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1.
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Cell cycle variations in the modification of histones and nonhistones by ADPribosylation were investigated. Proteins of HeLa interphase nuclei and metaphase chromosomes were radioactively labeled in vivo with [3H]adenosine. Histones of metaphase chromosomes were extensively modified by ADPribosylation, with H2B, H2A and H4 being predominant acceptors of [3H]adenosine label. For histones of interphase nuclei from synchronized cells, the highest level of 3H labeling was observed by two-dimensional gel electrophoresis to occur in S phase. The minimum level was noted in G1 phase. ADPribosylation of histones is, however, significant during all phases of the cell cycle. These conclusions were confirmed by experiments using [32P]NAD. The results with the specific inhibitor of ADPribosylation, 3-aminobenzamide, and with snake venom phosphodiesterase indicated that the radioactive isotopes were incorporated as ADPribose. Two-dimensional gels of HeLa nonhistones labeled with [3H]adenosine showed strikingly different patterns for interphase and metaphase samples. Over 100 ADPribosylated species were found for interphase nuclei, but poly(ADPribose) polymerase was the only major acceptor for metaphase chromosomes. A simple pattern was also revealed for nuclear scaffolds, with the 'lamins' and poly(ADPribose) polymerase being identifiable as modified species.  相似文献   

3.
Rat plasma low- and high-density lipoproteins were labeled with [3H]cholesteryl linoleyl ether and isolated by rate-zonal ultracentrifugation into apolipoprotein B-containing LDL, apolipoprotein E-containing HDL1 and apolipoprotein E-poor HDL2. These fractions were incubated with cultured rat hepatocytes and comparable amounts of all lipoproteins were taken up by the cells. Rat HDL was isolated at d 1.085-1.21 g/ml and apolipoprotein E-free HDL was prepared by heparin Sepharose chromatography. The original HDL and the apolipoprotein E-free HDL were labeled with 125I or with [3H]cholesteryl linoleyl ether and incubated with rat hepatocytes or adrenal cells in culture. The uptake of apolipoprotein E-free [3H]cholesterol linoleyl ether HDL by the cultured hepatocytes was 20-40% more than that of the original HDL. Comparison of uptake of cholesteryl ester moiety (represented by uptake of [3H]cholesteryl linoleyl ether) and of protein moiety (represented by metabolism of 125I-labeled protein) was carried out using both original and apolipoprotein E-free HDL. In experiments in which low concentrations of HDL were used, the ratio of 3H/125I exceeded 1.0. In cultured adrenal cells, the uptake of [3H]cholesteryl linoleyl ether-labeled HDL was stimulated 3-6-fold by 1 X 10(-7) M ACTH, while the uptake of 125I-labeled HDL increased about 2-fold. The ratio of 3H/125I representing cellular uptake was 2-3 and increased to 5 in ACTH-treated cells. The present results indicate that in cultured rat hepatocytes the uptake of homologous HDL does not depend on the presence of apolipoprotein E. Evidence was also presented for an uptake of cholesteryl ester independent of protein uptake in cultured rat adrenal cells and to a lesser extent in rat hepatocytes.  相似文献   

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Biochemical and autoradiographic methods were used to investigate the retrograde transport of labeled material after injection of [3H]norepinephrine ([3H]NE) in the olfactory bulb (OB) of rat. Mechanical obstruction of the ventricular recess prevented intraventricular diffusion. At different time intervals following bilateral [3H]NE injections, total radioactivity was measured in the OB, locus caeruleus (LC), raphe dorsalis and periaqueductal gray. Preferential accumulation occurred in the LC, and an approximate rate of retrograde transport of 1–6 mm/h could be calculated. Previous administration of 6-hydroxydopamine in the OB reduced this accumulation by 60%. Sixteen hours after [3H]NE injection, the radioactivity in LC was equally distributed between an ethanol-soluble and -insoluble fraction. A small proportion of the soluble material was recovered as [3H]NE and/or [3H]normetanephrine. Following unilateral injections of [3H]NE, light microscopic autoradiograms demonstrated nerve cell body labeling mainly in the ipsilateral LC and of greater intensity after 16 than 4 and 8 h. These data lead to the conclusion that the movement of radioactive material was indeed representative of retrograde axonal transport rather than of other mechanisms such as diffusion. The observation of a preferential labeling of noradrenergic perikarya in LC supports the hypothesis of a process mediated by specific binding and/or uptake of [3H]NE into noradrenergic axon terminals.  相似文献   

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In the present study the tissue distribution of [3H]methotrexate was studied after intravenous injection of [3H]methotrexate-containing liposomes in normal and macrophage-depleted mice. Elimination of macrophages was performed by treatment with dichloromethylene diphosphonate- (DMDP)-containing liposomes. After thorough elimination of the macrophages from spleen and liver, by two intravenous injections of DMDP liposomes 6 and 4 days before tissue distribution studies, we found dramatic changes in the localization pattern of [3H]methotrexate liposomes in the blood, due to a decreased uptake of [3H]methotrexate liposomes by the DMDP liposome-treated liver. Because of the absence of these macrophages that are able to clear the blood of liposomes, and because of the resulting higher blood level of liposomes, we found an enhanced uptake of [3H]methotrexate liposomes by the spleen. It may be concluded that, in the spleen, apart from uptake of liposomes by macrophages, at least one other mechanism is responsible for the clearance of liposomes from the circulation. When comparing cholesterol-rich with cholesterol-poor liposomes, we found basically the same results, although uptake of cholesterol-rich liposomes by macrophages was smaller than that of cholesterol-poor liposomes, as found in several other studies. We suggest that pretreatment with DMDP liposomes can help to maintain a high level of intravenous-injected liposome-entrapped material in the blood, which otherwise would be removed by macrophages.  相似文献   

8.
The tumor promoting phorbol diester, 12-O-tetradecanoyl-phorbol-13-acetate, stimulates MDCK cells to deacylate cellular phospholipids and to produce prostaglandins when measured as the release of arachidonic acid and its metabolites nto the culture fluid. Indomethacin, at levels of 2.8 × 10−8 to 2.8 × 10−6 M, inhibits the release of radioactivity from [3H]arachidonate labeled cells stimulated by 12-O-tetradecanoyl-phorbol-13-acetate treatment in a concentration dependent manner. At these concentrations, the conversion of released [3H]arachidonic acid into prostaglandins E2 and F and the production of PGE2 measured serologically also is suppressed in a concentration dependent manner.Indomethacin, at these levels, has no effect on the acylation of [3H]arachidonic acid into cellular lipids. The tumor promoting phorbol diester does not stimulate the release of radioactive materials from MDCK cells labeled with [14C]linoleic acid, although prostaglandin production by these cells is stimulated.  相似文献   

9.
[3H]Ouabain specifically bound at sublethal concentrations to Na,K-ATPase on the surface of HeLa cells is taken up (internalized) by the cells at a rate of three membrane equivalents of labeled sites per generation. Immediately following a pulse label with the glycoside, codistribution of radioactivity with the surface marker 5'-nucleotidase is found in both conventional sucrose-gradient fractionation and in fractionation following a digitonin treatment. At appropriate concentrations digitonin increases the buoyant density of the HeLa surface membrane and solubilizes the lysosomal marker beta-hexosaminidase (Tulkens et al., 1974). After internalization, [3H]ouabain is also solubilized by digitonin. A shear analysis is described which shows internalized ouabain and beta-hexosaminidase to be codistributed in a particulate fraction that is homogeneous with respect to shear; extrapolation to zero-shear shows that little or none of either marker is found in the soluble fraction of the cytosol. Both markers are coreleased from the particulate fraction by osmotic shock. Although internalized ouabain is subsequently released from these cells with a half-time of about 70 hr, apparently by exocytosis, the shear sensitivity of the remaining cell-associated ouabain does not change for up to 72 hr. Thus ouabain (together with Na,K-ATPase?) appears to be taken up from the surface into a lysosomal compartment and, by at least one criterion, this compartment does not change its physical properties with time, i.e., does not "age."  相似文献   

10.
[17-2H]Pregnenolone was incubated with the microsomal fraction of boar testis under an 18O2 atmosphere. The metabolites were analyzed by gas chromatography-mass spectrometry, and the following six metabolites labeled with 2H or 18O (or both) were identified: 17 alpha-[17-18O]hydroxypregnenolone, [17-18O]dehydroepiandrosterone, 5-[17-18O]androstene-3 beta, 17 beta-diol, 16 alpha-[16-18O]hydroxy[17-2H]pregnenolone, 5-[17 beta-2H, 17-18O]androstene-3 beta,17 alpha-diol, and 5,16-[17-2H]androstadien-3 beta-ol. The time course of the formation of these metabolites from pregnenolone was also studied using 14C-labeled substrate. The results obtained from these experiments suggest that the first three metabolites were synthesized by a well-documented pathway--pregnenolone yields 17 alpha-hydroxypregnenolone yields dehydroepiandrosterone yields 5-androstene-3 beta,17 beta-diol--, and that 16 alpha-hydroxypregnenolone, 5-androstene-3 beta,17 alpha-diol and 5,16-androstadien-3 beta-ol were synthesized from [17-2H]pregnenolone with retention of 17-2H.  相似文献   

11.
The incorporation of [3H]-glucosamine into polypeptides of three fractions of polysomes in MPC-11 cells was studied. After short term incubation greatest incorporation was observed in a fraction of membrane-bound polysomes, which after nitrogen cavitation of cells, remained bound to the endoplasmic reticulum (ER) associated with the nucleus (fraction 2). Polypeptide chains on membrane-bound polysomes in the microsomal fraction (fraction 1) and free polysomes contained much less radioactivity. Since nascent polypeptide chains contained within membrane-bound polysomes of fraction 2 are glycosylated at an earlier stage than those in fraction 1 it is likely that this represents a difference in type of proteins synthesized in the respective fractions of ER.  相似文献   

12.
Polycyclic aromatic hydrocarbons (PAHs) present in ambient air are considered as potential human carcinogens, but the detailed mechanism of action is still unknown. Our aim was to study the in vitro effect of exposure to dibenzo[a,l]pyrene (DB[a,l]P), the most potent carcinogenic PAH ever tested, and benzo[a]pyrene (B[a]P) in a normal human diploid lung fibroblast cells (HEL) using multiple endpoints. DNA adduct levels were measured by 32P-postlabelling, the expression of p53 and p21(WAF1) proteins by western blotting and the cell cycle distribution by flow cytometry. For both PAHs, the DNA adduct formation was proportional to the time of exposure and dependent on the stage of cell growth in culture. DNA binding was detectable even at the lowest concentration used (24h exposure, 0.01 microM for both PAHs). The highest DNA adduct levels were observed after 24h of exposure in near-confluent cells (>90% of cells at G0/G1 phase), but DNA damage induced by DB[a,l]P was approximately 8-10 times higher at a concentration one order of magnitude lower as compared with B[a]P (for B[a]P at 1 microM and for DB[a,l]P at 0.1 microM: 237+/-107 and 2360+/-798 adducts/10(8) nucleotides, respectively). The induction of p53 and p21(WAF1) protein occurred subsequent to the induction of DNA adducts. The DNA adduct levels correlated with both p53 (R=0.832, P<0.001 and R=0.859, P<0.001, for DB[a,l]P and B[a]P, respectively) and p21(WAF1) levels (R=0.808, P<0.001 and R=0.797, P=0.001, for DB[a,l]P and B[a]P, respectively), regardless of the PAH exposure and the phase of cell growth. The results showed that a detectable increase of p53 and p21(WAF1) proteins (> or = 1.5-fold as compared with controls) requires a minimal DNA adduct level of approximately 200-250 adducts/10(8) nucleotides for both PAHs tested and suggest that the level of adducts rather than their structure triggers the p53 and p21(WAF1) responses. The cell cycle was altered after 12-16h of treatment, and after 24h of exposure to 0.1 microM DB[a,l]P in growing cells, there was approximately 24% increase in S phase cells accompanied by a decrease in G1 and G2/mitosis (G2/M) cells. Cell treatment with 1.0 microM B[a]P resulted in more subtle alterations. We conclude that DB[a,l]P, and to a lesser degree B[a]P, are able to induce DNA adducts as well as p53 and p21(WAF1) without eliciting G1 or G2/M arrests but rather an S phase delay/arrest. Whether the S phase delay observed in our study is beneficial for the survival of the cells remains to be further established.  相似文献   

13.
Experiments about the interaction of herpes viruses with BHK-cells during the first 6 h after infection concerning uptake and incorporation of dThd have been reported. During adsorption and penetration, the inhibition of uptake and of incorporation of [3H] dThd is sensitive to heat, but not to ultraviolet irradiation or cycloheximide. The eclipse is characterized by a strongly increased uptake of [3H] dThd and by inhibition of cell DNA synthesis. Both are sensitive to ultraviolet irradiation of the particles and cycloheximid treatment of the cells. It is concluded that the events during adsorption and penetration are dependent on the particles themselves, whereas the events during the eclipse depend on the activity of the viral genome. The implications of the findings are discussed.  相似文献   

14.
15.
Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.  相似文献   

16.
It has previously been shown that anthranilamide-pyrazolo[1,5-a]pyrimidine conjugates activate p53 and cause apoptosis in cervical cancer cells such as HeLa and SiHa. Here we establish the role of these conjugates in activating p53 pathway by phosphorylation at Ser15, 20 and 46 residues and downregulate key oncogenic proteins such as MYCN and Mdm2 in IMR-32 neuroblastoma cells. Compounds decreased the proliferation rate of neuroblastoma cells such as IMR-32, Neuro-2a, SK-N-SH. Compound treatment resulted in G2/M cell cycle arrest. The expression of p53 dependent genes such as p21, Bax, caspases was increased with concomitant decrease of the survival proteins as well as anti-apoptotic proteins such as Akt1, E2F1 and Bcl2. In addition the expression of important microRNAs such as miR-34a, c, miR-200b, miR-107, miR-542-5p and miR-605 were significantly increased that eventually lead to the activation of apoptotic pathway. Our data revealed that conjugates of this nature cause cell cycle arrest and apoptosis in IMR-32 cells [MYCN (+) with intact wild-type p53] by activating p53 signalling and provides a lead for the development of anti-cancer therapeutics.  相似文献   

17.
Quinazolines are multitarget agents, which have broad spectrum of biological activity, and some of them are now in cancer clinical testing. 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline is a new synthetically prepared derivative, which in our previous study showed cytotoxic effects on cancer cell lines HeLa and B16. Quinazoline, at micromolar concentrations, induced morphological changes and necrosis of B16 cells, and at nanomolar concentrations it produced changes of F-actin cytoskeleton. It did not cause changes in the cell cycle, did not induce apoptotic cell death in B16 cells, did not have a mutagenic effect, and did not even behave as a typical intercalating agent. Little significant reduction of tumor volume in intramuscular transplanted B16 cells was observed. The aim of the present study was to examine the cytotoxic effect of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline on murine leukemia L1210 cells and fibroblast NIH-3T3 cells. Induction of cell morphology and cell cycle changes, induction of apoptosis and caspase 3 activity were studied. Quinazoline acted cytotoxically on both cell lines. The sensitivity of leukemia L1210 cells to the quinazoline was higher than that of fibroblast NIH-3T3. The IC(100) was 12 microM for L1210 cells and 24 microM for NIH-3T3 cells. No effect of quinazoline on the cell cycle profile of L1210 and NIH-3T3 was detected, however, quinazoline induced an increase of the sub-G(0) cell fraction, apoptotic DNA fragmentation, and apoptotic morphological changes at a concentration of 12 microM. This quinazoline concentration induced caspase 3 activity. Our results demonstrated that induction of apoptotic cell death via activation of caspase 3 contributed to the cytotoxic effects of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline in murine leukemia L1210 cells.  相似文献   

18.
The interaction of p-[14C] chloro-o-toluidine with hepatic macromolecules of rats and mice has been investigated. At all time points after single administration the extent of binding decreased in the order protein greater than RNA greater than DNA in both species. The level of binding to mouse liver DNA was greater than that to rat liver DNA after both single and repeated administration. In vitro studies showed that mouse liver fractions catalysed the binding of p-chloro-o-toluidine to calf thymus DNA more readily than rat liver fractions. Conversely, binding to protein and RNA was more marked in the rat than in the mouse. Species differences in DNA repair rates were not observed. The results failed to demonstrate a preferential persistence of binding to mouse liver nonparenchymal cell DNA. Autoradiographic determinations did not demonstrate any effect of p-chloro-o-toluidine upon the incorporation of [3H] thymidine into subcutaneous capillary endothelial cells. The results suggest that different reactive metabolites are responsible for binding to DNA and protein, and that the pattern of reactive metabolites formed from p-chloro-o-toluidine in the mouse differs from that formed in rats.  相似文献   

19.
The effects of dextrans of varying molecular weights and of pentosan sulfate on the distribution of 51Cr-labeled mouse lymph node cells were studied in vivo, i.e., in recipients treated with the sulfated polysaccharides, and in vitro, i.e., by following the fate of cells treated in vitro, in intact syngeneic recipients. Both types of experiments demonstrate that dextrans, especially dextran sulfate (DXS) and pentosan sulfate (PS), considerably reduce lymph node entry of lymphocytes, with concomitant increases in the blood and, in the case of DXS, in both the blood and lungs. A parallel quantitative autoradiographic analysis of the distribution of [3H]adenosine-labeled cells confirmed the data with the 51Cr-labeled cells and, in adidtion, indicated that DXS and PS slow down circulation of lymphocytes through the marginal zone and red pulp of the spleen and, in the case of DXS, in the pulmonary capillary bed. Unusually large numbers of unlabeled lymphocytes were found in the endothelial wall of the post-capillary venules in lymph nodes of PS-treated mice.  相似文献   

20.
Plasma membrane-enriched fractions from disrupted S49 lymphoma cells contained high affinity sites for [3H]nitrobenzylthioinosine, a potent and specific inhibitor of nucleoside transport. These sites were absent from similar preparations from AE1 cells, a nucleoside-transport deficient clone derived from the S49 cell line. Reversible binding of [3H]nitrobenzylthioinosine to the S49 membrane preparations was inhibited by adenosine, nitrobenzylthioguanosine, and dipyridamole. Exposure of S49 membrane preparations to UV light in the presence of [3H]nitrobenzylthioinosine resulted in the covalent radiolabeling of a membrane protein(s) which migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent Mr of 45,000 to 66,000. Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine and markedly reduced in the presence of adenosine and dipyridamole. AE1 membrane proteins were not covalently labeled under these conditions.  相似文献   

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