Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Cell-free synthesis and transport of precursors of mutant ornithine carbamoyltransferases into mitochondria

Synthesis, mitochondrial transport and processing of ornithine carbamoyltrasferase (EC 2.1.3.3) were studied in mutant mice strains (sparse-fur, spf, and sparse-fur with abnormal skin and hair, spf-ash) which exhibit a deficiency in this enzyme. Spf mice have an increased amount (about 150% of control) of the enzyme with abnormal kinetic properties, whereas spf-ash mice have a decreased amount (about 10% of control) of the enzyme with apparently normal kinetic properties. Precursors of the mutant enzymes were synthesized in a reticulocyte lysate cell-free system. The hepatic level of translatable mRNA coding for the enzyme and the rate of the enzyme synthesis in liver slices of spf mice were 58 and 60% of the controls, respectively. In the case of spf-ash mice the activity of translatable mRNA for the enzyme was 10% of the controls. These results indicate that the decreased amount of ornithine carbamoyltransferase protein in spf-ash mice is due mainly to a decreased level of translatable mRNA for the enzyme, whereas the increase in the enzyme amount in spf mice is presumably the result of a decreased rate of enzyme degradation. The subunit molecular weight of the spf enzyme precursor was practically the same as that of the normal enzyme precursor (M_r 40 000). Both precursors synthetized in vitro could be taken up and processed similary to an apparently mature form (M_r 37 000). In the case of spf-ash enzyme, two discrete in vitro products were observed on sodium dodecyl sulfate polyacrylamide gel; one comigrated with the normal enzyme precursor and the other moved slightly slower. Both products appeared to be taken up and processed to the mature form of the enzyme.     

玉米蔗糖磷酸合成酶(SPS)基因的克隆及表达载体的构建

利用RT-PCR方法从玉米幼苗叶片总RNA中克隆出玉米的蔗糖磷酸合成酶(SPS)基因的全长cDNA片段。该片段与文献报道的序列具有99%的同源性。并分别构建了以双CaMV35S为启动子,以Tnos为终止子的植物双元表达载体PBISPS和以Pcab为启动子,以T35S为终止子的植物表达载体PBSPS,其中PBISPS含有NPTⅡ选择标记基因,PBSPS不含选择标记基因。     

Translation in vitro and regulation of mouse liver S-adenosylmethionine synthetase messenger RNA

Total RNA was isolated from adult mouse liver tissues. The alpha- and beta-form isozymes of S-adenosylmethionine synthetase existing in liver were synthesized in a reticulocyte lysate cell-free system under the direction of total RNA and were immunoprecipitated with antibody to the beta-form. The newly synthesized and the in vivo labeled S-adenosylmethionine synthetase subunits were compared by SDS-polyacrylamide gel electrophoresis. Both the alpha- and beta-forms consist of the same size Mr 48 000 subunit. The level of the beta-form mRNA activity in mouse liver was shown to increase following intraperitoneal transplantation of Ehrlich ascites tumor cells and the changes in the mRNA activity parallel those in the cellular level of S-adenosylmethionine synthetase beta.     

Effect of protein deficiency on macroelement and trace element levels of weanling rats' small intestine and liver tissues

Protein energy malnutrition has become a major health issue in developing countries. In the present study, the effect of protein deficiency on the small intestine and liver tissue content of macroelements and trace elements was investigated in weanling rats. Forty-five male weanling Wistar albino rats were divided into three groups. The control group (C) was fed a standard diet containing 25% casein, whereas the two experimental groups E1 and E2 consumed 12% and 3% casein, respectively, over a period of 45 d. The tissue samples were analyzed for zinc, copper, iron, manganese, calcium, and magnesium by atomic absorption spectroscopy. The protein-deficient groups showed increased levels of iron in both tissues and decreased manganese in small intestine tissue from the E1 group. No other differences were found for the other elements. These results suggest that protein deficiency might cause iron accumulation in the liver and intestine and decreases of manganese in the small intestine.     

Developmental pattern of poly (ADP-ribose) synthetase and NAD glycohydrolase in the brain of the fetal and neonatal rat

Poly (ADP-ribose) synthetase and NAD glycohydrolase were examined in nuclear fractions from rat brain at sequential times during late fetal and the first two weeks of neonatal life. In whole brain, both enzymes were demonstrable at all stages of development, but followed separate patterns. Activity of the synthetase which was greatest in fetal life, fell steadily with fetal maturation from 3.90±0.06 nmol/mg DNA at 16 days, to reach a nadir of 1.36±0.09 nmol/mg DNA on the 4th postnatal day. Subsequently it underwent a non sustained neonatal rise reaching a peak of 2.46±0.07 nmol/mg DNA on the 8th day. By contrast, NAD glycohydrolase activity increased steadily throughout late fetal and during the first two weeks of neonatal life, from 12.77±0.40 nmol/mg DNA on day 16 of gestation to 25.80±.95 nmol/mg DNA on neonatal day 12. In neonatal cerebellum the activity of poly (ADP-ribose) synthetase was greater at 8 than at 4 days, could be stimulated with graded concentrations of sonicated DNA up to 100 g, but was inhibited by higher concentrations of DNA and by all concentrations of exogenous histone. In an in vitro culture system of fetal rat brain cells, the activity of poly (ADP-ribose) synthetase increased steadily over six days. Cycloheximide 10^–3 M completely inhibited the activity of this enzyme. NAD glycohydrolase activity increased progressively in vitro, and after 6 days in cycloheximide (10^–3 M), the cultures contained significantly greater levels of enzyme activity. It is suggested that changing activities of poly (ADP-ribose) synthetase and NAD glycohydrolase could both provide potential markers for brain cell differentiation in this system.     

Selective decrease of type I collagen synthesis in Fraser mice skin

Quantification and biosynthesis of type I and type III collagens were determined in skin of control and Fraser mice (Cat^Fraser mutation), which exhibit a genetically determined cataract. Skin organ cultures were labelled with [³H]proline. Pepsin-solubilized collagens were studied using three different approaches: (a) differential salt precipitation at neutral pH, followed by SDS-polyacrylamide gel electrophoresis; (b) differential salt precipitation at acid pH followed by SDS-polyacrylamide gel electrophoresis. (c) CNBr peptide analysis. These methods gave consistent and reproducible results, indicating a selective decrease of type I collagen in Fraser mouse skin as compared to control mouse skin. Metabolic labelling of skin organ cultures showed a decreased specific radioactivity of hydroxy[³H]proline in type I collagen of Fraser mouse skin. The concordant results of these experiments suggest a genetically determined alteration of interstitial collagen metabolism in the Fraser mutation apparently specifically concerning the expression of type I collagen gene(s).     

皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶活性及其基因表达的影响

采用高效液相色谱和原位杂交技术研究了皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶 (ODC)活性及ODCmRNA表达的影响。结果显示 ,大鼠完整肝脏中ODC水平较低 ,2 / 3肝切除 (PH)后 3h ,不同处理组ODC活性开始升高 ,6h达到最高值 ,其中 ,去肾上腺 NaCl组和糖皮质激素受体拮抗剂RU4 86处理组的酶活性高于对照组 (去肾上腺假手术组 ) ,而去肾上腺 皮质酮处理组的酶活性低于对照组 ,36h恢复到肝切除前水平 ;完整肝脏的ODCmRNA水平极低 ,PH后表达量迅速增加 ,5h达到最大值 ,不同处理组mRNA水平的高低顺序与酶活性一致 ,12h降至肝切除前水平 ;在PH前 12h给大鼠注射RU4 86 (10mg/kg体重 ) ,取得了与去肾上腺 NaCl处理鼠相似的结果。以上结果表明 ,在PH诱导的再生肝细胞中 ,ODCmRNA表达量的增加和 /或减少是造成ODC活性改变的原因之一 ,皮质酮对ODC活性及其mRNA的表达具有抑制作用 ,主要表现在肝再生的早期 ,该作用可能是通过受体实现的     

Hormonal control of ornithine decarboxylase in isolated liver cells and the effect of ethanol oxidation

The regulation of ornithine decarboxylase activity was studied in freshly isolated rat hepatocytes incubated in a chemically defined medium for 5 h. Glucagon, dibutyryl cyclic AMP, insulin and dexamethasone produced dramatic increases in ornithine decarboxylase activity, 6–100-times the basal activity. Actinomycin D inhibited completely the stimulatory action of these substances. With glucagon, dibutyryl cyclic AMP and insulin, the rise in ornithine decarboxylase activity was rapid but transient, peaking at 200 min and then declining rapidly. By contrast, the response to dexamethasone was gradual and sustained in the 5 h incubation. The transient nature of the response to glucagon was unaltered by repeated additions of optimally effective doses of glucagon suggesting the development of ‘refractoriness’ to the actions of this hormone. Ethanol oxidation inhibited by 50% the stimulation of ornithine decarboxylase by glucagon and dexamethasone and this effect was blocked by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase. Acetate (2.5–20 mM), the metabolic product of hepatic ethanol oxidation, was also effective. The data indicate that glucagon, insulin and glucocorticoids are all effective in stimulating the activity of ornithine decarboxylase in isolated hepatocytes but they differ in their duration and time of peak of action. Additionally, the inhibitory effect of ethanol on the hormonal stimulation of ornithine decarboxylase is dependent on its oxidation and may be mediated by acetate.     

Gender-specific alterations in gene expression and loss of liver sexual dimorphism in the long-lived Ames dwarf mice

Genetic mutations that increase lifespan in mice frequently involve alterations in the growth hormone/insulin-like growth factor-I signaling pathway. Although several of the effects of GH on gene expression are known to be sex-dependent, an understanding of the gender-specific vs. gender-independent effects of lifespan-extending mutations of the GH/IGF-I axis is currently lacking. The Ames dwarf mice (prop1(df/df)) are GH, prolactin and thyroid-stimulating hormone deficient and exhibit an increase in mean lifespan of 49% in males and 68% in females. We used oligonucleotide arrays containing over 14,000 genes to study the gender-specific vs. gender-independent effects of the prop1(df) mutation in liver of male and female Ames mice. We identified 381 gender-independent and 110 gender-specific alterations in gene expression produced by the Prop1(df/df) genotype. The gender-specific alterations corresponded to genes with a strong sexual dimorphism in wild-type mice and produced an almost complete loss of sex-specific gene expression in the liver of Ames dwarf mice: out of 123 genes that showed sexual dimorphism in wild-type mice only six maintained a gender difference in mutant mice. However, the Prop1(df/df) genotype did not introduce new sexually dimorphic patterns of gene expression in Ames dwarf mice that were not present in the wild-type animals. The gender-specific alterations accounted for a large fraction of the most significant changes in gene expression in male and female Ames mice livers and affected several metabolic processes, particularly fatty acid metabolism, steroid hormone metabolism, and xenobiotic metabolism.     

Expression of glutamine-synthetase genes in cotyledons of germinating Phaseolus vulgaris L.

In the legume Phaseolus vulgaris L., glutamine synthetase (GS; EC.6.3.1.2.) is encoded by four actively transcribed genes, gln-, gln-, gln- and gln-. We have studied the expression of these genes in cotyledons during seed germination and have studied the effect of light and nitrate on this process. An RNase-protection method, used to detect the abundances of GS mRNAs, revealed that the four GS genes are differentially expressed in the germinating cotyledons. The gln-. mRNA was present in dry seeds and was the most abundant GS mRNA during early stages of germination. The gln- and gln- mRNAs were first detectable 2 d after sowing and their abundances differed in light- and dark-grown cotyledons at later stages of germination. The gln- mRNA (which encodes the plastid-located GS) was detectable only in light-grown cotyledons, at a low abundance. A nitrate supply of 2 mM had only a minor effect on the expression of the GS genes. Western immunodetection and ion-exchange high-performance liquid chromatography demonstrated that the polypeptide and isoenzyme were present in extracts of dry seeds and represented the major GS products at 2 d and 4 d. Both the and polypeptides appeared at the 2-d stage. The role of differential GS gene expression in controlling cotyledonary GS activity is discussed.Abbreviations 1D, 2D one-, two-dimensional - GS glutamine synthetase - GS_t GS transferase activity - IEX-HPLC ion-exchange high-performance liquid chromatography - kDa kilodaltons - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We are grateful to the Association of Commonwealth Universities and the Science and Engineering Research Council for financially supporting R.S. and to the S.E.R.C. for a grant to support M.J.B. We would like to thank Dr K.J.F. Farnden (University of Otago, New Zealand) and Dr T.H.N. Ellis (John Innes Institute, Norwich) for scanning the autoradiographs for Fig. 2.     

The glutamine synthetase gene family of Arabidopsis thaliana: light-regulation and differential expression in leaves, roots and seeds.

Summary Glutamine synthetase (GS) plays an important role in the assimilation of nitrogen by higher plants. We present here a molecular analysis of the GS polypeptides, mRNAs, and genes of Arabidopsis thaliana. Western blot analysis of leaf and root protein extracts revealed at least two distinct GS polypeptides; 43 kDa and 39 kDa GS polypeptides were present in leaves, while only a 39 kDa GS was detected in roots. The 43 kDa GS polypeptide is light-inducible. In etiolated seedlings only the 39 kDa GS was detected. However, upon greening the 43 kDa GS increased to levels comparable to those observed in light-grown plants. Four distinct GS cDNA clones, Atgsl1, Atgsrl, Atgsr2 and Atk6 were isolated and characterized. Their complete nucleotide and deduced amino acid sequences are presented. The coding sequences of the four clones are 70–88% similar while their 5 and 3 untranslated regions exhibit less than 50% similarity. Northern blots of leaf, root and germinated seed RNA revealed that the four cDNAs hybridize to mRNAs which are differentially expressed in the organs of Arabidopsis thaliana. Atgsl1 is leaf-specific and hybridizes to a 1.6 kb mRNA. Both Atgsr1 and Atgskb6 hybridize to 1.4 kb mRNAs which are expressed in both roots and germinated seeds. Atgsr2 hybridizes to a 1.4 kb mRNA, which is primarily expressed in roots with low levels of expression in seeds and leaves. Atgsl1, which represents the leaf-specific mRNA, is induced by light. Atgsl1 mRNA levels increase during the greening of etiolated seedlings while Atgsr1 levels remain constant. Southern blot analysis indicated that the Arabidopsis genome contains at least four and possibly five distinct GS genes.     

Conjugal transfer of aminoglycoside and macrolide resistance between Enterococcus faecium isolates in the intestine of streptomycin-treated mice

The purpose was to study conjugal transfer of resistance genes between a multi-resistant Enterococcus faecium isolate and a sensitive E. faecium isolate. Co-transfer of erm(B)-Tn5405-like element and aac(6')-Ie-aph(2')-Ia was obtained in both in vivo and in vitro. Plasmid profiles and Southern blots showed that both the erm(B)-Tn5405-like element and aac(6')-Ie-aph(2')-Ia were placed on the same large plasmid (>147 kb). These data show to our knowledge the first co-transfer of the erm(B)-Tn5405-like element and aac(6')-Ie-aph(2')-Ia. The in vivo study also indicates that transfer of resistance genes between enterococci might occur under natural conditions in the gut of animals.     

Sequential up-regulation of thyroid hormone β receptor,ornithine transcarbamylase,and carbamyl phosphate synthetase mRNAs in the liver of Rana catesbeiana tadpoles during spontaneous and thyroid hormone-induced metamorphosis

During both spontaneous and thyroid hormone (TH)-induced metamorphosis, the Rana catesbeiana tadpole undergoes postembryonic developmental changes in its liver which are necessary for its transition from an ammonotelic larva to a ureotelic adult. Although this transition ultimately results from marked increases in the activities and/or de novo synthesis of the urea cycle enzymes, the precise molecular means by which TH exerts this tissue-specific response are presently unknown. Recent reports, using RNA from whole Xenopus laevis tadpole homogenates and indirect means of measuring TH receptor (TR) mRNAs, suggest a correlation between the up-regulation of TRβ-mRNAs and the general morphological changes occurring during amphibian metamorphosis. To assess whether or not this same relationship exists in a TH-responsive tissue, such as liver, we isolated and characterized a cDNA clone containing the complete nucleotide sequence for a R. catesbeiana urea cycle enzyme, ornithine transcarbamylase (OTC), as well as a genomic clone containing a portion of the hormone-binding domain of a R. catesbeiana TRβ gene. Through use of these homologous sequences and a heterologous cDNA fragment encoding rat carbamyl phosphate synthetase (CPS), we directly determined the relative levels of the TRβ, OTC, and CPS mRNAs in liver from spontaneous and TH-induced tadpoles. Our results establish that TH affects an up-regulation of mRNAs for its own receptor prior to up-regulating CPS and OTC mRNAs. Moreover, results with cultured tadpole liver demonstrate that TH, in the absence of any other hormonal influence, can affect an up-regulation of both the TRβ and OTC mRNAs. © 1992 Wiley-Liss, Inc.