Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The recovery of the enzyme poly(ADP-ribose) polymerase (pADPRp) in the nuclease- and 1.6 M NaCl-resistant nuclear subfraction prepared from a number of different sources was assessed by Western blotting. When rat liver nuclei were treated with DNase I and RNase A followed by 1.6 M NaCl, approximately 10% of the nuclear pADPRp was recovered in the sedimentable fraction. The proportion of pADPRp recovered with the residual fraction decreased to less than 5% of the total nuclear polymerase when nuclei were prepared in the presence of the sulfhydryl blocking reagent iodoacetamide and increased to approximately 50% of the total nuclear pADPRp when nuclei were treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) prior to fractionation. To determine whether this effect of disulfide bond formation was unique to rat liver nuclei, nuclear matrix/cytoskeleton structures were prepared in situ by sequentially treating monolayers of tissue culture cells with Nonidet-P40, DNase I and RNase A, and 1.6 M NaCl (S.H. Kaufmann and J.H. Shaper (1991) Exp. Cell Res. 192, 511-523). When nuclear monolayers were prepared from HTC rat hepatoma cells, CaLu-1 human lung carcinoma cells, and CHO hamster ovary cells in the absence of NaTT, pADPRp was undetectable in the nuclease- and 1.6 M NaCl-resistant fraction. In contrast, when nuclear monolayers were isolated in the presence of NaTT, from 5% (CaLu-1) to 26% (HTC cells) of the total nuclear pADPRp was recovered with the nuclease- and salt-resistant fraction. Examination of these residual structures by SDS-polyacrylamide gel electrophoresis under nonreducing conditions suggested that pADPRp was present as a component of disulfide cross-linked complexes. Further analysis by immunofluorescence revealed that the pADPRp was diffusely distributed throughout the CaLu-1 or CHO nuclear matrix. In addition, when matrices were prepared in the absence of RNase A, pADPRp was also observed in the residual nucleoli. These observations reveal that the recovery of pADPRp with a nuclease- and salt-resistant nuclear subfraction is dependent on the source of the nuclei and on the conditions used to fractionate those nuclei. In addition, these observations raise the possibility that there might be different functional classes of pADPRp molecules within the nucleus.
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PMID:Association of poly(ADP-ribose) polymerase with the nuclear matrix: the role of intermolecular disulfide bond formation, RNA retention, and cell type. 170 86

Previous studies have resulted in conflicting data regarding the recovery of the nuclear enzymes topoisomerase (topo) II and topo I in the nuclear matrix fraction. In the present study we have assessed the effect of systematically altering a single extraction procedure on the distribution of these enzymes during the subfractionation of nuclei from HTC hepatoma tissue culture cells. When nuclear monolayers (prepared by treating attached cells in situ with the neutral detergent Nonidet-P40 at 4 degrees C) were isolated in the presence of the irreversible sulfhydryl blocking reagent iodoacetamide, subsequent treatment with DNase I and RNase A followed by 1.6 M NaCl resulted in structures which were extensively depleted of intranuclear components as assessed by phase contrast microscopy and conventional transmission electron microscopy. These structures contained 12 +/- 4% of the total protein present in the original nuclear monolayers. The lamins and polypeptides with molecular weights comparable to those of actin and vimentin were the predominant polypeptides present on SDS-polyacrylamide gels. Western blotting revealed that less than 5% of the total nuclear topo II molecules were present in these structures. In contrast, when the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) was substituted for iodoacetamide, the same extraction procedure yielded structures containing components of the nucleolus and an extensive intranuclear network. These structures contained a wide variety of nonlamin, nonhistone nuclear polypeptides including 23 +/- 4% of the total nuclear topo II. SDS-polyacrylamide gel electrophoresis performed under nonreducing conditions revealed that topo II in these nuclear matrices was present as part of a large disulfide cross-linked complex. Treatment of these structures with reducing agents in 1.6 M NaCl released the topo II. In contrast, topo I did not form disulfide cross-linked oligomers and was not detectable in any of these nuclease- and salt-resistant structures prepared at 4 degrees C. To assess the effect of in vitro heat treatment on the distribution of the topoisomerases, nuclear monolayers (isolated in the absence of iodoacetamide and NaTT) were heated to 37 degrees C for 1 h prior to treatment with nucleases and 1.6 M NaCl. The resulting structures (which retained 26 +/- 5% of the total nuclear protein) were morphologically similar to the NaTT-stabilized nuclear matrices and contained 15 +/- 4% of the total nuclear topo II. High-molecular-weight disulfide cross-linked oligomers of topo II were again demonstrated. Attempts to demonstrate these disulfide cross-linked oligomers in intact cells were unsuccessful.
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PMID:Association of topoisomerase II with the hepatoma cell nuclear matrix: the role of intermolecular disulfide bond formation. 184 38

IL-12, a recently described cytokine, is an important mediator in the early production of IFN-gamma during infection. To evaluate the timing of IL-12 production, and its relationship to TNF-alpha, and IFN-gamma production during primary murine listeriosis, we measured cytokine mRNA and protein levels in C57B1/6 mice infected intravenously with Listeria monocytogenes (LM). IL-12 is a disulfide-linked heterodimer containing two chains (designated P35 and P40); however, bioactive cytokine production has been more closely linked with P40 expression. Consequently, we monitored mRNA and protein levels of P40 in the spleen as a marker for IL-12 production in vivo. Splenic P40 mRNA levels (assayed using RNase protection methods) were low in uninfected animals, but increased markedly beginning 15 to 18 hr after LM infection. In sublethally infected animals, P40 mRNA levels remained elevated for 5 days, returning to baseline with the resolution of infection. P40 protein (assayed using an antibody capture ELISA) could be detected in the spleens of LM-infected animals beginning around 18 hr postinfection confirming linkage between P40 mRNA accumulation and the generation of a protein product. In comparing P40 and IFN-gamma mRNA levels in vivo, we found in each case that substantial increases in mRNA accumulation did not appear until 15-18 hr postinfection. In comparable studies using BALB/c animals, cytokine production began slightly earlier (between 12 and 15 hr) but once again P40 and IFN-gamma mRNA levels increased in a coordinated manner. P40 mRNA (like IFN-gamma and TNF-alpha mRNA) only accumulated in animals infused with live, virulent bacteria. Although we could detect no obvious lag between the time of onset of IL-12 and IFN-gamma accumulation in vivo, infusions of anti-IL-12 antibodies markedly reduced IFN-gamma expression implying that IL-12 production precedes and directs IFN-gamma production. TNF-alpha production, on the other hand, was not diminished by anti-IL-12 treatment. Our studies demonstrate that IL-12 generation is an essential step in normal IFN-gamma production during listeriosis, and suggest that IL-12, once produced, may begin enhancing IFN-gamma production in vivo in less than 3 hr.
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PMID:Analysis of the interrelationship between IL-12, TNF-alpha, and IFN-gamma production during murine listeriosis. 760 97

P40 is the protein encoded by the first open reading frame (ORF1) of the human LINE-1 (L1Hs) retrotransposon; it is 338 amino acids long, has a leucine zipper motif and has been found in human teratocarcinoma cell lines and some tumor cells. In this report, we describe properties of p40 in the human teratocarcinoma cell lines NTera2D1 and 2102Ep. The results indicate that: (i) most of p40 occurs in large multimeric cytoplasmic complexes, (ii) L1Hs RNA is associated with the p40 complexes, (iii) the complexes are dissociated by ribonuclease and (iv) p40 is a novel RNA-binding protein. Cross-linking experiments with full-length and truncated p40 produced in Escherichia coli also showed that: (i) p40 itself can form a multimeric complex larger than 250 kDa, (ii) the leucine zipper motif and the region conserved among the predicted ORF1 polypeptides of mammalian LINE-1s participate in complex formation and (iii) the amino terminal region is important for the stability of complex formation. Analysis of the amino acid sequence of p40 suggests that long segments of the molecule can assume an alpha-helical configuration including the leucine zipper and the conserved region. The evidence presented here suggests that the p40 complex is a ribonucleoprotein complex containing L1Hs RNA(s) and that protein-protein interactions in which alpha-helix structures participate, for example coiled-coils, may occur in the complex.
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PMID:Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA. 859 46

P40 is encoded by the first open reading frame of the human LINE-1 retrotransposon and is found in a large cytoplasmic ribonucleoprotein (RNP) complex, the p40 RNP-complex, in association with LINE-1 RNA(s) in human teratocarcinoma cell lines. We report here investigations on the stability of the p40 RNP-complex against various nucleases and high salt (0.5 M NaCl) treatment. The results indicate that (1) the p40 RNP-complex is dissociated after ribonuclease or high salt treatment, (2) DNase I does not disrupt the complex, (3) after dissociation of the complex, p40 maintain protein-protein interactions but in smaller complexes, and (4) p40 is not associated with the LINE-1 RNA(s) after high salt treatment. These observations suggest that the RNA molecule(s) is(are) essential for the stability of the large p40 complex and that the complex has a structure which allows various nucleases to reach the RNA. These features are distinct from those of typical virus and virus-like particles of retroviruses and other retrotransposons, respectively. Together with the fact that no significant sequence homology exists between p40 and the gag and gag-like proteins, it is likely that the p40 RNP-complex is structurally different from the typical virus and virus-like particles.
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PMID:Ribonuclease and high salt sensitivity of the ribonucleoprotein complex formed by the human LINE-1 retrotransposon. 930 51

The release of GnRH peptide from neuroterminals in the median eminence increases during postnatal development. We were interested in determining the biosynthetic component contributing to the regulation of GnRH decapeptide levels, and ascertaining the molecular mechanism for these changes. Male and female C57bl/6 mice, from embryonic day (E)16 through postnatal day (P)60, were killed, and the preoptic area-anterior hypothalamus was dissected out. Cytoplasmic and nuclear RNA were extracted separately. Levels of GnRH messenger RNA (mRNA) and primary transcript were quantitated in individual preoptic area-anterior hypothalamus cytoplasmic and nuclear fractions, respectively, by ribonuclease protection assays. Serum LH levels were assayed by RIA. GnRH mRNA levels in the cytoplasm increased gradually and significantly during postnatal development in both males and females, reaching a peak at P55 in females and P40 in males. GnRH primary transcript levels in the nucleus, an index of GnRH gene transcription, changed in a completely different manner developmentally, and they differed between male and female mice. GnRH primary transcript levels in males were quite low until P5, when they underwent an increase of approximately 4-fold, between P5 and P7. They continued to increase through P15, at which time they reached adult levels. In females, GnRH primary transcript levels were high at E16, decreased to a nadir at P5, and then underwent an increase of approximately 5-fold to P7, which were comparable with adult levels. The large and sexually dimorphic changes in GnRH primary transcript between E16 and P7, in the absence of similar changes in GnRH mRNA, suggest that differential mechanisms, such as gene transcription and mRNA stability, play a role in determining levels of GnRH mRNA at different stages of development.
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PMID:Mechanisms for the regulation of gonadotropin-releasing hormone gene expression in the developing mouse. 1021 81

These studies were performed to determine the developmental expression pattern of neurotrophic factor (NTF: nerve growth factor (betaNGF), brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT-3) and NT-4 mRNA and NGF, NT-3 and NT-4 protein in the urinary bladder of the postnatal Wistar rat. It was hypothesized that NTFs may contribute to the development of the spinobulbospinal micturition reflex that represents the adult micturition pattern. Changes in NTF mRNA or protein expression in the urinary bladder at the time of development of the mature micturition reflex (postnatal days (P) 16-18) may suggest an involvement of target-derived NTFs in this maturation process. Developmental ages, prior to (P5, P10, P15) or following (P20, P30, adult P90) the development of the spinobulbospinal micturition reflex were selected and the urinary bladder was analyzed for levels of neurotrophic factor mRNA or protein. Results from ribonuclease protection assays demonstrated a similar developmental pattern among each neurotrophic factor examined. Neurotrophic factor mRNA levels increased by P10 and reach a maximum by P15. Subsequently, NTF mRNA levels declined to adult levels that were less than the earliest postnatal time examined (P5). NTF mRNA expression was significantly (p</=0.05-0.001) greater at P10, P15, P20 and P40 (NT-4 mRNA) compared to adult levels for each NTF examined except GDNF mRNA. In general, NGF, NT-3 and NT-4 urinary bladder protein levels in early postnatal development, as determined by ELISA, were similar when compared to the corresponding mRNA expression. Differences in the correlation between NT-3 and NT-4 mRNA and protein expression were demonstrated in the adult urinary bladder where significantly (p</=0. 001) greater levels of protein were revealed despite relatively low abundance of NT-3 and NT-4 mRNA. The developmental expression pattern (maximum expression at the second to third postnatal week) of NTFs in the urinary bladder is consistent with a potential role in the development of the spinobulbospinal reflex. Relatively high expression of NT-3 and NT-4 protein in the adult urinary bladder suggests a potential importance of these factors in the adult lower urinary tract.
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PMID:Developmental expression of urinary bladder neurotrophic factor mRNA and protein in the neonatal rat. 1067 71

We have used a quantitative RNase protection assay to characterize the relative accumulation and abundance of individual adeno-associated virus type 2 (AAV) RNAs throughout the course of AAV-adenovirus coinfections and preinfections. We have demonstrated that there is a previously unrecognized temporal order to the appearance of AAV RNAs. First, unspliced P5-generated transcripts, which encode Rep78, were detectable prior to the significant accumulation of other AAV RNAs. Ultimately, as previously demonstrated, P19-generated products accumulated to levels greater than those generated from P5, and P40-generated transcripts predominated in the total RNA pool. Second, the percentage of each class of AAV RNA that was spliced increased during infection, and the degree of this increase was different for the P5/P19 products than for those generated by P40. At late times postcoinfection, approximately 90% of P40 products, but only approximately 50% of RNAs generated by P5 and P19, were seen to be spliced; thus, the AAV intron was removed to different final levels from these different RNA species. We have shown that each of the AAV RNAs is quite stable; the majority of each RNA species persisted 6 h after treatment with actinomycin D. Quantification of the accumulation of individual AAV RNAs, over intervals during which degradation was negligible, allowed us to infer that at late times during infection the relative strength of P5, P19, and P40 was approximately 1:3:18, respectively, consistent with the steady-state accumulated levels of the RNAs generated by each promoter. All AAV RNAs exited to the cytoplasm with similar efficiencies in the presence or absence of adenovirus; however, adenovirus coinfection appeared to stimulate total splicing of AAV RNAs and the relative use of the downstream intron acceptor. Our results confirm and extend previous observations concerning the appearance and processing of AAV-generated RNAs.
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PMID:Adeno-associated virus RNAs appear in a temporal order and their splicing is stimulated during coinfection with adenovirus. 1102 14