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41.
Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death 总被引:10,自引:0,他引:10
A Yoshioka S Tanaka O Hiraoka Y Koyama Y Hirota D Ayusawa T Seno C Garrett Y Wataya 《The Journal of biological chemistry》1987,262(17):8235-8241
The mechanism of cytotoxic action of 5-fluorodeoxyuridine (FdUrd) in mouse FM3A cells was investigated. We observed the FdUrd-induced imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools and subsequent double strand breaks in mature DNA, accompanied by cell death. The imbalance of dNTP pools was maximal at 8 h after 1 microM FdUrd treatment; a depletion of dTTP and dGTP pools and an increase in the dATP pool were observed. The addition of FdUrd in culture medium induced strand breaks in DNA, giving rise to a 90 S peak by alkaline sucrose gradient sedimentation. The loss of cell viability and colony-forming ability occurred at about 10 h. DNA double strand breaks as measured by the neutral elution method were also observed in FdUrd-treated cells about 10 h after the addition. These results lead us to propose that DNA double strand breaks play an important role in the mechanism of FdUrd-mediated cell death. A comparison of the ratio of single and double strand breaks induced by FdUrd to that observed following radiation suggested that FdUrd produced double strand breaks exclusively. Cycloheximide inhibited both the production of DNA double strand breaks and the FdUrd-induced cell death. An activity that can induce DNA double strand breaks was detected in the lysate of FdUrd-treated FM3A cells but not in the untreated cells. This suggests that FdUrd induces the cellular DNA double strand breaking activity. The FdUrd-induced DNA strand breaks and cell death appear to occur in the S phase. Our results indicate that imbalance of the dNTP pools is a trigger for double strand DNA break and cell death. 相似文献
42.
Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat liver medium chain acyl coenzyme A dehydrogenase 总被引:14,自引:0,他引:14
Y Matsubara J P Kraus H Ozasa R Glassberg G Finocchiaro Y Ikeda J Mole L E Rosenberg K Tanaka 《The Journal of biological chemistry》1987,262(21):10104-10108
cDNA encoding the precursor of rat liver medium chain acyl-CoA dehydrogenase (EC 1.3.99.3) was cloned and sequenced. The longest cDNA insert isolated was 1866 bases in length. This cDNA encodes the entire protein of 421-amino acids including a 25-amino acid leader peptide and a 396-amino acid mature polypeptide. The identity of the medium chain acyl-CoA dehydrogenase clone was confirmed by matching the amino acid sequence predicted from the cDNA to the NH2-terminal and nine internal tryptic peptide sequences derived from pure rat liver medium chain acyl-CoA dehydrogenase. The calculated molecular masses of the precursor medium chain acyl-CoA dehydrogenase, the mature medium chain acyl-CoA dehydrogenase, and the leader peptide are 46,600, 43,700, and 2,900 daltons, respectively. The leader peptide contains five basic amino acids and only one acidic amino acid; thus, it is positively charged, overall. Cysteine residues are unevenly distributed in the mature portion of the protein; five of six are found within the NH2-terminal half of the polypeptide. Comparison of medium chain acyl-CoA dehydrogenase sequence to other flavoproteins and enzymes which act on coenzyme A ester substrates did not lead to unambiguous identification of a possible FAD-binding site nor a coenzyme A-binding domain. The sequencing of other homologous acyl-CoA dehydrogenases will be informative in this regard. 相似文献
43.
Molecular cloning and sequence analysis of cDNA for batroxobin, a thrombin-like snake venom enzyme 总被引:26,自引:0,他引:26
Determination of the nucleotide sequence of a cDNA for batroxobin, a thrombin-like enzyme from Bothrops atrox, moojeni venom, allowed elucidation of the complete amino acid sequence of batroxobin for the first time for a thrombin-like snake venom enzyme. The molecular weight of batroxobin is 25,503 (231 amino acids). The amino acid sequence of batroxobin exhibits significant homology with those of mammalian serine proteases (trypsin, pancreatic kallikrein, and thrombin), indicating that batroxobin is a member of the serine protease family. Based on this homology and enzymatic and chemical studies, the catalytic residues and disulfide bridges of batroxobin were deduced to be as follows: catalytic residues, His41, Asp86, and Ser178; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys230, Cys118-Cys184, Cys150-Cys163, and Cys174-Cys199. The amino-terminal amino acid residue of batroxobin, valine, is preceded by 24 amino acids. This may indicate that the amino-terminal hydrophobic peptide (18 amino acids) is a prepeptide and that the hydrophilic peptide (6 amino acids), preceded by the putative prepeptide, is a propeptide. 相似文献
44.
Immunochemical and kinetic evidence for two different prostaglandin H-prostaglandin E isomerases in sheep vesicular gland microsomes 总被引:1,自引:0,他引:1
Splenic lymphocytes from mice immunized with a partially purified prostaglandin (PG) H-PGE isomerase from sheep vesicular glands were fused with SP2/0-Ag14 myeloma cells. Two spleen cell-myeloma hybrids (hei-7 and hei-26) were selected and cloned. The mouse antibodies secreted by the two hybrids, IgG1 (hei-7) and IgG1 (hei-26), caused immunoprecipitation of a maximum of 45 and 22%, respectively, of the solubilized PGH-PGE isomerase activity of sheep vesicular gland; immunoprecipitation of activity by the two antibodies was additive. The antigens reactive with IgG1 (hei-7) and IgG1 (hei-26) were identified as proteins with Mr = 17,500 and 180,000, respectively, by Western transfer blotting or sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated 125I-labeled microsomes. The PGH-PGE isomerase activities precipitated by IgG1 (hei-7) and IgG1 (hei-26) exhibited different kinetic properties with respect to time course, Km for PGH2, and concentration dependence for GSH. No significant GSH-S-transferase activity was present in these immunoprecipitates. These data indicate that there are at least two different proteins in sheep vesicular gland microsomes capable of catalyzing GSH-dependent PGH-PGE isomerase reactions. IgG1 (hei-7), but not IgG1 (hei-26), caused coprecipitation of PGH synthase and PGH-PGE isomerase activities when incubated with intact right-side-out vesicular gland microsomes. Thus, the epitope for IgG1 (hei-7) is located on the cytoplasmic surface of those microsomal spheres which contain PGH synthase. This latter finding suggests that the isomerase reactive with IgG1 (hei-7) is involved in PGE synthesis in sheep vesicular glands. 相似文献
45.
Murine monoclonal antibodies to the myelin-associated glycoprotein (MAG) recognize Leu-7-reactive molecules on human mononuclear cells 总被引:2,自引:0,他引:2
M Arai M Nishizawa T Inuzuka M Tanaka H Baba S Sato T Miyatake 《Journal of immunology (Baltimore, Md. : 1950)》1987,138(10):3259-3263
It is known that the antibody to human myelin-associated glycoprotein (MAG) reacts with a subset of human mononuclear cells (MNC) mediating a natural killer (NK) activity. The properties of the target molecule of the anti-MAG antibody, however, have not yet been elucidated. Three (GC-J4, MC-P2, and MC-P4) of five murine monoclonal antibodies (mAb) to MAG bound to human MNC. Moreover, MC-P2 and MC-P4 inhibited the binding of 125I-labeled anti-Leu-7 to MNC in a dose-dependent fashion. Conversely, anti-Leu-7 inhibited the binding of MC-P2 and MC-P4 to MNC, but did not inhibit the binding of GC-J4. Therefore, it is possible that MC-P2 and MC-P4 bind directly to or close to the Leu-7 epitope, and that GC-J4 binds to the epitope which is distinct from the Leu-7 epitope. The electrophoretic patterns of immunoprecipitates with GC-J4, MC-P2 and anti-Leu-7 from detergent lysates of surface-labeled human MNC were very similar. The target molecules of anti-Leu-7 and anti-MAG mAb have apparent m.w. of 205, 170, 150, 135, 110, 85, 65, and 55 kDa. All of the molecules precipitated by these mAb are monomeric or noncovalently associated proteins, because the electrophoretic mobilities of the proteins remained unchanged whether the samples were reduced or not. MC-P4 may have a higher affinity for the 65 kDa molecule than the other mAb, and precipitates the 58 kDa molecule as well. Therefore, the fine antigenic specificity of MC-P4 is slightly different from those of anti-Leu-7 or MC-P2. The implication of these results is that mAb, whose specificity is directed to the carbohydrate part of human MAG, reacts with the Leu-7 reactive molecules on human MNC, and that at least two epitopes detected by anti-MAG mAb coexist on the surface molecules with various apparent m.w. 相似文献
46.
In order to investigate a role of proteinase in the pathogenesis of Candida infections, invasion of C. albicans strains of different proteinase activity into the chorioallantoic membrane (CAM) of developing chicks was studied. Eight strains were used after examining the inducible proteinase activity in the culture containing bovine serum albumin as the sole source of nitrogen. Six were proteinase-producing strains (type I) and two were proteinase-deficient ones (type II). Type I strains were subdivided into type Ia strains in which the proteinase activity persisted for a week in the in vitro culture and type Ib ones in which the enzyme activity was lost by the 7th day after inoculation. By inoculation onto CAM, the type I strains could invade the tissue in which secreted proteinase was detected on the periphery of the invading Candida cells by immunohistochemical method. At an early stage of the infection, proteinase secretion was detected on the surface of the yeast cells before their entry into the tissue. The type II strains remained on the surface of the CAM and did not invade the tissue where the secretion of the enzyme was not detected. The mortality rate of the chick embryo was not correlated with the degree of proteinase production of these strains. Two type Ib strains invaded the CAM tissue and elicited some tissue reactions by the host, yielding a low mortality rate of the chick embryos. These results suggested that the secretion of proteinase was an important factor for the invasion of CAM but other factors were also involved for the pathogenicity of C. albicans. 相似文献
47.
In 1979 and 1980 the Canadian Paediatric Society''s Nutrition Committee published guidelines for professionals counselling mothers of infants on feeding practices. The practices in 1984-85 of mothers in Toronto were determined for comparison with the practices identified in a similar study conducted in Toronto and Montreal in 1977-78 to ascertain if practices had changed in favour of the recommendations. Between July 1984 and February 1985, 404 metropolitan Toronto mothers of infants were interviewed. Compared with the 1977-78 group of mothers, more of the 1984-85 mothers had chosen to breast-feed and fewer had stopped breast-feeding in the first month. As well, fewer of the 1984-85 infants had been fed unmodified cow''s milk in the first 6 months of life and introduced to solid foods before 4 months of age. We conclude that major changes in infant feeding practices had occurred since 1977-78 and that the 1984-85 practices corresponded closely to the infant feeding guidelines. 相似文献
48.
Purification of bleomycin hydrolase with a monoclonal antibody and its characterization 总被引:1,自引:0,他引:1
We established a hybridoma clone that produced anti-bleomycin hydrolase antibody. The subclass of the monoclonal antibody was immunoglobulin M. The antibody significantly reacted with bleomycin hydrolase from rabbit tissues, mouse livers, sarcoma 180, and adenocarcinoma 755 but not significantly with that from MH 134 and Ehrlich carcinoma. The enzyme from L5178Y cells showed an intermediate reactivity. Bleomycin hydrolase was purified from rabbit liver by immunoaffinity with the monoclonal antibody and DEAE gel chromatography. Approximately 1300-fold-purified bleomycin hydrolase was obtained. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing on a polyacrylamide slab gel of purified bleomycin hydrolase showed a single band with an apparent Mr of 48K and an isoelectric pH of 5.2. The molecular weight of bleomycin hydrolase determined on gel filtration high-performance liquid chromatography was ca. 300K, suggesting a hexameric enzyme. The enzyme showed an optimum pH of 6.8-7.8 and gave a Vmax value of 6.72 mg min-1 mg-1 for peplomycin and 9.24 mg min-1 mg-1 for bleomycin B2 and a Km value of 0.79 mM for both substrates. The enzyme was inhibited by E-64, leupeptin, p-tosyl-L-lysine chloromethyl ketone, N-ethylmaleimide, Fe2+, Cu2+, and Zn2+ but was enhanced by dithiothreitol. The results suggest that bleomycin hydrolase is a thiol enzyme. 相似文献
49.
Lipid metabolism in various regions of squid giant nerve fiber 总被引:3,自引:0,他引:3
The purpose of this investigation was to compare the incorporation of radioactivity from various precursors into lipids of different regions of squid giant nerve fiber systems including axoplasm, axon sheath, giant fiber lobes which contain stellate ganglion cell bodies, and the remaining ganglion including giant synapses. To identify the labeled lipids, stellate ganglia including giant fiber lobes and the remaining tissue were first incubated separately with [14C]glucose, [32P]phosphate, [14C]serine, [14C]acetate and [3H]myristate. The radioactivity from glucose, after conversion to glycerol and fatty acids, was incorporated into most lipids, including triacylglycerol, free fatty acids, cardiolipin, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, sphingomyelin and ceramide 2-aminoethylphosphanate [corrected]. The radioactivity from serine was largely incorporated into phosphatidylserine and, to a lesser extent, into other phospholipids, mainly as the base component. The sphingoid bases of ceramide and sphingomyelin were also significantly labeled. Saturated and monounsaturated and, to a lesser extent, polyunsaturated fatty acids of these lipids were synthesized from acetate, glucose and myristate. Among the major lipids, cholesterol was not labeled by any of the radioactive compounds used. Ganglion residues incorporated the most radioactivity in total lipids from either [14C]glucose or [14C]serine, followed by giant fiber lobes and then sheath. Axoplasm incorporated the least. Among various lipids, phosphatidylethanolamine with shorter saturated fatty acids and phosphatidylglycerol contained the most radioactivity from glucose in all regions. Axoplasm was characterized by a higher proportion of glucose radioactivity in ceramide, sphingomyelin and phosphatidylglycerol. Axoplasm and sheath contained a higher proportion of serine radioactivity than did the other two regions in ceramide. Essentially no radioactivity from [14C]galactose was incorporated in any region. 相似文献
50.
C Shigemasa K Abe S Taniguchi Y Mitani Y Ueta K Urabe T Adachi T Tanaka A Yoshida H Mashiba 《Endocrinologia japonica》1987,34(6):821-829
Serum total thyroxine (T4), total triiodothyronine (T3), T4-binding globulin (TBG), free T4(FT4) and free T3(FT3) concentrations and the T3-uptake(T3-U) value were estimated in 11 patients with subacute thyroiditis, and compared with the same parameters in 11 patients with Graves' disease, whose serum T4 concentrations were similar to the former group. Seven patients with subacute thyroiditis, who were treated with dicrofenac sodium alone, were investigated as to the sequential changes in serum parameters during their clinical courses. The mean serum T3-U value and FT4, T3 and FT3 concentrations in patients with subacute thyroiditis were increased, but all were significantly lower than those in patients with Graves' disease (p less than 0.01, p less than 0.001, p less than 0.001 and p less than 0.001, respectively). Three patients with subacute thyroiditis, who showed shorter duration of symptoms than 10 days, had serum TBG excess. Thus the mean (+/- SD) serum TBG concentration (26.5 +/- 8.4 micrograms/ml) was significantly higher than that (18.3 +/- 2.9 micrograms/ml) in patients with Graves' disease (p less than 0.02). The ratios of serum T3 to T4 and FT3 to FT4 in patients with subacute thyroiditis were also significantly lower than those in patients with Graves' disease (p less than 0.001 and p less than 0.001, respectively). The serum FT4 in 7 patients treated with dicrofenac sodium alone decreased to the normal range after 3 to 8 weeks from the onset of the illness. In 3 patients with TBG excess and one patient (TBG; 29.0 micrograms/ml), serum TBG declined in consequence of the serum FT4 normalization.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献