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1.
Identification of estrogenresponsive genes is important to understand the molecular mechanisms of estrogen action. Suppression subtractive hybridization was employed to screen estrogenresponsive genes in chick liver. A single injection of estrogen into 6weekold chick induced upregulation of several known genes encoded for yolk proteins, such as Vitellogenin I and II and very low density lipoprotein II (apo-VLDL II). One novel sequence displayed a dramatic change (3fold increase) in response to estrogen treatment. This cDNA fragment was extended and the resultant sequence was analyzed. Translated amino acid sequence was 90, 88, 83 and 87% identical to the Larginine:glycine amidinotransferase of pig, rat, frog and human, respectively. The sequence has a conservative catalytic site of Larginine:glycine amidinotransferase. The expression pattern of this gene in organs is consistent with previous reports of Larginine:glycine amidinotransferase in chick. Thus, this clone represented the chicken Larginine:glycine amidinotransferase. It appeared that estrogeninduced alteration of arginine:glycine amidinotransferase was not dependent on protein synthesis, because concurrent administration of cycloheximide did not affect the estrogenmediated expression pattern. This is the first study demonstrating that Larginine:glycine amidinotransferase is a target of the estrogen receptor. 相似文献
2.
D[3H]mannoheptulose was recently reported to be poorly taken up by tumoral pancreatic islet cells of the RINm5F and INS1 lines. We have now investigated the effects of Dmannoheptulose upon Dglucose metabolism in these two cell lines. Dmannoheptulose (1.0–10.0 mM) only caused a minor decrease of Dglucose metabolism in RINm5F cells, whether at low (1.1 mM) or higher (8.3 mM) Dglucose concentration. A comparable situation was found in INS1 cells examined after more than 20 passages. In both cases, however, the hexaacetate ester of Dmannoheptulose (5.0 mM) efficiently inhibited Dglucose metabolism. In the INS1 cells, the relative extent of the inhibitory action of Dmannoheptulose upon Dglucose metabolism increased from 12.4 ± 2.6 to 38.3 ± 3.8% as the number of passages was decreased from more than 20 to 13–15 passages, the latter percentage remaining lower, however, than that recorded in INS1 cells also examined after 13–15 passages but exposed to Dmannoheptulose hexaacetate (66.9 ± 2.2%). These findings when compared to our recent measurements of D[3H]mannoheptulose uptake, reinforce the view that the entry of the heptose into cells and, hence, its inhibitory action on Dglucose metabolism are dictated by expression of the GLUT2 gene. 相似文献
3.
Hallab Nadim J. Mikecz Katalin Vermes Csaba Skipor Anastasia Jacobs Joshua J. 《Molecular and cellular biochemistry》2001,222(1-2):127-136
Metal toxicity from sources such as orthopaedic implants was investigated in terms of immune system hyper-reactivity to metal implant alloy degradation products. Lymphocyte response to serum protein complexed with metal from implant alloy degradation was investigated in this in vitro study using primary human lymphocytes from healthy volunteers (n = 10). Cobalt chromium molybdenum alloy (CoCrMo, ASTM F75) and titanium alloy (Ti6Al4V, ASTM F136) beads (70 m) were incubated in agitated human serum at 37 degrees Celsius to simulate naturally occurring metal implant alloy degradation processes. Particulate free serum samples, which were incubated with metal, were then separated into molecular weight based fractions. The amounts of soluble Cr and Ti within each serum fraction were measured and correlated with lymphocyte proliferation response to the individual serum fractions. Lymphocytes from each subject were cultured with 11 autologous molecular weight based serum fractions either with or without added metal. Two molecular weight ranges of human serum proteins were associated with the binding of Cr and Ti from CoCrMo and Ti implant alloy degradation (at < 30 and 180–330 kDa). High molecular weight serum proteins ( 180 kDa) demonstrated greater lymphocyte reactivity when complexed with metal released from CoCrMo alloy and Ti alloy than with low (5–30 kDa) and midrange (30–77 kDa) serum proteins. When the amount of lymphocyte stimulation was normalized to both the moles of metal and the moles of protein within each fraction (MetalProtein Complex Reactivity Index, MPCRI), Cr from CoCrMo alloy degradation demonstrated approximately 10 fold greater reactivity than Ti in the higher molecular weight serum proteins ( 180–250 kDa). This in vitro study demonstrated a lymphocyte proliferative response to both CoCrMo and Ti alloy metalloprotein degradation products. This response was greatest when the metals were complexed with high molecular weight proteins, and with metalprotein complexes formed from CoCrMo alloy degradation. 相似文献
4.
The lactone isolated from Fusarium termed L659,699 is a potent specific inhibitor of the enzyme 3hydroxi3methylglutaril coenzyme A (HMG-CoA) synthase. In cultures of smooth muscle cells (SMC) isolated from aortic-arch of control (CSMC) and 5% of cholesterol diet (Ch-SMC) treated chicks, the incorporation of (14C)acetate to lipids (cholesterol, triacylglycerides and cholesterol ester) were greater in ChSMC cultures than in CSMC and the presence of 0.05 M L659,699 for 2 h in the incubation medium decrease the synthesis of cholesterol however the triacylglycerides synthesis increase. The effect of inhibitor is stronger in young cultures (3–4 steps) than in the older ones (11–12 steps). In young CSMC and ChSMC cultures the inhibition of cholesterol and triacylglycerides synthesis by L659,699 was reversal. 相似文献
5.
Marzban Lucy Bhanot Sanjay McNeill John H. 《Molecular and cellular biochemistry》2001,223(1-2):147-157
In this study, the in vivo effects of insulin and chronic treatment with bis(maltolato)oxovanadium (IV) (BMOV) on protein kinase B (PKB) activity were examined in the liver and skeletal muscle from two animal models of diabetes, the STZdiabetic Wistar rat and the fatty Zucker rat. Animals were treated with BMOV in the drinking water (0.75–1 mg/ml) for 3 (or 8) weeks and sacrificed with or without insulin injection. Insulin (5 U/kg, i.v.) increased PKB activity more than 10fold and PKB activity more than 3fold in both animal models. Despite the development of insulin resistance, insulininduced activation of PKB was not impaired in the STZdiabetic rats up to 9 weeks of diabetes, excluding a role for PKB in the development of insulin resistance in type 1 diabetes. Insulin-induced PKB activity was markedly reduced in the skeletal muscle of fatty Zucker rats as compared to lean littermates (fatty: 7fold vs. lean: 14fold). In contrast, a significant increase in insulinstimulated PKBa activity was observed in the liver of fatty Zucker rats (fatty: 15.7fold vs. lean: 7.6fold). Chronic treatment with BMOV normalized plasma glucose levels in STZdiabetic rats and decreased plasma insulin levels in fatty Zucker rats but did not have any effect on basal or insulininduced PKB and PKB activities. In conclusion (i) in STZdiabetic rats PKB activity was normal up to 9 weeks of diabetes; (ii) in fatty Zucker rats insulininduced activation of PKB (but not PKB) was markedly altered in both tissues; (iii) changes in PKB activity were tissue specific; (iv) the glucoregulatory effects of BMOV were independent of PKB activity. 相似文献
6.
Yanagawa Yuchio Kobayashi Takashi Ohnishi Motoko Kobayashi Takayasu Tamura Shinri Tsuzuki Teruhisa Sanbo Makoto Yagi Takeshi Tashiro Fumi Miyazaki Jun‐ichi 《Transgenic research》1999,8(3):215-221
Gene targeting in embryonic stem (ES) cells via homologous recombination can occur at very low frequency. In order to enrich homologous recombinants before screening, a negative selection marker, such as thymidine kinase (TK) and diphtheria toxin A fragment (DTA), has been commonly used. In this study, we developed a negative selection marker using DTA gene with polyadenylation signal and it was designated DTApA. To determine the difference in targeting efficiency of the negative selections, we constructed three different targeting vectors for each negative selection (first, TK at the 3 end, second, TK at both the 5 and 3 ends <2 X TK>, and third, DTApA at the 5 end of the homologous sequences). Gene targeting experiments using these constructs clearly showed that negative selection using DTApA was more efficient than that using TK for homologous recombination and that negative selection using DTApA was as efficient as that using 2 X TK. Considering the fact that the use of DTApA is more convenient for construction of targeting vectors than that of 2 X TK, DTApA is an efficient negative selection marker.In addition, we examined long and accurate PCR (LAPCR) for screening gene targeted clones. The use of LAPCR with genomic DNAs from ES cell clones facilitated simple detection of homologous recombinants, suggesting that the screening with LAPCR is compatible with the use of longer homologous sequences of both arms in vector design. Our results indicate that the use of DTApA for negative selection together with the application of LAPCR for screening ensures efficient and timesaving screening for homologous recombinants. 相似文献
7.
Bearne SL White RL MacDonnell JE Bahrami S Grønlund J 《Molecular and cellular biochemistry》2001,221(1-2):117-126
Methylaspartase (EC 4.3.1.2) was purified 20fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAESepharose. The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg2+ (Km = 0.27 ± 0.01 mM) and K+ (Km = 3.3 ± 0.8 mM) for maximum activity. Methylaspartasecatalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)3methylaspartate (major product) and (2S,3R)3methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)3methylaspartate (Km = 0.51 ± 0.04 mM) was observed at pH 9.7. The Nterminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme. 相似文献
8.
Effects of losartan on the collagen degradative enzymes in hypertrophic and congestive types of cardiomyopathic hamsters 总被引:4,自引:0,他引:4
Masutomo Kazuhiro Makino Naoki Fushiki Masahiro Sugano M. Shinji 《Molecular and cellular biochemistry》2001,224(1-2):19-27
The present study was undertaken to determine the effects of AT1 receptor blockade which occurred in response to losartan, on the extracellular matrix (ECM) degradation process in the Bio 14.6 (n = 12) and Bio 53.58 (n = 12) strains which are referred as models of hypertrophic and dilated cardiomyopathy, respectively. The administration of losartan (30 mg/kg/day) in hamsters from 10–20 weeks of age reduced the accumulation of the left ventricular collagen matrix in both of the Bio 14.6 and the Bio 53.58 strains. According to the RTPCR, the levels of mRNA for matrix metalloproteinase (MMP) and the tissue inhibitor of MMP (TIMP) were examined. MMP1, 2, 3, and 9 were more enhanced in both myopathic strains than in the control F1 strains. With losartan, the levels of MMP1, 2, 9, TIMP1 and 2 decreased in the both strains but those for MMP3 did not in Bio 14.6 strains. TIMP3 and 4 mRNA levels did not change in any of the experimental hamsters, whether treated or untreated with losartan. The Western blots also showed similar observations in the both strains as seen in mRNA expressions although MMP2 in the Bio 53.58 strains did not differ between treated and untreated with losartan. Although losartan has an inhibitory effect on collagen accumulation in the development of cardiomyopathy, MMPs (1, 2, 9) and TIMPs (1, 2) seem to be susceptible to responding to losartan in Bio cardiomyopathic hamsters. 相似文献
9.
Type II fish antifreeze protein accumulation in transgenic tobacco does not confer frost resistance 总被引:3,自引:0,他引:3
Kenward Kimberly D. Brandle James McPherson Joan Davies Peter L. 《Transgenic research》1999,8(2):105-117
Type II fish antifreeze protein (AFP) is active in both freezing point depression and the inhibition of ice recrystallization. This extensively disulfidebonded 14 kDa protein was targeted for accumulation in its pro and mature forms in the cytosol and apoplast of transgenic tobacco plants. Type II AFP gene constructs under control of a duplicate cauliflower mosaic virus 35S promoter, both with and without a native plant transit peptide sequence, were introduced into tobacco by Agrobacterium tumefaciensmediated transformation. AFP did not accumulate in the cytosol of transgenic plants, but active AFP was present as 2% of the total protein present in the apoplast. Plantproduced AFP was the same size as mature Type II AFP isolated from fish, and was comparable to wildtype AFP in thermal hysteresis activity and its effect on ice crystal morphology. Field trials conducted in late summer on R1 generation transgenic plants showed similar AFP accumulation in plants under field conditions at levels suitable for largescale production: but no difference in frost resistance was observed between transgenic and wildtype plants during the onset of early fall frosts. 相似文献
10.
Tappia PS Okumura K Kawabata K Shah KR Nijjar MS Panagia V Dhalla NS 《Molecular and cellular biochemistry》2001,221(1-2):89-98
Evidence indicates that, in addition to the Ltype Ca2+ channel blockade, Ca2+antagonists target other functions including the Ca2+pumps. This study was conducted to test the possibility that the reported inhibition of heart sarcolemmal (SL) and sarcoplasmic reticular (SR) Ca2+pumps by verapamil and diltiazem could be due to druginduced depression of phosphatidylethanolamine (PE) Nmethylation which modulates these Ca2+transport systems. Three catalytic sites individually responsible for the synthesis of PE monomethyl (site I), dimethyl (site II) and trimethyl (phosphatidylcholine (PC), site III) derivates were examined in SL and SR membranes by employing different concentrations of SadenosylLmethionine (AdoMet). Total methyl group incorporation into SL PE, in vitro, was significantly depressed by 10–6–10–3 M verapamil or diltiazem at site III. The catalytic activity of site I was inhibited by 10–3 M verapamil only, whereas the site II activity was not affected by these drugs. The inhibition induced by verapamil or diltiazem (10–5 M) was associated with a depression of the Vmax value without any change in the apparent affinity for AdoMet. Both drugs decreased the SR as well as mitochondrial PE Nmethylation at site III. A selective depression of site III activity was also observed in SL isolated from hearts of rats treated with verapamil in vivo. Furthermore, administration of [3H-methyl]methionine following the treatment of animals with verapamil, reduced the synthesis of PC by Nmethyltransferase. Verapamil also depressed the N-methylation-dependent positive inotropic effect induced by methionine in the isolated Langendorff heart. Both agents depressed the SL Ca2+pump and although diltiazem also inhibited the SR Ca2+pump, verapamil exerted a stimulatory effect. In addition, verapamil decreased SR Ca2+-release. These results suggest that verapamil and diltiazem alter the cardiac PE Nmethyltransferase system. This action is apparently additional to the drugs' effect on Ltype Ca2+ channels and may serve as a biochemical mechanism for the drugs' inhibition of the cardiac Ca2+pumps and altered cardiac function. 相似文献
11.
A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno TOplant were obtained and, except one Combi TOplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_oplants. Two of the respective T1progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation. 相似文献
12.
Heme oxygenase1, the major inducible isoform of heme oxygenase (HO), can be induced by heme and numerous other physical and chemical factors, many of which cause cellular stress. This has led to the realization that HO1 is a major highly conserved stress or heat shock protein. Recent work has implicated activation of mitogenactivated protein kinases and other kinases in the mechanism of induction of HO1, and suggested that signal transduction pathways through tyrosine kinases are involved in induction of HO1 gene expression by stress inducers. We hypothesized that phenylarsine oxide (PAO), an inhibitor of protein tyrosine phosphatases (PTPs), might up-regulate the HO1 gene. Here, we show that a remarkably brief (1–15 min) exposure of normal hepatocytes to low concentrations (0.5–3 M) of PAO produces a marked increase in mRNA and protein of HO1. This increase is comparable to the level obtained by addition of heme (20 M), and occurs without producing changes in cellular glutathione levels or stabilization of HO1 message. Preincubation of cells with inhibitors of protein synthesis decreased the ability of PAO to increase levels of HO1 mRNA, suggesting that the inductive effect requires de novo protein synthesis. Addition of thiol donors abrogated the PAOmediated induction of HO1 in a dose dependent fashion. Addition of genistein, a tyrosine kinase inhibitor, blunted the induction produced by both PAO and heme. After brief incubations with PAO or heme, cell extracts showed comparable increases in levels of protein tyrosine phosphorylation in general, and specifically in ZAP70 kinase. Our results are consistent with the proposition that induction of HO1 by PAO involves inhibition of specific PTP(s), and that the mechanisms of induction of HO1 by PAO and by heme may share some common pathways. 相似文献
13.
Homocysteine stimulates the expression of monocyte chemoattractant protein-1 in endothelial cells leading to enhanced monocyte chemotaxis 总被引:12,自引:0,他引:12
The effect of intraperitoneal administration of tocopherol (100 mg/kg wt/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of mitochondria and microsomes isolated from rat liver and testis was studied. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:6 n3 in liver and C20:4 n6 and C22:5 n6 in testis. The lipid peroxidation of liver mitochondria or microsomes produced a significant decrease of C20:4 n6 and C22:6 n3 in the control group, whereas changes in the fatty acid composition of the tocopherol treated group were not observed. The light emission was significantly higher in the control than in the tocopherol treated group. The lipid peroxidation of testis microsomes isolated from the tocopherol group produced a significant decrease of C20:4 n6 , C22:5 n6 and C22:6 n3, these changes were not observed in testis mitochondria. The light emission of both groups was similar. The treatment with tocopherol at the dose and times indicated showed a protector effect on the polyunsaturated fatty acids of liver mitochondria, microsomes and testis mitochondria, whereas those fatty acids situated in testis microsomes were not protected during non enzymatic ascorbateFe2+ lipid peroxidation. The protector effect observed by tocopherol treatment in the fatty acid composition of rat testis mitochondria but not in microsomes could be explained if we consider that the sum of C20:4 n6 + C22:5 n6 in testis microsomes is 2-fold than that present in mitochondria. 相似文献
14.
Keshava Nagalakshmi Zhou Gu Spruill Michelle Ensell Mang Ong Tong-man 《Molecular and cellular biochemistry》2001,222(1-2):69-76
Occupational exposure to beryllium (Be) and Be compounds occurs in a wide range of industrial processes. A large number of workers are potentially exposed to this metal during manufacturing and processing, so there is a concern regarding the potential carcinogenic hazard of Be. Studies were performed to determine the carcinogenic potential of beryllium sulfate (BeSO4) in cultured mammalian cells. BALB/c3T3 cells were treated with varying concentrations of BeSO4 for 72 h and the transformation frequency was determined after 4 weeks of culturing. Concentrations from 50–200 g BeSO4/ml, caused a concentrationdependent increase (9–41 fold) in transformation frequency. Nontransformed BALB/c3T3 cells and cells from transformed foci induced by BeSO4 were injected into both axillary regions of nude mice. All ten Beinduced transformed cell lines injected into nude mice produced fibrosarcomas within 50 days after cell injection. No tumors were found in nude mice receiving nontransformed BALB/c3T3 cells 90 days postinjection. Gene amplification was investigated in Kras, cmyc, cfos, cjun, csis, erbB2 and p53 using differential PCR while random amplified polymorphic DNA fingerprinting was employed to detect genomic instability. Gene amplification was found in Kras and cjun, however no change in gene expression or protein level was observed in any of the genes by Western blotting. Five of the 10 transformed cell lines showed genetic instability using different random primers. In conclusion, these results indicate that BeSO4 is capable of inducing morphological cell transformation in mammalian cells and that transformed cells induced by BeSO4 are potentially tumorigenic. Also, cell transformation induced by BeSO4 may be attributed, in part, to the gene amplification of Kras and cjun and some BeSO4induced transformed cells possess neoplastic potential resulting from genomic instability. 相似文献
15.
Laclau Muriel Lu Feng MacDonald Michael J. 《Molecular and cellular biochemistry》2001,225(1-2):151-160
Recent evidence of a pyruvate malate shuttle capable of transporting a large amount of NADPH equivalents out of mitochondria in pancreatic islets suggests that cytosolic NADP(H) plays a role in beta cell metabolism. To obtain clues about these processes the activities of several NADPHutilizing enzymes were estimated in pancreatic islets. Low levels of pyrroquinolone quinone (PQQ) and low levels of enzyme activity that reduce PQQ were found in islets. Low activities of palmitoylCoA and stearoylCoA desaturases were also detected. Significant activities of glutathione reductase, aldose reductase (EC.1.1.1.21) and aldehyde reductase (EC.1.1.1.2) were present in islets. Potent inhibitors of aldehyde and aldose reductases inhibited neither glucoseinduced insulin release nor glucose metabolism in islets indicating that these reductases are not directly involved in glucoseinduced insulin reaction. Over 90% of aldose reductase plus aldehyde reductase enzyme activity was present in the cytosol. Kinetic and chromatographic studies indicated that 60–70% of this activity in cytosol was due to aldehyde reductase and the remainder due to aldose reductase. Aldehyde reductaselike enzyme activity, as well as aldose reductase immunoreactivity, was detected in rat islet plasma membrane fractions purified by a polyethylene glycolDextran gradient or by a sucrose gradient. This is interesting in view of the fact that voltagegated potassium channel beta subunits that contain aldehyde and aldose reductaselike NADPH-binding motifs have been detected in plasma membrane fractions of islets [Receptors and Channels 7: 237–243, 2000] and suggests that NADPH might have a yet unknown function in regulating activity of these potassium channels. Reductases may be present in cytosol to protect the insulin cell from molecules that cause oxidative injury. 相似文献
16.
Groot Nanda de Kuik‐Romeijn Petra van Lee Sang He de Boer Herman A. 《Transgenic research》1999,8(2):125-135
The polymeric immunoglobulin receptor (pIgR), a transmembrane protein, transports dimeric IgA (dIgA) across the epithelial cells of the mucosal surfaces into the external secretions, for example milk from the mammary glands. The pIgR is consumed during the transcytosis of dIgA and is cleaved at the apical side of the epithelial cells, regardless of the binding to its ligand (dIgA), to form secretory component (SC). We hypothesize that the expression level of the endogenous murine pIgR gene in the epithelial cells is ratelimiting for the transport of dIgA across the epithelial cells into the secretions. We address this key issue by generating transgenic mice overexpressing the pIgR gene in their mammary glands in order to examine the effect on dIgA levels in the milk. Here we report on the generation of transgenic mice and analysis of the expression level of pIgR in their mammary glands. We cloned and characterized the murine pIgR gene and constructed an expression cassette bearing the pIgR gene under the control of the regulatory sequences of the bovine s1casein gene. Four transgenic lines were made, expressing the pIgR construct at RNA and protein level only in their mammary glands. The levels of the SC protein in the milk ranged from 0.1 to 2.7mg/ml during midlactation. These levels are 10–270 times higher than wildtype SC levels (0.01mg/ml). 相似文献
17.
We investigated the application of cryopreserved pronuclearstage zygotes for the production of transgenic rats. Most of the pronuclearstage zygotes cryopreserved by conventional twostep freezing or vitrification appeared morphologically normal, but the proportion of frozen zygotes that developed into fetuses following transfer (59.7–60.2%) was higher than that of vitrified zygotes (5.5–22.1%). When the frozenthawed zygotes were used for DNA microinjection, 97.5% survived after DNA microinjection and 25.1% of the transferred zygotes developed into fetuses. These proportions were comparable to those of the fresh control zygotes (97.0% and 30.0%, respectively). The integration efficiency of the exogenous DNA into fetuses was similar between the frozen group (3.3% per injected zygote) and the control group (3.5%). These results indicate that pronuclearstage rat zygotes can be successfully cryopreserved by conventional twostep freezing for production of transgenic rats. 相似文献
18.
Involvement of Erks activation in cadmium‐induced AP‐1 transactivation in vitro and in vivo 总被引:2,自引:0,他引:2
Huang Chuanshu Zhang Qunwei Li Jingxia Shi Xianglin Castranova Vincent Ju Gong Costa Max Dong Zigang 《Molecular and cellular biochemistry》2001,222(1-2):141-147
Cadmium is a potent and effective carcinogen in rodents and has recently been accepted by IARC (International Agency for Research on Cancer) as a category 1 carcinogen. Cadmium-induced upregulation of intracellular signaling pathways leading to increased mitogenesis is thought to be a major mechanism for the carcinogenic activity following chronic cadmium exposure. In the present study, we found that exposure of cells to cadmium induced significant activation of AP1 and all three members of the MAP kinase family in mouse epidermal JB6 cells. The induction of AP1 activity by cadmium appears to involve activation of Erks, since the induction of AP1 activity by cadmium was blocked by pretreatment of cells with PD98058. Interestingly, the induction of AP1 by cadmium was greatly enhanced by the chemical tumor promoter, TPA and the growth factor EGF, but not by ultraviolet C radiation. In vivo studies demonstrated that cadmium could also induce transactivation of AP1 in AP1luciferase report transgenic mice. Considering the role of AP1 activation in tumor promotion, the results presented in this study provide a possible molecular mechanism for cadmiuminduced carcinogenesis. 相似文献
19.
Mohammad Askar Bhanot Sanjay McNeill John H. 《Molecular and cellular biochemistry》2001,223(1-2):103-108
The PI3 kinase signalling pathway is an important pathway in mediating the glucoregulatory effects of insulin and skeletal muscle (SKM) is the major tissue involved in glucose utilization. In diabetes this pathway is impaired, either due to lack of insulin as in Type 1 diabetes, or due to insulin resistance as in Type 2 diabetes. Bis(maltolato)oxovanadium IV (BMOV), an insulin mimetic/enhancing agent, produces a marked glucose lowering effect in models of both types of diabetes. Some in vitro studies have shown that phosphatidylinositol 3 kinase (PI3 kinase) activity is enhanced by vanadium. In the present study we looked at changes in PI3 kinase expression and activity in SKM from STZdiabetic and fa/fa Zucker rats treated with BMOV for 3 weeks. Although BMOV treatment completely normalized glucose levels in STZdiabetic rats, no effect was observed on basal or insulin-stimulated PI3 kinase activity. In fatty Zucker rats, activation of PI3 kinase activity after insulin injection was impaired as compared to age matched lean controls, but BMOV again did not affect the activity. These results suggest that although PI3 kinase is an important signalling factor in glucose utilization, vanadium treatment does not reduce hyperglycemia through activation of SKM PI3 kinase in vivo. 相似文献
20.
O'Brien Travis Xu Jian Patierno Steven R. 《Molecular and cellular biochemistry》2001,222(1-2):173-182
Hexavalent chromium (Cr (VI)) is reduced intracellularly to Cr (V), Cr (IV) and Cr (III) by ascorbate (Asc), cysteine and glutathione (GSH). These metabolites induce a spectrum of genomic DNA damage resulting in the inhibition of DNA replication. Our previous studies have shown that treatment of DNA with Cr (III) or Cr (VI) plus Asc results in the formation of DNACrDNA crosslinks (CrDDC) and guaninespecific arrests of both prokaryotic and mammalian DNA polymerases. GSH not only acts as a reductant of Cr (VI) but also becomes crosslinked to DNA by Cr, thus, the focus of the present study was to examine the role of GSH in Crinduced DNA damage and polymerase arrests. Coincubation of Cr (III) with plasmid DNA in the presence of GSH led to the crosslinking of GSH to DNA. GSH cotreatment with Cr (III) also led to a decrease in the degree of Crinduced DNA interstrand crosslinks relative to Cr (III) alone, without affecting total Cr DNA binding. DNA polymerase arrests were observed following treatment of DNA with Cr (III) alone, but were markedly reduced when GSH was added to the reaction mixture. Preformed polymerasearresting lesions (CrDDC) were not removed by subsequent addition of GSH. Treatment of DNA with Cr (VI), in the presence of GSH, resulted in crosslinking of GSH to DNA, but failed to produce detectable DNA interstrand crosslinks or polymerase arrests. The inhibitory effect of GSH on Crinduced polymerase arrest was further confirmed in human genomic DNA using quantitative PCR (QPCR) analysis. Treatment of genomic DNA with Cr (III) resulted in a marked inhibition of the amplification of a 1.6 kb target fragment of the p53 gene by Taq polymerase. This was almost completely prevented by cotreatment with GSH and Cr (III). These results indicate that Crinduced DNA interstrand crosslinks, and not DNACrGSH crosslinks, are the principal lesions responsible for blocking DNA replication. Moreover, the formation of DNACrGSH crosslinks may actually preclude the formation of the polymerase arresting lesions. 相似文献