首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 47 毫秒
1.
Induction of metallothionein-I (MT-I) and metallothionein-II (MT-II) by glucocorticoids was determined by h.p.l.c. analysis of proteins and Northern-blot analysis of MT mRNAs. Rats were injected with dexamethasone (0.03-10 mumol/kg) and hepatic concentrations of MTs were determined 24 h later. In control rats, only MT-II was detected (9.4 +/- 2.5 micrograms/g of liver), whereas the hepatic concentration of MT-I was below the detection limit (5 micrograms of MT/g). Dexamethasone did not increase MT-I above the detection limit at any dosage tested, but MT-II increased to 2.5 times control values at dosages of 0.30 mumol/kg and higher. Time-course experiments indicated that MT-II reached a maximum at 24 h after a single dosage of dexamethasone and returned to control values by 48 h. To determine whether dexamethasone increased MT-I in liver, samples were saturated with 109Cd, after which the amount of 109Cd in MT-I and MT-II was determined. Results indicated that, by this approach, MT-I and MT-II could be detected in control rats, and there was approx. 1.8 times more 109Cd in MT-II than in MT-I. At 24 h after administration of dexamethasone (1 mumol/kg), there was a small increase in the amount of 109Cd bound to MT-I, whereas the amount of 109Cd bound to MT-II increased to more than 2 times control values. Northern-blot hybridization with mouse cRNA probes indicated that MT-I and MT-II mRNAs increased co-ordinately after administration of dexamethasone. Thus, although glucocorticoids increase both MT-I and MT-II mRNAs, MT-II preferentially accumulates after administration of dexamethasone.  相似文献   

2.
3.
4.
A rapid, reproducible, and sensitive high-performance liquid chromatography (HPLC) method for the determination of the concentrations of metallothionein-I (MT-I) and metallothionein-II (MT-II) in rat liver has been developed. Metallothioneins (MTs) were separated and quantitated by anion-exchange high-performance liquid chromatography coupled with atomic absorption spectrophotometry (AAS). Purified rat liver MT-I and MT-II, used as standards for developing the method, were easily resolved, eluting at 7.5 and 10.4 min, respectively. To establish standard curves, protein concentrations of solutions of the purified MTs were determined by the Kjeldahl method for the determination of nitrogen, after which the standards were saturated with Cd (final concentration of 50 ppm Cd). Rat liver cytosols obtained from untreated and Cd- or Zn-treated rats were prepared for HPLC-AAS analysis by saturation with Cd (50 ppm Cd) followed by heat denaturation (placing in a boiling water bath for 1 min). Based on the method of standard additions, recovery of MTs exceeded 95% and repeated injection of a sample yielded a coefficient of variance of approximately 2%. A detection limit of 5 micrograms MT/g liver was established for the method. Only MT-II was detected in untreated rats, whereas following exposure to Cd or Zn, both forms of MTs were detected. Concentrations of total MTs in liver of untreated and Cd- or Zn-treated rats were also determined by the Cd/hemoglobin radioassay (which fails to distinguish MT-I from MT-II) and indicated that results obtained with the HPLC-AAS method compared favorably to the Cd/hemoglobin radioassay. Thus, the HPLC-AAS method for quantitating MT-I and MT-II offers the advantage of determining the concentrations of both proteins in tissues and should be useful for studying the regulation of MT-I and MT-II.  相似文献   

5.
The induction of metallothionein (MT) isoform synthesis was investigated in mouse cerebral cortex 18 h after oral ethanol administration. The expression of MT-I isoform mRNA increased in a dose-dependent manner after ethanol loading at doses between 2 g/kg (ethanol/body weight) and 8 g/kg. Lipid peroxide formation, measured as the amount of malondialdehyde-reactive substances, remained at the control level after all of the administered ethanol doses. The expression of MT-III isoform mRNA remained at the control level up until an ethanol loading dose of 4 g/kg and then finally increased to a significant level at a dose of 8 g/kg, which is almost the LD50 for oral ethanol in mice. The different patterns of MT synthesis induction among MT isoforms suggests that the MT-I isoform, which is ubiquitous in mammalian tissues, plays a significant role as an antioxidant. On the other hand, the MT-III isoform, which has a limited tissue distribution, especially in the central nervous system, seems to be implicated in tissue repair and/or protection against critical tissue injury.  相似文献   

6.
In this study, we investigated the expression of metallothionein (MT)-I and MT-II in the rat brain following traumatic brain injury (TBI). In the early stage, significant induction of MT-I and MT-II were observed in various regions including ventricle walls, pia mater, and dentate gyrus. At 12-24 h after TBI, strong induction of MT-I mRNA was observed in cerebral cortical layer II/III, amygdala, and piriform cortex where neurons reside. On the other hand, MT-II appeared to be expressed mainly in glial cells localized in the cerebral cortex and hippocampal formation. Three days after TBI, MTs were observed in the vimentin-positive astrocytes in the penumbra as revealed by double immunohistochemistry. The differences in expression of MT-I and MT-II in different brain regions and cell types (neuron vs. glial cells) suggests that multiple regulatory mechanisms are involved in the control of MT expression following brain injury.  相似文献   

7.
Abstract: Metallothionein (MT) protein and mRNA levels were monitored following exposure of rat neonatal primary astrocyte cultures to methylmercury (MeHg). MT-I and MT-II mRNAs were probed on northern blots with an [α-32P]dCTP-labeled synthetic cDNA probe specific for rat MT mRNA. MT-I and MT-II mRNAs were detected in untreated cells, suggesting constitutive MT expression in these cells. The probes hybridize to a single mRNA with a size appropriate for MT, ∼550 and 350 bp for MT-I and MT-II, respectively. Expression of MT-I and MT-II mRNA in astrocyte monolayers exposed to 2 × 10−6 M MeHg for 6 h was increased over MT-I and MT-II mRNA levels in controls. Western blot analysis revealed a time-dependent increase in MT protein synthesis through 96 h of exposure to MeHg. Consistent with the constitutive expression of MTs at both the mRNA level and the protein level, we have also demonstrated a time-dependent increase in MT immunoreactivity in astrocytes exposed to MeHg. The cytotoxic effects of MeHg were measured by the rate of astrocytic d -[3H]aspartate uptake. Preexposure of astrocytes to CdCl2, a potent inducer of MTs, completely reversed the inhibitory effect of MeHg on d -[3H]aspartate uptake that occurs in MeHg-treated astrocytes with constitutive MT levels. Associated with CdCl2 treatment was a time-dependent increase in astrocytic MT levels. In summary, astrocytes constitutively express MTs; treatment with MeHg increases astrocytic MT expression, and increased MT levels (by means of CdCl2 pretreatment) attenuate MeHg-induced toxicity. Increased MT expression may represent a generalized response to heavy metal exposure, thus protecting astrocytes and perhaps also, indirectly, juxtaposed neurons from the neurotoxic effects of heavy metals.  相似文献   

8.
Preparative human liver metallothionein (MT) isolation is described. MT was saturated with cadmium to follow MT purification spectrophotometrically instead of by metal content and to increase the stability of the protein. A concentrated, MT-rich fraction of the liver cytosol was prepared by selective organic solvent (acetone or acetone/methanol) fractionation. Conventional gel filtration and ion-exchange chromatographies resolved two MT isoforms, MT-I and MT-II. When needed, purification of MT from other low-molecular weight proteins was further increased by gel filtration chromatography at zero ionic strength, i.e., in distilled water. Reversed-phase high performance liquid chromatography of both MT isoforms resolved further peaks sharing MT properties not only from the MT-I but also from the MT-II ion-exchange isoform. The results show that it is feasible to perform a human liver MT isolation from an entire human liver with a reasonable laboratory capability.  相似文献   

9.
10.
11.
12.
Cocaine-induced hepatotoxicity was examined in vivo in a dose-responsive manner in C57BL/6Ibg, DBA/2Ibg, C3H/2Ibg, and Balb/cJ mice. Serum glutamic-pyruvic transaminase (SGPT) activities were determined 24 hours after intraperitoneal (IP) administration of cocaine (20 to 100 mg/kg). Significant elevations (100- to 150-fold) in SGPT were observed in male mice receiving cocaine. Significant differences in sensitivity to cocaine-induced hepatotoxicity were found among males of the inbred strains, with Balb being most sensitive and C57BL being least sensitive and C3H and DBA strains exhibiting intermediate sensitivity. Female mice of the four inbred strains were more resistant than males to cocainemediated hepatotoxicity, as indicated by only twofold to tenfold elevations in SGPT values. Among the females, sensitivity of the four inbred strains—as indicated by dose response curves—fell into two categories: the sensitive strains (C3H and C57BL) and the resistant strains (Balb and DBA). Pretreatment of males of the four inbred strains with the P-450 inducer phenobarbital resulted in enhancement of cocaine-mediated hepatotoxicity in the C57BL and Balb but not the C3H and DBA mice. Phenobarbital pretreatment of females of the four inbred strains resulted in enhancement of the hepatotoxic response to cocaine in the C3H, DBA, and Balb mice. Phenobarbital-pretreated C57BL females exhibited a 100% mortality rate after the acute cocaine dose, and thus no determination of hepatotoxicity could be established for them. These data demonstrate sex and strain differences in cocaine-induced hepatotoxicity and suggest that phenobarbital pretreatment does not uniformly enhance the hepatotoxicity of cocaine.  相似文献   

13.
Northern blot analysis revealed that metallothionein (MT) mRNAs accumulate after inhibition of protein synthesis with cycloheximide (CHX) in primary cultures of chick embryo hepatocytes and fibroblasts, as well as in an established mouse hepatoma cell line. Inhibition of RNA synthesis with actinomycin D (AMD) led to rapid loss of MT mRNAs in these cells, whereas CHX dramatically retarded the rate of MT mRNA decay (t1/2 greater than 24 h). These results suggest that CHX causes MT mRNA accumulation primarily by increasing stability of MT mRNA. Thus, changes in MT mRNA turn-over rates may play an important role in regulating the accumulation of MT mRNA. The half-lives of MT mRNAs in chicken and mouse cells were determined by oligodeoxyribonucleotide excess solution hybridization with RNA samples extracted after different periods of exposure to AMD. The half-life of chicken MT (cMT) mRNA in uninduced chicken embryo hepatocytes was 3.6 h. Induction of cMT mRNA by pretreatment of these cells with zinc (Zn) prior to exposure to AMD, did not alter the half-life of cMT mRNA significantly. In contrast, cadmium (Cd) induction led to a 2.5-fold increase in the stability of this mRNA. In uninduced chicken embryo fibroblasts, cMT mRNA levels were too low to allow accurate determination of half-life using the methods employed here. However, the half-life of this mRNA in Zn-induced chicken embryo fibroblasts was 6.2 h, whereas it was 9.3 h in Cd-induced cells. Thus, the turn-over rate of cMT mRNA after Cd-induction is very similar in chick embryo fibroblasts and hepatocytes. These data suggest that the accumulation of MT mRNA in chicken cells may reflect, in part, metal-specific effects on MT mRNA stability. The half-lives of mouse MT-I and MT-II (mMT-I and mMT-II) mRNAs in uninduced BNL hepatoma cells were identical (9.2 h), and were not effectively altered after induction by metals (Zn, Cd) or interleukin-1 beta (IL-1 beta). However, mMT mRNAs in pachytene spermatocytes and round spermatids, freshly isolated from the adult testes, were 2.2- to 4.5-fold more stable than in hepatoma cells. These results suggest that cell-type specific accumulation of mMT mRNAs may be regulated, in part, by mRNA stability.  相似文献   

14.
Shi YB  Fang JL  Liu XY  Du L  Tang WX 《Biopolymers》2002,65(2):81-88
The secondary structures of porcine brain Cu(4)Zn(3)-metallothionein (MT)-III and Cd(5)Zn(2)MT-I, Cd(5)Zn(2)MT-II, and Zn(7)MT-I from rabbit livers in the solid state are investigated by Fourier transform IR spectroscopy (FTIR) and Fourier transform Raman spectroscopy (FT-Raman). The Cu(4)Zn(3)MT-III contains 26-28% beta-turns and half-turns, 13-14% 3(10)-helices, 47-49% random coils, and 11-12% beta-extended chains. The structural comparison of porcine brain Cu(4)Zn(3)MT-III with rabbit liver Cd(5)Zn(2)MT-I (II) and Zn(7)MT-I shows that the contents of the random coil structure are obviously increased. The results indicate that the insert of an acidic hexapeptide in the alpha domain of Cu(4)Zn(3)MT-III possibly forms an alpha helix. However, because the bands assigned to the alpha-helix and random coil structures are overlapped in the spectra, the content of random coil structures in Cu(4)Zn(3)MT-III is therefore higher than those in Cd(5)Zn(2)MT-I, Cd(5)Zn(2)MT-II, and Zn(7)MT-I.  相似文献   

15.
In the present study we report on the effects of commonly used nonsteroidal antiinflammatory drugs on metallothionein (MT) and MT-I mRNA levels. A single dose of chloroquine (100 mg/kg), diclofenac (100 mg/kg), indomethacin (10 mg/kg), or piroxicam (100 mg/kg) was administered ip to C57B1 mice. After 18 h, MT levels were determined with a Cd-saturation radioassay. MT-I mRNA levels were measured by Northern Blot analyses using a probe containing the mouse MT-I gene. All drugs tested caused an increase in the MT content of the liver but not of the kidneys and lung. The lowest and highest effects were observed with chloroquine (8 times the control value) and diclofenac (18 times), respectively. In accordance with the stimulation of MT synthesis, increased accumulation of hepatic MT-I mRNA could be demonstrated. These results indicate that elevated MT levels may contribute to the effectiveness of nonsteroidal antiinflammatory drugs in the treatment of rheumatoid arthritis (RA).  相似文献   

16.
17.
镉中毒大鼠睾丸与肝脏金属硫蛋白表达的时相研究   总被引:6,自引:1,他引:5  
啮齿目动物睾丸对镉毒性较肝脏更敏感.为阐明睾丸的镉毒性分子机制,比较了肝脏与睾丸金属硫蛋白(MT)表达的时相变化.mRNA采用RT-PCR技术分析并用光密度扫描定量;蛋白质定量用ELISA方法.结果显示,睾丸中存在MT,镉中毒后MT1与MT2 mRNA明显升高,但MT没有相应增加;肝脏镉中毒后MTmRNA与MT均明显升高.结果提示:镉虽然能诱导睾丸MTmRNA的转录,但没有促进其MT的合成,这可能是睾丸对镉毒性与致癌作用较肝脏更敏感的重要原因.  相似文献   

18.
19.
Metallothionein (MT) can be induced in mouse liver by a bacterial exotoxin, toxic shock syndrome toxin-1 (TSST-1). Hepatic MT was induced by TSST-1 in a dose-dependent manner from 100 μg/kg through 3 mg/kg in CF-1 mice, and by 6 h the induction was almost maximal. The increase of hepatic MT occurred at the mRNA level, also, and both MT-I and II mRNAs increased coordinately. Because TSST-1 is a superantigen, it was investigated whether TSST-1 induces MT through cytokines as a consequences of immunostimulation. In low-cytokine-producing mice (C3H/HeJ), up to a dose of 1 mg/kg of TSST-1, there was only 2- to 3-fold increase of hepatic MT. In contrast, in normal-cytokine-producing mice (C3Heb/FeJ), TSST-1 increased MT in a dose-dependent manner, and at a dose of 1 mg/kg, there was a 25-fold increase in hepatic MT. This suggests that activation of the immune system is probably involved in the induction of MT by TSST-1. Studies on the role of specific hepatic cytokines (IL-1, TNF-α, and IL-6) in TSST-mediated hepatic MT induction showed that TSST-1 did not increase hepatic IL-1 or TNF-α significantly over controls in any of the mouse strains studied. In contrast, TSST-1 induced hepatic IL-6 in all three strains of mice. However, in CF-1 and C3Heb/FeJ mice (normal-cytokine-producing) IL-6 induction preceded MT mRNA induction, but in C3H/HeJ mice (low-cytokine-producing), IL-6 induction did not precede MT and mRNA induction.  相似文献   

20.
We have examined the chromatin structure of the metallothionein (MT) gene region in MT- S49 mouse lymphoma cells and in derivatives which express MT-I alone, MT-II alone, or both genes. In all lines, these genes are contained in a 16-kilobase pair region between two DNase I sensitive sites: one site located 5.3 kilobase pairs 5' of MT-II (the 5' gene) is present in naked DNA and retained in the chromatin of all lines; the other site located 3.1 kilobase pairs 3' of MT-I is hypersensitive. Hypersensitivity at three other sites is dependent on the expression of MT genes. Two sites 5' of MT-II disappear, and a site 3' of MT-I appears regardless of which gene is activated. The fact that these sites respond when either gene is activated suggests that the regulation of the two genes is interdependent and that the region undergoes a general change in conformation with MT activation. In addition, a single site in the 5' region of MT-II becomes hypersensitive with activation of the gene and may be related directly to expression.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号