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1.
Earlier work of several laboratories established that the yields of radiation-induced ring and dicentric chromosomes are greater when human peripheral blood lymphocytes are irradiated in GH1 some hours after phytohemagglutinin stimulation than if they are irradiated in G0 before stimulation. Post-treatment of lymphocytes irradiated in G0 with the DNA polymerase inhibitor aphidicolin, which is effective against both pol α and pol δ, produces a similar increase in ring and dicentric yield. We found that aphidicolin post-treatment was much less effective in increasing ring and dicentric yield increases in cells irradiated in G1 four to five hours after stimulation. Because we had earlier found specific inhibitors of DNA pol α ineffective in producing increased yields in either G0 or G1 lymphocytes, we conclude that much of the G0 to G1 increase in yields is mediated by pol δ. 相似文献
2.
A comparison has been made of dicentric yields in G0 lymphocytes between man and crab-eating monkey, Macaca fascicularis, after acute and chronic γ-irradiations. With acute irradiation (49.6 rad/min) there was no significant difference between them, but for the chronic irradiation (17.1 rad/h) a significant difference was observed between the species. When the dose-response relations were fitted to the linear-quadratic model (Y = D + βD2), the species-difference observed for chronic irradiation was almost entirely due to change in the value of β. In addition, after chronic irradiation the β-value for monkey was almost negligible, but that for man was significant. Post-irradiation incubation experiment showed that cells with dicentrics were partly eliminated during the course of chronic irradiation, because there were appreciable reductions of dicentric yields (ca. 25% for both man and monkey at 400 rad) together with mitotic indices (ca. 30% and 60% for man and monkey, respectively, at 400 rad). Accordingly, it would be reasonable to postulate that G0 repair for dicentrics other than selection mechanism must play a major role in the effects of low dose rate. It can be further suggested that G0-repair capacity for chromosal damages leading to dicentrics may be different among different primate species. 相似文献
3.
Whole-blood cultures of human lymphocytes were exposed in the G2-phase to caffeine and to 4 inhibitors of DNA synthesis, hydroxyurea (HU), 2′-deoxyadenosine (dAdo), 1-β-
-arabinofuranosylcytosine (araC) and aphidicolin (Aph), either individually or in pairs resulting in 10 different possible combinations. Since dAdo is rapidly deaminated in whole-blood cultures, all treatments involving dAdo were carried out in the presence of an inhibitor of the enzyme adenosine deaminase (ADA). The G2-treatments were carried out on 3 different types of culture, (1) cultures that had not previously been exposed to any mutagenic treatment, (2) cultures that had been irradiated with 0.4 Gy of X-rays 3.5 h before harvesting, and (3) cultures that had been exposed for 2 h to 4 × 10−5 M thiotepa (TT) in G0 immediately before stimulation with phytohaemagglutinin. The aim of the study was to find out in which combinations the inhibitors enhanced synergistically the frequencies of spontaneous and induced chromatid aberrations. In all 3 types of experiment, synergistic effects were observed with most of the 10 combinations, those involving HU being particularly effective. A very strong synergistic enhancement was also obtained when dAdo was combined with Aph. 相似文献
4.
Enhancement of radiation-induced apoptosis by 6-formylpterin 总被引:7,自引:0,他引:7
Cui ZG Kondo T Ogawa R Feril LB Zhao QL Wada S Arai T Makino K 《Free radical research》2004,38(4):363-373
Radiation-induced apoptosis and its possible enhancement in the presence of 6-formylpterin (6-FP), a metabolite of folic acid, were examined in human myelomonocytic lymphoma U937 cells. When cells were treated with 6-FP at a nontoxic concentration of 300 μM, and then exposed to X-rays at a dose of 10 Gy, significant enhancement of radiation-induced apoptosis as determined by nuclear morphological change, phosphatidylserine (PS) externalization and DNA fragmentation were observed. Flow cytometry for the detection of intracellular hydrogen peroxide (H2O2) revealed that 6-FP increased the formation of intracellular H2O2, which further increased when the cells were irradiated. Decrease of mitochondria trans-membrane potential (MMP), release of cytochrome c from mitochondria, and activation of caspase-3 were enhanced after the combined treatment. Remarkable activation of protein kinase C δ (PKC δ) and its translocation from cytosol to mitochondria were detected in combined treatment. Increase of intracellular Ca2+ concentrations ([Ca2+]i) was also observed, however, neither calpain I nor calpain II could inhibit the apoptosis. In addition, c-Jun NH2-terminal kinase ( JNK) activation was not enhanced in the combined treatment. A protein involved in a caspase-independent apoptosis pathway, apoptosis inducing factor (AIF), remained unchanged even 3 h after treatment. These results indicate that intracellular H2O2 generated by 6-FP enhances radiation-induced apoptosis via the mitochondria-mediated caspase-dependent pathway, with the active involvement of PKC δ. 相似文献
5.
Experiments were carried out using human lymphocytes in order to test the effect of pH shifts on radiosensitivity of cells irradiated in the G2 stage. In our culture conditions constant variations in medium pH over the range of 7.4–6.8 were observed as a function of incubation time after PHA stimulation; in addition the pH of the medium was adjusted in parallel cultures over the range of 5.9–8.2. Then we exposed the cultures with different pH to a treatment of X-rays delivered 2 h before fixation.
The pH of the medium was found to greatly affect the yield of induced chromatid aberrations. 相似文献
6.
目的: 构建α1亚基诱导表达、β2和γ2L亚基稳定表达的人源α1β2γ2L-GABAAR-CHO(Chinese hamster ovary)细胞株。方法: 从人cDNA文库中扩增α1、β2、γ2L亚基编码基因,分别构建亚基表达载体;将三个亚基表达载体共转染CHO-K1细胞,通过抗性筛选、膜电位检测法进行稳定表达克隆筛选;通过qPCR、Western blot对亚基表达进行鉴定;以激动剂GABA、阳性变构调节剂地西泮(diazepam,Dia)、拮抗剂荷包牡丹碱(bicuculine)为工具药,采用全细胞膜片钳方法及膜电位检测法对稳定表达细胞的药理学功能进行鉴定。结果: 经克隆筛选获得表达量较高的α1β2γ2L-GABAAR-CHO并对其亚基表达鉴定,结果显示该细胞稳定表达α1、β2、γ2L亚基,构建的α1β2γ2L-GABAAR-CHO细胞仅在加入四环素(tetracyclin)诱导的情况下表达α1亚基并与β2、γ2L组装成具有功能活性的α1β2γ2L-GABAAR;对其进行全细胞膜片钳检测研究发现,GABA可对其产生激动效应,引起α1β2γ2L-GABAAR-CHO细胞产生氯离子通道特征性电流变化,Dia可剂量依赖性地增强GABA对α1β2γ2L-GABAAR的激动效应;在膜电位检测研究中,获得GABA激动效应EC50为(177.72 ± 15.92)nmol/L,Dia变构效应EC50为(3.63±0.52)μmol/L,拮抗剂Bicuculine拮抗效应IC50为(538.83±29.55)nmol/L。结论: 通过采用诱导表达策略,成功构建了α1β2γ2L-GABAAR-CHO稳定表达细胞株,该细胞株具有对激动剂、阳性变构剂、拮抗剂特异性检测的药理学功能。 相似文献
7.
A microbial process for the production of optically-active γ-decalactone from the ricinoleic acid present as triglycerides in castor oil has been developed, γ-decalactone (γDL) is a component of some fruit flavours, being an important organoleptic component of peach flavours. Screening showed two red yeast microorganisms, Rhodotorula glutinis and Sporobolomyces odortts to be especially suitable for this biotransformation. The process involves lipase-mediated hydrolysis of the castor oil to give free ricinoleic acid, uptake of the acid by the cells and aerobic fermentation to achieve abbreviated β-oxidation of the ricinoleic acid (12-hydroxyoleic acid) into 4-hydroxydecanoic acid (4HDA), lactonisation of the acid into γ-DL, followed by solvent extraction and distillation. γ-DL broth concentrations of 0.5-1.2g · 1-t were obtained after 3-5 days from fermentation media containing 10 g · 1-1 castor oil, representing an 8.3-20.0% theoretical yield. Intermediates detected were consistent with the operation of the β-oxidation pathway. Appreciable amounts of novel metabolites identified as cis and trans isomers of a tetrahydrofuran (C10) were also produced. Their formation from 4HDA appeared to be non-enzymic and was favoured by anaerobic conditions. Yields of γ-DL were inversely proportional to the concentration of castor oil present in the medium, indicating that substrate inhibition takes place. The highest yields of γ-DL were obtained when castor oil was present from the beginning of the fermentation, rather than when added once the fermentation had become established, demonstrating that the β-oxidation pathway and/or transport system require continual induction. Significant amounts of γ-DL were not produced from other fatty acids, including ricinelaidic acid, the trans isomer of ricinoleic acid. γ-DL formation was dramatically inhibited by antibiotic inhibitors of oxidative phosphorylation, indicating the importance of intact β-oxidation pathways, whereas inhibitors of protein synthesis and cell-wall synthesis had much less marked effects. Selective extraction of 4HDA from the fermentation broths, and of γDL from broth lactonised by heating at low pH, could be achieved by adsorption to Amberlite XAD-1 and XAD-7 resins respectively. Some product could be recovered from the exit gases of the fermenter by passing through propylene glycol traps. This pathway is unusual in that it is a rare example of the truncated β-oxidation of a fatty acid by microorganisms. This effect probably occurs because of partial inhibition of one or more enzymes of the β-oxidation pathway by the C10 hydroxylated fatty acid intermediate(s) allowing intracellular accumulation of the 4HDA, followed by leakage out of the cell; although further metabolism of this C10 intermediate does take place slowly. 相似文献
8.
The cytotoxic and mutagenic effect of X-irradiation was determined with Chinese hamster ovary cells arrested in the G0/G1 phase of the cell cycle through 9 days incubation in serum-free medium. In comparison with exponential phase cultures, the arrested cells showed increased cytotoxicity and mutation induction over the dose range of 50–800 rad. Exponential cultures showed a linear mutant frequency-survival relationship while the arrested cells showed a biphasic linear relationship. A post irradiation holding period of 24 h does not result in any change in the mutant frequency. The increased sensitivity of the arrested cells to the mutagenic effects of X-rays appears to be a cell-cycle phase phenomenon. Upon readdition of serum, the arrested cells re-enter the cell cycle in a synchronous manner, reaching S phase at 10–12 h. Cells irradiated at 5 h after serum addition, i.e. in G1, show a similar does response for mutant frequency, while those irradiated at 10 h or later, i.e. in late G1, S or G2, show lower mutation induction. These observations are consistent with a chromosome interchange mechanism of mutation induction by X-rays, possibly through interactions between repairing regions of the DNA. Irradiation of cells in the G0/G1 phase allow more time for such interactions in the absence of semiconservative DNA replication. 相似文献
9.
《FEBS letters》1988,230(1-2):109-115
We investigated the binding of subunit δ to solubilized chloroplast ATPase. Purified δ was covalently labeled with eosin 5-isothiocyanate and its rotational correlation time was determined by a photoselection technique as a function of added CF1 (containing δ) and of CF1(−δ) (lacking δ). In aqueous buffer the rotational correlation time of labeled δ was 33 ns. This is compatible with a rather elongated shape with the dimensions 2b = 100 Å/2a = 28 Å. Binding of δ to CF1 decreased the rotational correlation time about 10-fold. The result was a biphasic decay of the laser flash-induced absorption anisotropy which was analyzed to yield the proportion of δ (bound to CF1) relative to δ (free). CF1(−δ), which completely lacked the δ-subunit, bound one δ (mol/mol) with high affinity (Kd ≈ 100 nM) and at least another δ with about 20-fold lower affinity. The δ-containing CF1, revealed only the low-affinity site(s) for δ. This was compatible with a 1:1 stoichiometry of δ in isolated CF1. 相似文献
10.
Heng Xu John S. Partilla Brian R. de Costa Kenner C. Rice Richard B. Rothman 《Peptides》1992,13(6):1207-1213
Recent pharmacological data strongly support the hypothesis of δ receptor subtypes as mediators of both supraspinal and spinal antinociception (δ1 and δ2 receptors). In vitro ligand binding data, which are fully supportive of the in vivo data, are still lacking. A previous study indicated that [3H][
-Ala2,
-Leu5]enkephalin labels two binding sites in membranes depleted of μ binding sites by pretreatment with the site-directed acylating agent, 2-(p-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimidazole-HCI (BIT). The main goal of the present study was to develop a ligand-selectivity profile of the two δncx binding sites. The data indicated that naltrindole and oxymorphindole were relatively selective for site 1 (20-fold). [
-Ser2,Thr6]Enkephalin and deltorphin-II were only 2.7-fold and 2.2-fold selective for site 1. [
-Pen2,
-Pen5]Enkephalin and deltorphin-I were 80-fold and 38-fold selective for site 2.3-Iodo-Tyr-
-Ala-Gly-Phe-
-Leu was 52-fold selective for site 1. Morphine had moderate affinity for site 1 (Ki = 16 nM), and was about 11-fold selective for site 1. Thus, of the 10 drugs studied, only DPDPE and DELT-I were selective for site 2. Viewed collectively with other data, it is likely that the δ1 receptor and the δncx binding site are synonymous. 相似文献
11.
Cellamare S Stefanachi A Stolfa DA Basile T Catto M Campagna F Sotelo E Acquafredda P Carotti A 《Bioorganic & medicinal chemistry》2008,16(9):4810-4822
A series of N-terminus benzamides of glycine-based symmetric peptides, linked to m-xylylenediamine and 3,4′-oxydianiline spacers, were prepared and tested as inhibitors of β-amyloid peptide Aβ1–40 aggregation in vitro. Compounds with good anti-aggregating activity were detected. Polyphenolic amides showed the highest anti-aggregating activity, with IC50 values in the micromolar range. Structure–activity relationships suggested that π–π stacking and hydrogen-bonding interactions play a key role in the inhibition of Aβ1–40 self-assembly leading to amyloid fibrils. 相似文献
12.
Hideyuki Suzuki Nobukazu Miyakawa Hidehiko Kumagai 《Enzyme and microbial technology》2002,30(7):883-888
γ-L-Glutamyltaurine is a naturally occurring peptide and known to have several physiological functions in mammals. This paper describes a new method for the enzymatic production of γ-L-glutamyltaurine from L-glutamine and taurine through the transpeptidation reaction of γ-glutamyltranspeptidase (EC 2.3.2.2) of Escherichia coli K-12. The optimum conditions for the production of γ-L-glutamyltaurine were 200 mM L-glutamine, 200 mM taurine and 0.2 U/ml γ-glutamyltranspeptidase, pH 10, and 1-h incubation at 37°C. Forty-five mM γ-L-glutamyltaurine was obtained, the yield being 22.5%. γ-L-Glutamyltaurine was purified on Dowex 1 × 8 and C18 columns, and identified by means of NMR and a polarimeter. 相似文献
13.
High-efficiency expression of rat protein kinase C-gamma in baculovirus-infected insect cells 总被引:1,自引:0,他引:1
Effects of cyclopentenone prostaglandins, Δ12-prostaglandin (PG) J2 and PGA2 on the expression of N-myc in relation to the effects on cell cycle progression were investigated using human neuroblastoma cell line GOTO. Both PGs suppressed M-myc expression within several hours prior to inducing G1 arrest. The N-myc suppression with Δ12-PGJ2 was continued but with PGA2 it was gradually released, followed by the release of G1 arrest. These results suggest that Δ12-PGJ2 and PGA2 inhibit cell cycle progression in strong association with N-myc suppression and Δ12-PGJ2 is more potent and has a longer effect than PGA2. 相似文献
14.
It has been suggested that the F1-ATPase β-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, β-casomorphin1–7, in the absence and presence of cold enterostatin. 125I-β-casomorphin1–7 weakly binds to the rat F1-ATPase β-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect β-casomorphin1–7 binding to the F1-ATPase β-subunit. Peptides that suppressed food intake promoted β-casomorphin1–7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced β-casomorphin1–7 binding. Surface plasmon resonance measurements show that the β-subunit of F1-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F1-ATPase complex. Western blot analysis showed the F1-ATPase β-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F1-ATPase β-subunit is the enterostatin receptor and suggests that enterostatin and β-casomorphin1–7 bind to distinct sites on the protein. 相似文献
15.
Bruce E. Elliott Peter Ekblom Hugh Pross Audrey Niemann Kristofer Rubin 《Cell communication & adhesion》1994,1(4):319-332
In the present report, we investigated the possible importance of β1 integrins in the growth and metastasis of a murine mammary carcinoma, SP1, and a metastatic variant, SP1-3M in vivo. CBA/J female mice bearing SP1 tumor transplants were injected with anti-β1 integrin IgG or control nonimmune IgG (200 μg per mouse; i.p.) every two days. Animals received anti-CD4 antibody (100 μg per mouse) at time zero to suppress immunity against rabbit IgG. Outgrowth of macroscopic metastases from SP1, but not from SP1-3M primary tumors, was markedly inhibited in animals receiving anti-β1 integrin IgG but not nonimmune IgG. To assess the stage(s) in the metastatic cascade affected, we examined the number and diameter of micrometastatic nodules in treated and untreated groups. The diameter of micrometastases was significantly reduced in SP1-tumor-bearing mice treated with anti-β1 integrin IgG compared to control IgG, although the number of nodules per cm2 of lung sections examined remained unchanged. No change in the number or size of micrometastases in SP1-3M tumor-bearing mice was observed. No difference in the binding, or complement-mediated and antibody-dependent cell-mediated cytotoxicity of anti-β1 integrin IgG with SP1 and SP1-3M cells was detected. The results suggest that under these conditions anti-β1 integrin inhibits metastatic tumor growth in lung tissue, but has minimal effect on intravasation, adhesion to target organs and extravasation. 相似文献
16.
Alpha-Chymotrypsin was found to show a 119% increase in activity after three phase partitioning. The kcat/Km of the partitioned enzyme (TPP-C) for hydrolysis of Bz-Tyr-OEt in aqueous medium at 25°C was found to be 48.3×104 mM-1 min-1 as compared to the corresponding value of 17.7×104 mM-1 min-1 for the untreated control (C). The λmax of the fluorescence emission spectrum of TPP-C showed 178% increase in the quantum yield when compared to C. TPP-C showed a 2.94 and 3.58 fold increase (as compared to C) in initial rates for formation of the ester Ac-Phe-OEt (from Ac-Phe and ethanol) in low water containing toluene and n-octane, respectively. It was found that TPP-C also showed the phenomenon of pH memory. At 5% (v v-1) water (in t-amyl alcohol), while no esterification was observed with C, TPP-C still showed significant level of esterification activity. 相似文献
17.
Maria Marino Silvana Spagnuolo Matteo Cavallini Fulvia Terenzi Maria Teresa Mangiantini Silvia Leoni 《Cellular signalling》1996,8(8):555-559
We investigate the effect of EGF on IP3 production, PLCγ phosphorylation, calcium transients in rat hepatocytes isolated in quiescent liver (G0 phase of cell cycle) and at 4 h (G1 phase of cell cycle) and 24 h (M phase of cell cycle) after partial hepatectomy. Our results show that EGF does not utilize IP3 and calcium as its signal transduction molecules when the hepatocytes are in vivo stimulated to entry in the cell cycle. In particular the growth factor does not phosphorylate PLCγ and induces a decrease in IP3 content. These data suggest that EGF utilizes different signal transduction to send information from receptor to nucleus during PH with respect to the quiescent liver. 相似文献
18.
Recently, it has been shown that PKA-mediated phosphorylation of the β2-adrenergic receptor (β2-AR) by the cyclic AMP-dependent protein kinase (PKA) reduces its affinity for Gs and increases its affinity for Gi. Here we demonstrate that, like the β2-AR, the β1-AR is also capable of “switching” its coupling from Gs to Gi in a PKA-dependent manner. The β1-AR is capable of activating adenylate cyclase via Gs, and can also activate the extracellular-regulated kinases, p44 and p42 (ERK1/2). In transfected CHO cells, the observed β1-AR-mediated activation of ERK is both sensitive to pertussis toxin (PTX), indicating involvement of Gi/Go, and to the PKA inhibitor, H-89. β1-ARs with PKA phosphorylation sites mutated to alanines are unable to activate ERK. Mutating these same residues to aspartic acid, mimicking PKA phosphorylation, leads to a decrease in Gs-stimulated cAMP accumulation and an increase in PTX-sensitive ERK activation. These results strongly support the hypothesis that the β1-AR, like the β2-AR, can undergo PKA-dependent “Gs/Gi switching”. 相似文献
19.
Contrary to an earlier report, peripheral lymphocytes from 4 AT patients were not found to exhibit higher yields of unequivocal chromosome type aberrations following irradiation in the G0 phase of the cell cycle, providing that only first post-irradiation metaphases were included in the samples (ensured by 5-bromodeoxyuridine (BrdU) incorporation and differential fluorescence or Giemsa staining). We were able, however, to confirm the earlier-reported increase in chromatid-type aberrations in the G0-irradiated cells. AT lymphocytes were found to experience more cell-cycle delay following G0 irradiation than normal cells. These observations appear consistent with the damaged base excision DNA-repair defect reported for AT cells. 相似文献
20.
Marcus Moreno-Horn Enriqueta Martinez-Rojas Helmut Grisch Roland Tressl Leif Alexander Garbe 《Journal of Molecular Catalysis .B, Enzymatic》2007,49(1-4):24-27
Whole cells of Rhodococcus erythropolis DSM 44534 grown on ethanol, (R)- and (S)-1,2-propanediol were used for biotransformation of racemic 1,4-alkanediols into γ-lactones. The cells oxidized 1,4-decanediol (1a) and 1,4-nonanediol (2a) into the corresponding γ-lactones 5-hexyl-dihydro-2(3H)-furanone (γ-decalactone, 1c) and 5-pentyl-dihydro-2(3H)-furanone (γ-nonalactone, 2c), respectively, with an EE(R) of 40–75%. The transient formation of the γ-lactols 5-hexyl-tetrahydro-2-furanol (γ-decalactol, 1b) and 5-pentyl-tetrahydro-2-furanol (γ-nonalactol, 2b) as intermediates was observed by GC–MS. 1,4-Pentanediol (3a) was transformed into 5-methyl-dihydro-2(3H)-furanone (γ-valerolactone, 3c) whereas (R)- and (S)-2-methyl-1,4-butanediol (4a) was converted to the methyl-substituted γ-butyrolactones 4-methyl-dihydro-2(3H)-furanone (4c1) and 3-methyl-dihydro-2(3H)-furanone (4c2) in a ratio of 80:20 with a yield of 55%. Also cis-2-buten-1,4-diol (5a) was transformed resulting in the formation of 2(5H)-furanone (γ-crotonolactone, 5c). At the higher pH values of 8.8 the yield of lactone formed was improved; however, the enatiomeric excesses were slightly higher at the lower pH of 5.2. 相似文献