaneous), thigh (subcutaneous) and omentum were incubated for 2 h with [35S]methionine. Then glycosylation of lipoprotein lipase (LPL) was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of endoglycosidase H (endo H)-digested subunits of the 35S-labeled lipase. Adipose tissues from the abdomen, thigh, and omentum all synthesized LPL subunits with Mr = 57,000 composed of two types of subunits. One type was partially endo H-sensitive yielding a product with Mr = 55,000, indicating that it had one endo H-resistant and one endo H-sensitive oligosaccharide chain. The other type of subunit was totally endo H-sensitive yielding a product with Mr = 52,000. Subcutaneous adipose tissues contained nearly equal amounts of partially and totally endo H-sensitive subunits of LPL, whereas omental adipose tissues contained mainly partially endo H-sensitive subunits of LPL.
Adipose Tissue↗
Phenol-treatment and a homologous pairing-assay.
Homologous pairing is a key step in homologous genetic recombination. In the early stage of trials for the identification of homologous pairing-promoting proteins from a fission yeast, Schizosaccharomyces pombe, we treated DNA products with phenol in the presence of a salt for the removal of tightly bound proteins from DNA before the assay, but we found that this treatment caused very efficient protein-independent double-strand formation from complementary single-stranded DNAs. Using an assay including the phenol treatment, we detected another species of apparent homologous pairing-promoting proteins in the nuclei, in addition to a homologous pairing-promoting protein consisting of three components which we reported previously. However, studies involving the use of an assay without the phenol-treatments revealed that the second one was not really a homologous pairing-protein. Thus, the protein-independent double-strand formation by phenol-treatment in the presence of a salt could cause the erroneous identification of homologous pairing-promoting proteins.
Bacteriophages↗
Simultaneous assay of adenosine deaminase and purine nucleoside phosphorylase activity as possible biochemical means to detect non-Hodgkin lymphomas of the oral cavity.
BACKGROUND AND METHODS: This study proposes the usefulness of simultaneous assay of adenosine deaminase (enzyme code [EC] 3.5.4.4; ADA) and purine nucleoside phosphorylase (EC 2.4.2.1; PNP) activities in a biopsy specimen as a biochemical test adjunctive to the histologic diagnosis of oral non-Hodgkin lymphomas (NHL). RESULTS: NHL tissues showed an ADA activity of more than 1428 nmol/h/mg of protein, and more than 3451 nmol/h/mg of protein was the sum of ADA and PNP activity. In contrast to this finding, all the 106 lesional tissues affected by various carcinomas, various benign tumors, various cysts, ameloblastomas, osteosarcomas, gingival hyperplasia, and inflammation showed an ADA activity of less than 1000 nmol/h/mg of protein, except for 2. The sum of ADA and PNP activity was less than 2249 nmol/h/mg of protein in these diseases, except for squamous carcinoma. Different control tissues of the oral cavity showed less than 766 nmol/h/mg of protein in ADA activity, and the highest sum of ADA and PNP activity in these was only 1384 nmol/h/mg of protein. CONCLUSIONS: These findings suggest that a strong suspicion of oral NHL is justified when the sum of ADA and PNP activity in a biopsy specimen exceeds 3000 nmol/h/mg of protein and ADA activity is greater than 1000 nmol/h/mg of protein.
Adenosine Deaminase↗
Identification of two types of homologous DNA pairing activity in mouse cells.
We have identified two types of homologous DNA pairing activity in mouse cell extracts by a strand-transfer assay. Both activities are separated from each other by anion-exchange chromatography; neither of them needs ATP. One requires magnesium ion and is stimulated by Escherichia coli single-stranded DNA binding protein, whereas the other does not require the ion and shows a higher affinity for a left-handed Z-DNA.
Adenosine Triphosphate↗
Murine autoimmune hemolytic anemia resulting from Fc gamma receptor-mediated erythrophagocytosis: protection by erythropoietin but not by interleukin-3, and aggravation by granulocyte-macrophage colony-stimulating factor.
We have evaluated the therapeutic activity of recombinant erythropoietin (rEpo), in comparison with recombinant interleukin-3 (rIL-3) and granulocyte-macrophage colony-stimulating factor (rGM-CSF), on a lethal form of acute anemia resulting from Fc gamma receptor-mediated erythrophagocytosis after a single injection (500 micrograms) of a monoclonal anti-mouse red blood cell (MRBC) autoantibody. Continuous perfusion of rEpo before the administration of anti-MRBC monoclonal antibody completely protected animals from death due to anemia with a rapid recovery, while no protection was obtained by rIL-3 perfusion. In contrast, rGM-CSF perfusion markedly accelerated the progression of anemia and the mortality rate. This was found to result from an enhancement of erythrophagocytosis by Kupffer cells and by polymorphonuclear leukocytes that massively infiltrated the livers. Even after the injection of a sublethal dose (100 micrograms) of anti-MRBC monoclonal antibody, rGM-CSF-perfused mice died of a severe form of acute anemia. Furthermore, we have shown that rEpo was able to treat efficiently a spontaneous form of autoimmune hemolytic anemia in a majority of anemic NZB mice, whereas rGM-CSF markedly aggravated anemia. This may be of clinical importance, because GM-CSF administration could exhibit an adverse effect in some autoimmune diseases that involve autoimmune anemia.
Anemia, Hemolytic, Autoimmune↗
Epitope mapping of anti-recA protein IgGs by region specified polymerase chain reaction mutagenesis.
Monoclonal IgGs were shown to be useful for the specific inhibition of a set of activities of the recA protein, a key protein in homologous genetic recombination. The mapping of the epitopes for these IgGs and site-directed mutagenesis based on the mapping will facilitate location of the functionally active sites on the tertiary structure of the protein, which is being solved by means of physicochemical techniques. We developed a novel technique for region-specified mutagenesis and applied the technique to epitope mapping. Using the polymerase chain reaction in the presence of deoxyinosine triphosphate, we introduced random base substitutions specifically into a region of the recA gene defined by a pair of primers. RecA mutants exhibiting altered antigenicity were selected, in plaque-immunoblotting experiments, from libraries of mutagenized recA genes constructed on the lambda gt11 expression vector. Mutant recA genes were obtained at the frequency of about 10(-2) among the plaques expressing fused recA genes and then each one was expressed as a whole protein, which was characterized by enzyme-linked immunosorbent assay. Analyzing the DNA sequences of the mutant recA genes, we located at the amino acid sequence level the epitopes for two anti-recA IgGs which could not be located in previous studies. One of the antibodies was shown to prevent self-assembly of the recA protein and the other was suggested to inhibit the binding of double-stranded DNA. Thus, the active sites involved in these functions would be located in the space around or near the relevant epitope.
Amino Acid Sequence↗
A multicomponent protein of a fission yeast that promotes joint molecule formation from homologous DNAs.
We developed a quantitative assay ("homologous pairing gel assay") adequate for the purification of the activity promoting the formation of joint molecules, an intermediate of homologous recombination ("homologous pairing"). With this assay, one can measure the extent of homologous pairing between a single-stranded DNA and a strand of 3H-labeled double-stranded DNA by crude enzyme preparations. Since the total activity did not significantly change during the sporulation process, we tried to purify the activity from a whole cell extract of mitotic cells of a fission yeast, (Schizosaccharomyces pombe). Through quantitative assaying of a single fraction or of mixed fractions, we obtained three fractions, all of which were required for the maximum level of the ATP-independent homologous pairing: Fractions 65, 100, and 30. In Fractions 100 and 30, polypeptides of approximately 100 and approximately 30 kDa (the 100- and 30-kDa polypeptides), respectively, were the sole detectable components. Fraction 65 contained a polypeptide of approximately 65 kDa (the 65-kDa polypeptide) as the major component and also small amounts of the 30- and 100-kDa polypeptides. Fraction 65 by itself promoted homologous pairing, but the reaction was saturated at a level of approximately 20% of the maximum level achieved with the recA protein. Even when added in excess, Fraction 30 or 100 alone did not promote detectable homologous pairing. A mixture of Fractions 65 and 100 at a rather strict optimum ratio only promoted homologous pairing, the level being 50-70% that with the recA protein, suggesting a stoichiometric complex of these polypeptides as the active form. Fraction 30 alone did not enhance the reaction with Fraction 65, but stimulated homologous pairing promoted by the optimum mixture of Fractions 65 and 100 to the maximum level achieved with the recA protein. Therefore, the homologous pairing-promoting protein from the fission yeast is likely to be a multicomponent protein.
Chromatography, Affinity↗
Acute megakaryoblastic leukemia with translocation t(1;22)(p13;q13) in a 10-week-old infant.
A 10-week-old girl without Down syndrome developed an acute megakaryoblastic leukemia (AMKL). Bone marrow aspirates and biopsy showed megakaryoblastic infiltration with myelofibrosis. The diagnosis was made based on the findings that the positive reactions of leukemic cells to platelet peroxidase and to monoclonal antibodies which recognize platelet-specific surface glycoprotein (GP) IIb/IIIa and GP78. The blasts also showed myeloid and monocytoid differentiation antigens. The leukemic cells had a karyotype of 46,XX,t(1;22)(p13;q13). Our case and two other infantile cases reported by other investigators establish the novel association of the t(1;22) with AMKL.
Antibodies, Monoclonal↗
An endonuclease with multiple cutting sites, Endo.SceI, initiates genetic recombination at its cutting site in yeast mitochondria.
Endo.SceI is a mitochondrial sequence-specific endonuclease which has multiple cutting sites. In order to examine the possible role of Endo.SceI in homologous recombination, we analyzed the mode of recombination upon mating using antibiotic resistance markers on the mitochondrial genome. The segregation of a marker located very close to one of the Endo.SceI cutting sites showed a disparity (polarized segregation, i.e. gene conversion). This gene conversion depended on the presence of the functional Endo.SceI gene. In vivo cutting of mitochondrial DNA upon mating was detected at the cutting site in the antibiotic marker region, which also depended on the Endo.SceI activity. These results suggest that mitochondrial recombination is induced by cleavage of mitochondrial DNA by this sequence-specific endonuclease. This is the first demonstration that a sequence-specific endonuclease with multiple cutting sites induces genetic recombination.
Base Sequence↗
Enzyme immunoassay and immunochemical characterization of pancreatic stone protein in human serum.
Monoclonal antibodies were raised against human pancreatic stone protein (PSP) and used for one-step enzyme immunoassay (EIA). PSP-S2-5 was employed as the standard in the assay. The assay's measurable range was 25-1,500 ng/ml and within run coefficient of variation was 3.7-6.4%. Analytical recovery of the assay was 101.5 +/- 5.65% (mean +/- SD). The results of experiments in which serum was fractionated by Mono S (cation exchange chromatography) suggested that most of immunoreactive material in human serum is PSP-S2-5. The EIA offers simple, rapid, and specific analysis of serum PSP level for clinical diagnosis.
Antibodies, Monoclonal↗
Enzyme immunoassay for specific analysis of pancreatic stone proteins in human pancreatic juice.
In order to study the concentration of pancreatic stone protein (PSP) in human pancreatic juice, we investigated the influence of the insoluble form of PSP-S1 converted from PSP-S2-5 on PSP determination and the assay method for PSP-S1 precipitate after solubilizing PSP-S1. When bovine trypsin was added to pancreatic juice, PSP-S1 was converted from PSP-S2-5 and precipitated about 45-85% after 1 h. The precipitated PSP-S1 was dissolved in 0.1 M sodium acetate buffer, pH 4.0, and the concentration was measured by the enzyme immunoassay, with similar reactivity to PSP-S1 and PSP-S2-5. The proposed method can offer accurate and specific analysis of the PSP level in pancreatic juice. The results of the fractionation of pancreatic juice and duodenal juice on Mono S cation-exchange chromatography suggested that the major component of PSP was PSP-S2-5 in pancreatic juice and PSP-S1 in duodenal juice.
Calcium-Binding Proteins↗
Cellular immunosuppression in children with acute lymphoblastic leukemia: effect of consolidation chemotherapy.
The present study was designed to evaluate the chemotherapy-induced cellular immunosuppression in 20 children with acute lymphoblastic leukemia (ALL) in remission and receiving maintenance chemotherapy. Peripheral blood was serially obtained from leukemic children during vincristine/cyclophosphamide/6-mercaptopurine/prednisone combined consolidation chemotherapy. The mean absolute number of peripheral blood lymphocytes as well as the mean absolute numbers of lymphocyte subsets (T cells, T cell subsets, B cells, and natural killer cells) from leukemic children before consolidation chemotherapy were all significantly lower than in control subjects; however, the percentages of lymphocyte subsets were similar in both groups. After consolidation chemotherapy, the percentages of CD4+ T lymphocytes and natural killer (NK) cells were significantly decreased and the percentages of monocytes and CD8+ T lymphocytes were significantly increased. Phytohemagglutinin- and 12-O-tetradecanoylphorbol-13-acetate-induced production of interleukin-2 (IL-2) and NK-cell-mediated cytotoxic activity by peripheral blood mononuclear cells (PBMC) were also substantially decreased in the post-therapy groups. NK activity correlated with the percentage of NK cells in PBMC. In contrast, OK432-induced production of tumor necrosis factor alpha (TNF alpha) and killer activity against NK-resistant target cells were significantly increased after therapy as compared with the pre-therapy and control groups. TNF alpha production correlated with the percentage of monocytes in PBMC. These results demonstrate that substantial quantitative and qualitative chemotherapy-induced abnormalities of the cellular immune system are present in the majority of patients treated with ALL. It is also suggested that the increased TNF alpha production by monocytes and the appearance of potent killing activity against NK-resistant targets might compensate for the defects of IL-2 production and NK activity during intensive consolidation chemotherapy.
Animals↗
Acute and subacute inhalation toxicity of silane 1000 ppm in mice.
Male ICR mice were exposed to silane 1000 ppm, a concentration 200 or 2000 times higher than the recommended occupational exposure limits by many countries and academic associations, for 1, 2, 4, and 8 h (phase I study) and for 6 h/day, 5 days/week, over 2 and 4 weeks (phase II study). Hematological and biochemical studies were performed, and the animals were examined for histopathological lesions of the cornea, nasal cavity, respiratory tract, lung, liver, kidney, spleen, pancreas, thymus, thyroid, bone marrow, salivary glands, esophagus, and testis. All mice in both studies survived until they were sacrificed. In the phase I study, no exposure-related changes were found as a result of the hematological, biochemical, or histopathological examinations. In the phase II study, hematological and biochemical examinations failed to reveal any exposure-related changes, but mild irritation, manifested in the form of a small amount of exudate (eight out of ten animals), and inflammatory cells and/or necrotic cells on the nasal mucosa (six out of ten animals) was observed in the mice exposed to silane for 4 weeks. These findings suggest that silane toxicity and irritation are not severe.
Administration, Oral↗
Peritoneal absorption of pancreatic enzymes in dogs.
To elucidate peritoneal absorption of pancreatic enzymes, plasma levels of amylase, lipase, and trypsinogen were measured after the intraperitoneal injection of 10 ml human pancreatic juice in dogs. Plasma pancreatic amylase, lipase, and trypsinogen were determined using the immunoassay specific to the corresponding human pancreatic enzyme to exclude cross-reaction with the endogenous enzyme activities of the canine plasma. Plasma immunoreactivity of human amylase persistently rose during 24 h after the injection, whereas elevation of plasma amylase enzyme activity become significant only at 24 h. Increase of plasma lipase was not remarkable. Significant increase of the immunoreactivity was observed only at 24 h but there was no significant increase of the enzyme activity during this period. Plasma trypsinogen immunoreactivity peaked at 2 h remained significantly elevated during 24 h. The transperitoneal absorption of pancreatic enzymes in dogs was confirmed using intraperitoneal injection of human pancreatic juice combined with immunoassay specific to human pancreatic enzymes.
Amylases↗
Molecular form of pancreatic elastase 1 in human plasma.
In order to study the molecular forms of immunoreactive pancreatic elastase 1 (IRE) in human plasma, we investigated the characteristics of proelastase 1, elastase 1, and their alpha 1-protease inhibitor (alpha 1-PI) complexes on anion-exchange (Mono Q) chromatography, gel (Superose 12) chromatography, and enzyme immunoassay systems with monoclonal antielastase 1 antibodies of different affinities for alpha 1-PI-elastase 1 and alpha 1-PI-proelastase 1 complexes. The rate of complex formation between proelastase 1 and alpha 1-PI was dependent on temperature of incubation and concentration of alpha 1-PI. At 37 degrees C, 90% of proelastase 1 was bound to alpha 1-PI during 2 h of incubation of purified human proelastase 1 with human alpha 1-PI. Similar results were also obtained by 2 h of incubation of proelastase 1 with human plasma at 37 degrees C. Therefore, under physiological conditions, neither free proelastase 1 nor elastase 1 can be detected in the human blood stream, and most IRE exists as the complex of proelastase 1 with alpha 1-PI in human plasma.
Chromatography, Ion Exchange↗