大鼠肝生长发育过程中蛋白水解酶和ADAMs的变化(英文)

用SDS-PAGE、SDS-G-PAGE、Western等方法研究了大鼠肝从胚胎到成体生长发育过程中酸性、中性和碱性蛋白水解酶和ADAMs变化。结果表明:30和32kD酸性蛋白水解酶在开始吃奶和出生后第五周各出现一个活性高峰;75/80kD酸性蛋白水解酶仅在出生后第三周有活性。71和75kD的中性蛋白酶在出生后第三周活性最强;15kD碱性蛋白水解酶在开始吃奶时有活性。75kD的ADAMs在出生前至出生后第20天和出生后第四周到成体的含量呈两次正态分布;40kD的ADAM主要在胚胎肝和吃奶时检测到,但30kD ADAMs主要出现在吃饲料以后各个时期;50/47、73和140kD MDC15变化和75kD的ADAM相似,30kD的MDC15仅出现在出生后15~35天的肝脏内,58kD的MDC15主要在出生后7~45天之间。实验表明,营养来源和方式能有效影响肝脏的生理生化过程,揭示营养、肝功能、生长发育的密切关系。     

Microheterogeneity of molecular forms of arginase in mammalian tissues

Two isoforms of arginase, A1 and A2, were found in rat liver, submaxillary gland and kidney as well as beef kidney. In beef liver, however, A2 was the only detectable form. Two additional forms, A3 and A4, found only in rat kidney were probably artifactitious. A1 and A2 exhibited chromatographic and immunological microheterogeneity. While A1 in rat liver and submaxillary gland was excluded by DEAE-cellulose (pH 8.3) and retained on CM-cellulose (pH 7.5), that (A'1) in beef and rat kidneys was excluded by both ion-exchangers. A2 in all tissues was retained on DEAE-cellulose, but not on CM-cellulose. Both A1 and A2 in rat liver and beef kidney, A1 from rat submaxillary gland and A2 from beef liver were precipitated by antibodies to rat and beef liver arginases. None of the forms in rat kidney (A1, A2, A3 and A4) showed any cross-reactivity to either antibody. Rat submaxillary gland A2 was precipitated by anti-rat liver arginase, but activated by anti-beef liver arginase. While the major molecular forms were A1 in rat liver and submaxillary gland and A2 in beef liver and rat kidney, the two forms occurred in equal proportions in beef kidney. It appears that different isoforms might function as components of the urea cycle in the liver of different mammals and of the arginine catabolic pathway in different extrahepatic tissues.     

Anti-catechol-O-methyltransferase: Demonstration of specificity and immunological cross-reactivity with the enzyme from rat liver,kidney, brain,and choroid plexuses

An antiserum to rat liver catechol-O-methyltransferase (COMT) was utilized in the immunological characterization of COMT from rat kidney, brain, and choroid plexuses, in addition to rat liver. The presence of anti-COMT activity was confirmed by the direct inhibition of the activity of the enzyme from rat liver by small quantities of the antiserum and by the inhibition of the activity of the enzyme from rat brain. The specificity of the antiserum was demonstrated both by immunoelectrophoresis of rat liver COMT, and by a partial purification of rat liver COMT in which changes in COMT specific activity were correlated with the appearance of a precipitin line in double-immunodiffusion experiments. The antigenic similarity of the enzyme derived from rat liver, kidney, brain, and choroid plexuses was demonstrated by the formation of a precipitin line of identity when preparations from these four tissues were diffused against the antiserum.     

Fraction from human and rat liver which is inhibitory for proliferation of liver cells

A comparative study was undertaken with human and rat liver of a fraction reported to have growth inhibitory activity when prepared from rat liver. Fractions which were soluble in 70% ethanol and insoluble in 87% ethanol were prepared from liver cytosols. Electrophoretic analysis under denaturing conditions indicated that there were several quantitative or qualitative differences in the fractions from the two species. Fractions from both human and rat liver were found to be inhibitory for the incorporation of 3H-thymidine into DNA of foetal chick hepatocytes. Under conditions in which the rat fraction inhibited precursor incorporation into DNA of rat liver epithelial cells there was not a significant inhibitory effect with the fraction from human liver. DNA synthesis in a rat hepatoma cell line was not significantly inhibited by preparations from either species. The data suggested that corresponding fractions from both rat and human liver could have inhibitory effects on precursor incorporation into DNA but the magnitude of the effects and target cell specificity may differ.     

Responsibility of tRNA(Ile) for spermine stimulation of rat liver Ile-tRNA formation

To determine whether tRNA or aminoacyl-tRNA synthetase is responsible for spermine stimulation of rat liver Ile-tRNA formation, homologous and heterologous Ile-tRNA formations were carried out with Escherichia coli and rat liver tRNA(Ile) and their respective purified Ile-tRNA synthetases. Spermine stimulation was observed only when tRNA from the rat liver was used. Spermine bound to rat liver tRNA(Ile) but not to the purified aminoacyl-tRNA synthetase complex. Kinetic analysis of Ile-tRNA formation revealed that spermine increased the Vmax and Km values for rat liver tRNA(Ile). The Km value for ATP and isoleucine did not change significantly in the presence of spermine. Furthermore, higher concentrations of rat liver tRNA(Ile) tended to inhibit Ile-tRNA formation if spermine was absent. Spermine restored isoleucine-dependent PPi-ATP exchange in the presence of rat liver tRNA(Ile), an inhibitor of this exchange. The nucleotide sequence of rat liver tRNA(Ile) was determined and compared with that of E. coli tRNA(Ile). Differences in nucleotide sequences of the two tRNAs(Ile) were observed mainly in the acceptor and anticodon stems. Limited ribonuclease V1 digestion of the 3'-32P-labeled rat liver tRNA(Ile) showed that both the anticodon and acceptor stems were structurally changed by spermine, and that the structural change by spermine was different from that by Mg2+. The influence of spermine on the ribonuclease V1 digestion of E. coli tRNA(Ile) was different from that of rat liver tRNA(Ile). The results suggest that the interaction of spermine with the acceptor and anticodon stems may be important for spermine stimulation of rat liver Ile-tRNA formation.     

A difference in the effect of rat liver extract (chalone) on the mitotic index of chick embryo liver when extracted at different times of day.

Adult and neonatal rat liver was used as a source for liver chalone. Extracts of rat liver were prepared from two groups of rats: one group was killed at 0900 and a second group was killed at 2100. The rat liver extracts were injected into the air sacs of 15-day old chick embryos and the effect on the mitotic index of the chick liver was studied. All rat liver extracts prepared from rats killed at 2100 demonstrated significantly more inhibition of the mitotic index in the chick liver than did the extracts obtained at 0900.     

The level of beta-alanine aminotransferase activity in regenerating and differentiating rat liver

beta-Alanine aminotransferase from rat liver was purified to electrophoretic homogeneity. The immunological and kinetic properties of this enzyme were similar to those of the enzyme from rat brain. However, the liver enzyme transaminates from beta-alanine to 2-oxoglutaric acid, while the brain enzyme transaminates from gamma-aminobutyric acid. beta-Alanine aminotransferase activity in regenerating rat liver was lower than that in control rat liver. Activity of this enzyme, as well as of other uracil-catabolizing enzymes (Weber, G., Queener, S.F. and Ferdinandus, A. (1970) in Advances in Enzyme Regulation (Weber, G., ed.), Vol. 9, pp. 63-95, Pergamon Press, Oxford), was low in newborn rat liver and increased about 5-fold, reaching the level observed in adult rat liver. beta-Alanine and prednisolone induced beta-alanine aminotransferase in rat liver.     

Antibodies to dehistonized chromatin of normal and fetal rat liver

Antisera to dehistonized adult or fetal rat liver chromatin were treated with electrophoretically separated chromosomal proteins of adult and fetal liver, Novikoff hepatoma and adult rat kidney. Both types of antisera reacted with numerous antigens in both tissue chromatins. However, immunoabsorption experiments have shown that while adult rat liver shared most of its nuclear antigens with other tissues, antisera to dehistonized fetal liver chromatins were more specific. In terms of antigenic specificity, the fetal rat liver and Novikoff hepatoma chromatins were similar; however, the former contained several antigens which could not be absorbed with Novikoff hepatoma chromatin.     

Effects of dithiothreitol and thioredoxines on the in vitro activity of enzymes of the urea cycle in rats

The five urea cycle enzymes were studied in desactivated extracts of rat liver. After reduction by dithiothreitol (DTT) and in presence of Mg2+ ions, thioredoxines isolated from rat liver were able to activate carbamyl phosphate synthetase-I (CPS-I) and argininosuccinate synthetase (ASS) respectively by 468% and by 370%. Thioredoxines were purified from adult rat liver and an antiserum was raised to these proteins. After immunologic quantitation, their level in adult rat was 0.103 mg/g liver.     

猪苓及猪苓多糖对BBN联合糖精作用Fisher-344大鼠肝脏代谢酶的影响

以BBN联合糖精作用Fisher-344大鼠,分别使用ELISA及RT-PCR方法测定样本中CYP450、GST和NQO1酶活性及Gstpi和NQO1 mRNA相对表达含量。结果表明,猪苓及猪苓多糖均可使样本中CYP450酶活性显著降低,其中猪苓对CYP450酶活性抑制呈剂量依赖性;猪苓及猪苓多糖对样本中GST无明显影响,猪苓可显著升高样本中NQO1酶活性;猪苓高剂量及猪苓多糖可上调GSTPi mRNA表达,猪苓及猪苓多糖对NQO1 mRNA表达无明显作用。研究表明猪苓及猪苓多糖抗癌作用与降低CYP450与升高NQO1酶活性有关,但机制有待进一步研究。     

Comparative study on vertebrate liver AMP deaminases

Similar activity of AMP deaminase was found in rat, hen, turtle and flounder liver when estimated at high AMP concentration. The enzyme activity was of an order of magnitude higher in frog liver. Simple step by step phosphocellulose column chromatography revealed two forms of AMP deaminase in chicken and flounder liver and one form in the liver of rat and turtle. All enzymes (except for frog liver AMP deaminase) were activated by ATP. The enzymes from rat, frog and both forms from flounder were also activated by ADP. GTP exhibited a variety of effects. The enzyme from rat and turtle was inhibited, both forms from hen and flounder were activated and frog liver enzyme was not influenced.     

Comparative studies on nucleoside diphosphatase of rat ascites hepatoma and rat liver: activity level, purification and properties

1. The activities of nucleoside diphosphatase in various rat ascites cells of hepatoma, and fetal and neonatal rat liver were much lower than that of normal adult rat liver. 2. The enzyme was purified from ascites hepatoma (AH-66 cell lines) to an apparently homogeneous state and the enzymatic properties were studied in comparison with the enzyme from rat liver microsomes. 3. The hepatoma enzyme had less stability based on the results of heat-inactivation experiments. 4. However, the other properties of hepatoma enzyme; Km value, molecular weight, optimal pH, isoelectric point, substrate specificity and antigenicity, were similar to those of rat liver enzyme.     

The level of β-alanine aminotransferase activity in regenerating and differentiating rat liver

β-Alanine aminotransferase from rat liver was purified to electrophoretic homogeneity. The immunological and kinetic properties of this enzyme were similar to those of the enzyme from rat brain. However, the liver enzyme transaminates from β-alanine to 2-oxoglutaric acid, while the brain enzyme transaminates from γ-aminobutyric acid. β-Alanine aminotransferase activity in regenerating rat liver was lower than that in control rat liver. Activity of this enzyme, as well as of other uracil-catabolizing enzymes (Weber, G., Queener, S.F. and Ferdinandus, A. (1970) in Advances in Enzyme Regulation (Weber, G., ed.), Vol. 9, pp. 63–95, Pergamon Press, Oxford), was low in newborn rat liver and increased about 5-fold, reaching the level observed in adult rat liver. β-Alanine and prednisolone induced β-alanine aminotransferase in rat liver.     

Separation and quantitative determination of dolichol and dolichyl phosphate in rat and trout liver

The dolichol concentrations in rat and trout liver were found respectively to be 50-59 and 16-21 micrograms/g using three experimental methods: densitometric scanning of thin-layer plates, colorimetric assay and HPLC analysis. By HPLC of benzoylated dolichols, the distribution of the dolichols according to the number of their isoprene residues, was determined in rat and trout liver. The major component was dolichol -18 in rat and dolichol -19 in trout liver. Dolichyl phosphate concentrations were found to be 6-7 micrograms/g of rat liver and 8-9 micrograms/g of trout liver by densitometric scanning of thin-layer plates.     

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4'-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6beta-hydroxylation

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6beta-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6beta-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.     

Isoenzyme patterns in parenchymal and non-parenchymal cells isolated from regenerating and regenerated rat liver

Parenchymal and non-parenchymal cells were isolated from adult rat liver that had been fully regenerated after a 70% partial hepatectomy. The characteristics of the parenchymal cell preparations from regenerated rat liver indicated that they were a homogeneous population and comparable with parenchymal cells isolated from intact liver. The parenchymal cells from regenerated adult rat liver contain glucokinase, hexokinase, pyruvate kinase type I and aldolase B. The non-parenchymal cells contain hexokinase, pyruvate kinase type III and aldolase B. When cells were isolated at different times of the day from rats on controlled feeding schedules, variation of tyrosine aminotransferase activity and liver glycogen content were observed in the parenchymal cells in keeping with the reported diurnal oscillations found in whole liver extracts. When parenchymal cells were isolated from rats 48 and 72h after partial hepatectomy, different isoenzyme patterns were observed. These cells appeared to synthesize pyruvate kinase type III, a function that was assigned previously to non-parenchymal cells or to foetal rat liver hepatocytes.     

Side-chain metabolism of propranolol: involvement of monoamine oxidase and mitochondrial aldehyde dehydrogenase in the metabolism of N-desisopropylpropranolol to naphthoxylactic acid in rat liver

We examined the metabolism of N-desisopropylpropranolol (NDP), which is generated from propranolol (PL) by side-chain N-desisopropylation, to naphthoxylactic acid (NLA) in rat liver. S(-)-NDP (S-NDP) and R(+)-NDP (R-NDP) were enantioselectively metabolized to NLA in isolated rat hepatocytes and in an enzyme reaction system of rat liver mitochondria with cofactor NAD+. Furthermore, the clearance profiles of NDP enantiomers were examined in an enzyme reaction system of rat liver mitochondria without NAD+. The amounts of S-NDP remaining in the incubation medium were similar to those of R-NDP, suggesting that monoamine oxidase (MAO) catalyzes the deamination of NDP to the aldehyde intermediate, but fails to deaminate enantioselectively S-NDP or R-NDP. Cyanamide, a potent inhibitor of aldehyde dehydrogenase (ALDH), markedly decreased the formation of NLA from racemic NDP in the enzyme reaction system of rat liver mitochondria with NAD+. When rat liver cytosol and microsomes were added to this enzyme reaction system, no significant alterations were observed in the amount of NLA generated from racemic NDP. We concluded that MAO deaminates NDP to an aldehyde intermediate, and that mitochondrial ALDH subsequently catalyzes the enantioselective metabolism of the aldehyde intermediate to NLA in rat liver.     

Epoxy derivatives of aromatic polycyclic hydrocarbons. The preparation and metabolism of epoxides related to benzo[a]pyrene and to 7,8- and 9,10-dihydrobenzo[a]pyrene

Products that appeared to be mainly benzo[a]pyrene 7,8-oxide and benzo[a]pyrene 9,10-oxide were synthesized and their chemical and biochemical properties were investigated. The oxides were unstable and readily rearranged to phenols. They were converted by rat liver homogenates and microsomal preparations into phenols and dihydrodiols, but glutathione conjugates were not formed in appreciable amounts. The dihydrodiols formed from benzo[a]pyrene 7,8- and 9,10-oxide by rat liver microsomal preparations were identical in their chromatographic and spectrographic properties with dihydrodiols formed when benzo[a]pyrene was metabolized by rat liver homogenates. 9,10-Dihydrobenzo[a]pyrene 7,8-oxide and 7,8-dihydrobenzo[a]pyrene 9,10-oxide were also synthesized. They were converted by rat liver homogenates and microsomal preparations into the related cis- and trans-dihydroxy compounds. Glutathione conjugates were formed from the oxides by rat liver homogenates. Both 7,8- and 9,10-dihydrobenzo[a]pyrene were metabolized by rat liver homogenates to mainly the trans-isomers of the related dihydroxy compounds. In experiments with boiled homogenates, the benzo[a]pyrene oxides were converted into phenols, whereas the dihydrobenzo[a]pyrene oxides yielded small amounts of the related dihydroxy compounds.     

基于超声射频信号的大鼠脂肪肝分级诊断研究

目的：从分析肝脏超声射频信号的角度,探究一种对脂肪肝进行分级定量诊断的新方法。方法：80只Wistar大鼠,通过喂养高脂饲料建立大鼠脂肪肝模型,其中正常肝的大鼠28只,轻度脂肪肝的大鼠21只,中度脂肪肝的18只,重度脂肪肝的13只。然后采集大鼠左右肝的超声射频信号,选取大鼠肝脏部位感兴趣区域的超声射频信号,分析大鼠肝脏超声射频信号的脂肪肝特征信息,提取了射频信号幅度包络值的均值／标准差（MSR）、偏度（SK）、峰度（Ku）这三个统计特征量,再利用BP神经网络对大鼠脂肪肝进行分类识别。结果：该方法对大鼠正常肝的识别率达92．5％,轻度脂肪肝的识别率达87．5％,中度脂肪肝的识别率达76．7％．重度脂肪肝的识别率达77．3％。结论：本文研究的方法可对大鼠脂肪肝进行分级定量诊断,且证明了超声射频信号在脂肪肝诊断中是有价值的,为对分级诊断脂肪肝疾病的研究提供了新的方向。     

Monoclonal antibodies against salt-resistant rat liver lipase. Cross-reactivity with lipases from rat adrenals and ovaries

To obtain monoclonal antibodies against rat salt-resistant liver lipase, mice were immunized with enzyme purified from heparin-containing rat liver perfusates. Hybridomas were screened for antibody production by means of an enzyme-linked immunosorbent assay (ELISA) and an immunoprecipitation assay. Five hybridoma cell lines secreting antibodies against rat liver lipase indicated as A, B, C, D and E, have been obtained. All antibodies possess gamma one (gamma 1) heavy chains and kappa (kappa) light chains. The antibodies precipitate salt-resistant lipase from rat post-heparin plasma, are positive in ELISA, inhibit liver lipase activity and bind monospecifically with the enzyme as shown by immunoblotting. The monoclonal antibodies showed no significant reactivity with human liver lipase. The salt-resistant lipases of rat adrenals and ovaries are also precipitated by the monoclonal antibodies directed against the liver enzyme. Therefore, the heparin-releasable lipases of the liver, adrenals and ovaries possess identical epitopes.