Effect of hypophysectomy on liver nuclear ribonucleic acid synthesis in aging rats.

Changes in RNA synthesis in liver nuclei were observed at different ages and after hypophysectomy and hormone replacement in female Sprague-Dawley rats. As determined by the incorporation of [3H]UMP into an acid-insoluble product, RNA synthesis decreased by about 75% in intact rats from 6 months to 24 months of age. This decline with age was not observed in liver nuclei from 24-month-old rats that had been hypophysectomized at 12 months and maintained on a minimal hormone-replacement therapy. Thyroid hormones and somatotropin (growth hormone) had an additive effect on RNA synthesis in liver nuclei from these hypophysectomized rats. The same hormones had no significant effect on intact, age-matched rats. With advancing age, nuclei of intact rats had an increase in the pool of free RNA polymerase and an apparent decrease in the enzyme activity bound to nuclear chromatin. There was no change in total enzyme with age. In hypophysectomized, hormone-treated rats, free RNA polymerase activity decreased and chromatin-bound activity increased. There was no difference in total nuclear RNA polymerase activity between operated or intact rats. However, the ratio of the bound to the free activity was different. These results suggest that the ability of RNA polymerase to bind to chromatin may be involved in the age-related decrease in liver nuclear RNA synthesis of intact rats.     

Bile acids via FXR initiate the expression of major transporters involved in the enterohepatic circulation of bile acids in newborn mice

The enterohepatic circulation (EHC) of bile acids (BAs) plays a pivotal role in facilitating lipid absorption. Therefore, initiation of the EHC in newborns is of crucial importance for lipid absorption from milk. The purpose of this study was to determine at what age BA transporters in liver are expressed, and the mechanism for their initiation. Serum and liver samples were collected from C57BL/6 mice at 2 days before birth and various postnatal ages. Messenger RNA assays revealed a dramatic increase at birth in the expression of the BA transporters (Ntcp, Bsep, Mrp4, Ostβ), as well as the phospholipid floppase Mdr2 in mouse liver, with the highest expression at 1 day of age. The mRNA expression of the ileal BA transporters (Ostα and Ostβ) also markedly increased at birth. Meanwhile, taurine-conjugated cholic acid markedly increased in both serum and liver of newborns, correlated with upregulation of the classic pathway of BA biosynthesis in newborn liver. The mRNA levels of the major BA sensors, FXR and PXR, were increased at 1 day of age, and their prototypical target genes were upregulated in liver. The mRNA expression of transporters involved in the EHC of BAs was similar in wild-type and PXR-null mice. In contrast, in FXR-null mice, the "day 1 surge" pattern of Ntcp, Bsep, Ostβ, and Mdr2 was blocked in newborn mouse liver, and the induction of Ostα and Ostβ was also abolished in ileums of FXR-null mice. In conclusion, at birth, BAs from the classic pathway of synthesis trigger the induction of transporters involved in EHC of BAs in mice, through activation of the nuclear receptor FXR.     

Age-associated changes in initiation of ribonucleic acid synthesis in isolated rat liver nuclei.

Initiation of RNA synthesis was studied in an attempt to determine a possible molecular mechanism for age-related biochemical and physiological changes. Initiation of RNA synthesis was determined by incorporation of [gamma-32 P]ATP and of [gamma-32P]GTP into an acid-insoluble product by intact nuclei isolated from livers of Sprague-Dawley CD-strain rats of various ages. When the rats were grouped into young (0.75-9 months) and old (12-30 months) rats, a significant decrease (P less than or equal to 0.001) in incorporation of initiating nucleotides was observed. The rat population was divided into five age groups (0.75-3 months, 4-9 months, 12-18 months, 19-23 months and 30 months) for further analysis of the effect of age on the initiation of RNA synthesis. Analysis of data from these groups indicated a significant trend for an age-related decrease in RNA-synthesis initiation (correlation coefficient = 0.94). Long-term hypophysectomy coupled with minimal hormone-replacement therapy was shown to have a significant effect on the reversal of the age-related decrease in initiation of RNA synthesis. It was observed that initiation of RNA synthesis in nuclei from 19-month-old rats, hypophysectomized at 12 months of age, was closest to that in 3-month-old intact rats and was not significantly different from that in liver nuclei of 0.75-9-month-old intact rats.     

Enhancement of in vitro ribonucleic acid synthesis on chromium(III)-bound chromatin

Z chromatin-chromium (Cr) complex, prepared from mouse liver chromatin and CrCl3, showed a significantly enhanced template activity for in vitro RNA synthesis. Digestion experiments with this complex using micrococcal nuclease and DNase I suggested that Cr(III) preferentially binds to linker regions rather than core regions of chromatin. Further, it was found that Cr(III) binds to DNA and nonhistone proteins (NHP), but hardly to histones. Moreover, the template activity of an NHP-Cr complex, when added to a DNA-histones complex, was inhibited remarkably. The template activity of the chromatin-Cr complex was not significantly altered by proteinase K digestion. Furthermore, experiments using rifampicin and [gamma-32P]guanosine 5'-triphosphate (GTP) demonstrated an increase in the number of initiation sites in the chromatin-Cr complex. These results suggest that, in this in vitro system, Cr(III) preferentially binds to DNA in chromatin and causes an increase in the number of initiation sites, thus enhancing RNA synthesis.     

Template requirements for RNA synthesis by a recombinant hepatitis C virus RNA-dependent RNA polymerase

The RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV), nonstructural protein 5B (NS5B), has recently been shown to direct de novo initiation using a number of complex RNA templates. In this study, we analyzed the features in simple RNA templates that are required to direct de novo initiation of RNA synthesis by HCV NS5B. NS5B was found to protect RNA fragments of 8 to 10 nucleotides (nt) from RNase digestion. However, NS5B could not direct RNA synthesis unless the template contained a stable secondary structure and a single-stranded sequence that contained at least one 3' cytidylate. The structure of a 25-nt template, named SLD3, was determined by nuclear magnetic resonance spectroscopy to contain an 8-bp stem and a 6-nt single-stranded sequence. Systematic analysis of changes in SLD3 revealed which features in the stem, loop, and 3' single-stranded sequence were required for efficient RNA synthesis. Also, chimeric molecules composed of DNA and RNA demonstrated that a DNA molecule containing a 3'-terminal ribocytidylate was able to direct RNA synthesis as efficiently as a sequence composed entirely of RNA. These results define the template sequence and structure sufficient to direct the de novo initiation of RNA synthesis by HCV RdRp.     

大鼠衰老过程中肝细胞核及染色质的体外转录活性

用同位素掺入法研究不同年龄大鼠的肝细胞核及染色质体外转录活性,所得结果表明:(1)老年大鼠肝细胞核的转录起始能力较断乳鼠及青年鼠分别下降68％及56％。(2)大鼠肝细胞核内与染色质结合的RNA聚合酶所致的转录活性随增龄呈近似线性下降,而不与染色质结合的RNA聚合酶所致的转录活性随增龄则无变化。(3)老年大鼠肝染色质体外转录活性较断乳鼠及青年鼠分别下降52％及35％。这些结果提示。老年大鼠肝染色质功能的改变可能是转录活性改变的主要原因。     

Sequence of molecular events involved in induction of allophanate hydrolase.

Addition of urea to an uninduced culture of Saccharomyces at 22 C results in appearance of allophanate hydrolase activity after a lag of 12 min. We have previously demonstrated that both ribonucleic acid (RNA) and protein synthesis are needed for this induction to occur. To elucidate the time intervals occupied by known processes involved in induction, temperature-sensitive mutants defective in messenger RNA transport from nucleus to cytoplasm (rna1) and in protein synthesis initiation (prt1) were employed along with an RNA polymerase inhibitor in experiments that measure cumulative synthetic capacity to produce allophanate hydrolase. These measurements identify the time within the lag period at which each of the above processes is completed. We observed that RNA synthesis, rna1 gene product function, and protein synthesis initiation are completed at 1 to 1.5, 4, and 9 to 10 min, respectively.     

Fibrillar nucleolar remnants do not contain macromolecular precursors of ribosomal RNA : Demonstration by the effects of -galactosamine

Administration of -galactosamine to rats produces inhibition of liver nuclear RNA synthesis and associated alterations in the structure of the nucleolus. Polyacrylamide gel electrophoretic analysis of liver nuclear RNA from galactosamine-treated rats has shown the virtual complete absence of ribosomal RNA (rRNA) precursor molecules at a time when the nucleolus consists solely of a dense fibrillar core devoid of granules. No evidence for an artefactual, preferential breakdown of nuclear RNA during extraction could be obtained from either 2.7 or 8% acrylamide gels. Furthermore, the almost complete cessation of nuclear RNA synthesis makes the possibility of there being rapid synthesis and degradation of ribosomal precursor molecules in vivo unlikely. With toluidine blue stains for RNA with nuclei isolated from galactosamine-treated animals, the large, brightly staining area associated with the normal nucleolus was not seen. On the basis of these observations, it is concluded that an RNA-depleted nucleolus appears fibrillar. It is suggested that the fibrillar material of a normal nucleolus may not itself be RNA even though this region does contain RNA precursor molecules.     

Role of endogenous regucalcin in nuclear regulation of regenerating rat liver: suppression of the enhanced ribonucleic acid synthesis activity

The role of endogenous regucalcin in the regulation of ribonucleic acid (RNA) synthesis activity in the nucleus of normal and regenerating rat livers was investigated. Nuclear RNA synthesis was measured by the incorporation of [(3)H]-uridine 5'-triphosphate into the nuclear RNA in vitro. The presence of regucalcin (0.25 or 0.5 microM) in the reaction mixture caused a significant decrease in nuclear RNA synthesis of normal rat liver. alpha-Amanitin (10(-8)-10(-6) M), an inhibitor of RNA polymerase II and III, decreased significantly nuclear RNA synthesis activity. The effect of regucalcin (0.25 microM) in decreasing nuclear RNA synthesis activity was not seen in the presence of alpha-amanitin (10(-6) M). The calcium chloride (10 microM)-increased nuclear RNA synthesis activity was significantly suppressed by the addition of regucalcin (0.25 microM). RNA synthesis activity was significantly enhanced in the nuclei of regenating rat liver obtained at 24, 48, or 72 h after partial hepatectomy. This enhancement was significantly inhibited in the presence of PD98059 (10(-5) M), staurosporine (10(-6) M), or vanadate (10(-3) M). Western analysis of the nuclei of regenerating liver obtained at 24, 48, or 72 h after partial hepatectomy showed a significant increase in regucalcin protein as compared with that of sham-operated rats. The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the reaction mixture caused a significant increase in nuclear RNA synthesis activity of normal rat liver. This increase was completely blocked by the addition of regucalcin (1.0 microM). The effect of anti-regucalcin monoclonal antibody (50 ng/ml) in increasing nuclear RNA synthesis activity was significantly enhanced in the nuclei of regenerating liver obtained at 24, 48, or 72 h after partial hepatectomy. This enhancement was significantly suppressed by the addition of alpha-amanitin (10(-6) M), PD98059 (10(-5) M), staurosporine (10(-6) M), or vanadate (10(-3) M) in the reaction mixture. The present study demonstrates that endogenous regucalcin has a suppressive effect on the enhancement of RNA synthesis activity in the nucleus of regenerating rat liver with proliferative cells.     

Inhibition of the reactions of DNA hydrolysis and RNA synthesis by the alkaloid sanguinarine

The increase of the contour length of the low molecular linear duplex DNA in the complex with an alkaloid sanguinarine has been evidenced by the viscometric method. The enzymatic hydrolysis of modified DNA by pancreatic deoxyribonuclease I and RNA synthesis of DNA by rat liver nuclear RNA polymerase were studied. Sanguinarine has been shown to inhibit the first stages of DNA hydrolysis. This alkaloid is a weaker inhibitor than ethidium bromide, a more potent inhibitor than actinomycin D and exerts an inhibiting effect similar to that of distamycin A. Sanguinarine also decreases the rate of the labelled precursor incorporation into the acid-insoluble fractions by nuclear RNA polymerase from rat liver. A 50% inhibition by sanguinarine was observed at the same alkaloid concentration as that of ethidium bromide.     

Sigma factor is not released during transcription in Bacillus subtilis.

The relationship between sigma (sigma) and delta (delta) factors of Bacillus subtilis RNA polymerase has been analyzed during initiation of RNA synthesis. When core enzyme (E) containing delta factor (E delta) binds to DNA, the delta factor is released with the formation of an E-DNA complex. The addition of sigma to the E-DNA complex results in the formation of a stable E sigma-DNA complex which can synthesize RNA upon addition of nucleoside triphosphates. Sigma factor, significantly, is not released from the core during RNA synthesis. These results suggest that delta and sigma factors can act sequentially during initiation of RNA synthesis with delta acting as a DNA recognition factor and sigma acting as an initiation factor. The results do not preclude the possibility that E sigma can initiate RNA synthesis correctly since E sigma alone can bind to DNA and initiate RNA synthesis.