Metabolism of ribosomal precursor ribonucleic acid in kidney.

The labile precursors of ribosomal RNA in mouse kidney are preserved when nuclei rapidly isolated after sieving through multiple screens are swollen and cleansed in the presence of an RNase inhibitor before digestion with DNase and phenol extraction. The kinetics of nucleolar labeling analyzed on polyacrylamide gels show that 36S RNA is the major intermediate product in the catabolism of the original 45S RNA precursor to 32S RNA, from which 28S RNA is derived. Each kidney nucleus contains about 200-600 molecules of 45S RNA; the turnover time of the 45S pool is about 3 +/- 2 min. Compared with HeLa cells, kidney nuclei have a different major intermediate product and a much smaller and more rapidly turning-over pool of ribosomal precursor RNA.

Can we predict a delirium after cardiac surgery? A validation study of a delirium risk checklist.

BACKGROUND: Delirium is a common temporary mental disorder that often occurs in patients who undergo cardiac surgery. It is important to prevent the negative side effects of delirium by identifying high-risk patients before surgery. Koster and colleagues designed a risk model to identify patients with an increased risk of postoperative delirium after cardiac surgery. AIM: The aim of this study was to validate the risk model for delirium and further improve the risk model. METHODS: A delirium risk checklist containing predictors associated with postoperative delirium was used during the preoperative outpatient screening in 329 patients. The delirium observation screening scale was used preoperatively and postoperatively to assess delirium. RESULTS: Compared with the model of Koster and colleagues age greater than 70 years and a history of delirium were confirmed as statistically significant predictors of postoperative delirium, while cognitive impairment and alcohol abuse were almost significant factors. The European system for cardiac operative risk evaluation (EuroSCORE), comorbidity and type of surgery could not predict a postoperative delirium again. The area under the curve of this model was 0.79 (95% confidence interval (CI) 0.73-0.86; P<0.001). Based on the data of this study the model was improved with the following independent predictors of postoperative delirium: age, more than one comorbidity, history of delirium and a lower standardised mini mental state examination score as with an area under the curve of 0.79 (95% CI 0.73-0.85; P<0.001). CONCLUSION: The risk model could not be fully validated. It is difficult to validate a risk model over time; there are different circumstances such as the increased focus on the prevention of delirium.

Estrogen-inducible binding of a nuclear factor to the vitellogenin upstream region.

The estrogen-dependent binding of a protein to the upstream region of the chicken vitellogenin gene was detected by using in vivo dimethyl sulfate, genomic DNase I, and in vitro exonuclease III footprinting. The site is located between base pairs -848 and -824, and its sequence resembles that of the nuclear factor I binding site. The results suggest that a nuclear factor binding to this site is involved in the regulation of the vitellogenin gene.

A European Multicenter Study of 616 Patients Receiving the Freedom Solo Stentless Bioprosthesis.

BACKGROUND: The purpose of this study was to evaluate the safety and performance of the Freedom Solo valve in aortic valve replacement by clinical and hemodynamic outcomes. METHODS: Six hundred sixteen patients underwent aortic valve replacement in 18 European centers; mean age was 74.5 ± 5.9 years, 54.1% of the patients were male, and concomitant procedures were performed in 43.2% of the patients. The majority (69%) of the implanted sizes were 23 mm and 25 mm. RESULTS: At 1 year, overall survival was 94.0%, whereas freedom from valve-related death was 98.6%. There were 9 (1.5%) early (≤ 30 days) and 27 (4.4%) late (>30 days) deaths. Early and late valve-related mortality was 0.3% (n = 2) and 1.1% (n = 7), respectively. Freedom from explant was 97.6%; 10 valves were explanted for endocarditis and 4 for paravalvular leak. There were 10 (1.6%) early and 5 (0.8%) late strokes. Atrioventricular block requiring pacemaker implant occurred in 8 (1.3%) and 1 (0.2%) patients in the early and late postoperative period, respectively. Thrombocytopenia was seen in 27 cases (4.4%) in the early postoperative period. Preoperatively, 93.8% of patients were in New York Heart Association functional classes II through IV, whereas at 1 year 96.9% of patients were in New York Heart Association functional classes I and II. At 1-year follow-up, mean and peak pressure gradients were 7.2 and 14.6 mm Hg, respectively. Indexed left ventricular mass decreased by 12% from 138 g/m(2) at discharge to 122 g/m(2) at 1 year. CONCLUSIONS: At 1-year follow-up after Freedom Solo implantation, we found acceptable clinical results with low mortality and morbidity and good hemodynamic performance, confirming safety and effectiveness in this multicenter experience.

Expression-linked demethylation of 5-methylcytosines in the chicken vitellogenin gene region.

We have studied the methylation status of the estradiol-controlled chicken vitellogenin (Vtg) gene, which is expressed in the liver. A 30-kb region was investigated, containing 17 HpaII and 18 HhaI sites, of which 21 are in the 22-kb gene. Of these 21 sites, 9 were found to be demethylated in laying-hen liver relative to immature chicken liver. Outside the transcribed region, only one site was found to be relatively undermethylated in laying-hen liver. This site, at 0.6 kb in front of the gene, is, as shown earlier, also demethylated in rooster or immature chicken liver upon primary hormone stimulation, as well as in the non-expressing estradiol target organ oviduct. In this respect, this site sharply contrasts with those in the transcribed region, which appear to become demethylated only upon prolonged transcription of the gene.

Estradiol-induced synthesis of vitellogenin. IV. The isolation of non-degraded polysomes from avian liver using an endogenous ribonuclease inhibitor.

A procedure allowing the isolation of intact polysomes from rooster liver is described. Good recovery of polysomes is achieved by the presence of Triton X-100 in the homogenization and centrifugation steps since the detergent prevents the sedimentation of microsomes with the nuclear fraction. This sedimentation of microsomes leads to considerable losses of polysomes, especially the larger ones. In the detergent-treated homogenate the integrity of the polysomes is threatened by various ribonucleases, some of which can be effectively inhibited by the addition of both heparin and yeast RNA. The remaining nuclease activity is counteracted by the endogenous ribonuclease inhibitor of the liver. In estradiol-treated roosters, sufficient endogenous inhibitor is present to inhibit its specific ribonuclease, but in control roosters there is not. This difference is due to a hormone-mediated increase in inhibitor level and decrease in nuclease level. Consequently, for an estrogenized rooster, the addition of both heparin and yeast RNA to the homogenate suffices to stabilize the polysomes, whereas control rooster liver homogenate needs supplementation with endogenous ribonuclease inhibitor. The cytosol of estrogenized rooster liver can be used as a crude inhibitor preparation. Rat liver cytosol is only partially effective; this may indicate a certain degree of species specificity of the inhibitor. The isolation procedure described also yields large polysomes from the livers of duck and Xenopus.

Terminal strand-switching of E. coli RNA polymerase transcribing a truncated DNA fragment.

When transcribing a restriction fragment containing the promoters and the first part of the rrnE operon of Escherichia coli, RNA polymerase holoenzyme starts exclusively on the promoters. Besides run-off transcripts, molecules longer than template-size are formed by terminal strand switch.

Estradiol-induced vitellogenin synthesis in duck liver.

We have studied the induction of vitellogenin by estradiol in duck liver. From the accumulation of vitellogenin in blood plasma we calculated that the rate of vitellogenin synthesis increases linearly with time for about 4 days after estradiol administration. Vitellogenin from chicken and duck cross-react immunologically and their mRNAs show only 7% sequence divergence. We could therefore determine vitellogenin mRNA content of duck liver using chicken vitellogenin cDNA as a hybridization probe. The number of vitellogenin mRNA molecules per hepatocyte increases from less than one in normal duck liver to 18 000 at 4 days after estradiol injection. The rate of vitellogenin synthesis in vivo is roughly proportional to vitellogenin mRNA content, although the data suggest a somewhat enhanced translation of vitellogenin mRNA at later times after hormone administration. Vitellogenin mRNA levels had returned to control values after 4 weeks after hormone administration. In the first 11 h after secondary administration of hormone vitellogenin mRNA accumulates at an only slightly higher rate than is observed after primary hormonal stimulation.

Binding of a bZip protein to the estrogen-inducible apoVLDL II promoter.

Activation of the very low density apolipoprotein II (apoVLDL II) gene in chicken liver by estrogen results in the binding of a variety of nuclear proteins including members of the steroid receptor superfamily and the bZip superfamily to the immediate 5' flanking region. In the present study, we have identified a bZip protein from chicken liver as one of the potential binding activities. Its cognate cDNA was cloned from an expression library using a recognition site DNA probe corresponding to part of the apoVLDL II promoter region. By footprinting and gel shift analysis with the recombinant protein from a prokaryotic expression system we have established that the protein binds to at least three different sites in the apoVLDLII promoter region. One of these sites partially overlaps with the major estrogen response element of the gene. Despite the proximity of their binding sites, the estrogen receptor and the bZip protein can bind simultaneously to the very region. Possible implications of this intimate arrangement of binding sites for the activation of the apoVLDL II promoter are discussed.

Estradiol-induced synthesis of vitellogenin. I. The isolation of large polysomes from estrogenized rooster liver.

Conventional procedures for the isolation of polysomes, applied to estrogenized rooster liver, fail to yield polysomes containing 30 or more ribosomes, the size expected for polysomes synthesizing the estradiol-induced protein vitellogenin. A new procedure characterized by early and selective removal of cell components ribonucleases allowed the isolation of polysomes with up to 55 ribosomes. Electron microscopy was used for the determination of polysome size and showed that the large polysomes were not aggregates.

Estradiol-induced synthesis of vitellogenin. III. The isolation and characterization of vitellogenin messenger RNA from avian liver.

The messenger RNA of the hormone-induced protein vitellogenin was isolated from the liver of estrogen-treated roosters. Starting from total polysomal RNA, the vitellogenin messenger was purified 67-fold by oligo (dT)-cellulose chromatography and sizing on a sucrose gradient. The messenger was translated in vitro into a 170 000 dalton polypeptide chain, having the immunochemical characteristics of vitellogenin. From electrophoretic and immunochemical analysis of the in vitro product of translation at least 63% of the messenger activity of the RNA preparation could be attributed to vitellogenin mRNA. Gel electrophoresis of the most purified fraction revealed residual contamination with the larger ribosomal RNA species. The molecular weight of the messenger RNA molecule, obtained by contour length measurements in the electron microscope, lies between 2.5 - 10(6) and 2.8 - 10(6).

Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha.

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.

Homology of Drosophila yolk proteins and the triacylglycerol lipase family.

Drosophila yolk proteins consist of a set of related proteins of 50,000 Mr. They are derived from slightly larger precursors by cleavage of a signal peptide. In this respect, they differ from the yolk proteins of other insects which are proteolytic fragments of precursors of 200,000 Mr or larger, termed vitellogenins and probably homologous to the vitellogenins of other egg-laying species. We report here a comparative amino acid analysis demonstrating that the Drosophila yolk proteins are non-homologous to the vitellogenin group of yolk proteins, but surprisingly are related to the triacylglycerol lipase family.

Nucleotide sequence of a chicken vitellogenin gene and derived amino acid sequence of the encoded yolk precursor protein.

The gene encoding the major vitellogenin from chicken has been completely sequenced and its exon-intron organization has been established. The gene is 20,342 base-pairs long and contains 35 exons with a combined length of 5787 base-pairs. They encode the 1850-amino acid pre-peptide of vitellogenin, which is the precursor of the mature yolk proteins, the serine-rich and heavily phosphorylated phosvitin and the lipovitellin. The 217-amino acid phosvitin polypeptide occupies an internal position (residue 1112 through 1328) within the vitellogenin molecule. The 125,000 and 30,000 Mr lipovitellin polypeptides are encoded by the sequences at the N-terminal and the C-terminal sides of the phosvitin section, respectively. The main features of the gene and protein sequences, and the evolutionary implications, are discussed.

Estrogen receptor in avian embryo and adult liver: estrogen receptor activation and dissociation kinetics of estradiol, ethynylestradiol, and moxestrol.

Estrogen receptor-enriched fractions from cockerel and embryo liver cytosol were obtained by precipitation at 0--35% ammonium sulfate saturation. At low ionic strength conditions, the embryo receptor sediments in sucrose density gradients mainly as an 8S entity. The cockerel receptor, however, sediments at 4.9S, unless an inhibitor of thiol proteases, iodoacetate, is included in the isolation medium. Moreover, the affinity of the cockerel receptor for moxestrol is doubled in the presence of iodoacetate and equals that of the embryo receptor (Kd = 1.4--1.8 x 10(-10) M). We conclude that thiol cathepsins from cockerel liver cause proteolytic degradation of the estrogen receptor. At temperatures between 0--28 C, the dissociations of moxestrol, ethynylestradiol, and estradiol follow monophasic kinetics. At 28 C, the half-times for the moxestrol-, ethynylestradiol-, and estradiol-receptor complexes are 65, 11, and 6 min, respectively. The chaotropic salt, NaSCN, reduces 6-fold the half-time of the moxestrol-receptor complex at 28 C (t 1/2 = 10 min). In 0.15 M KCl, the estrogen-receptor complex from embryo liver sediments at 4S. Incubation at 28 C, before the sucrose gradient analysis at 0 C, does not change the sedimentation coefficient. Sucrose gradient centrifugation carried out at 20 C results in the transformation to a 5S form of the moxestrol- as well as the estradiol-receptor complex. Our findings suggest that the estrogen receptors in the cytosol of avian liver and mammalian reproductive tissues are very similar. From both tissues, the native 8--10S form is extracted, and the receptors are transformed into the 5S form upon incubation with hormone at 20--30 C.

The chicken CCAAT/enhancer binding protein alpha gene. Cloning, characterisation and tissue distribution.

We present the cloning and sequencing of the gene encoding the chicken CCAAT/Enhancer Binding Protein alpha (cC/EBP alpha). The coding region and 1.5 kb of 5' flanking DNA from a CpG island. Comparison of the chicken C/EBP alpha sequence to the homologous proteins of other species reveals several evolutionary conserved regions. cC/EBP alpha mRNA expression is restricted to a subset of tissues with high expression in liver, lung and small intestine. Recombinant cC/EBP alpha binds to its cognate C/EBP binding site as a homodimer or as a heterodimer with the related cC/EBP beta/NF-M.

Estradiol-dependent transcription initiation upstream from the chicken apoVLDLII gene coding for the very-low-density apolipoprotein II.

We have investigated RNAs originating from the 5'-flanking region of the chicken very-low-density apolipoprotein II (apoVLDLII) gene. S1 nuclease mapping and primer extension experiments revealed two minor upstream transcription start points located 1105 and 1530 nucleotides in front of the apoVLDLII gene. Transcription starting at these points is dependent upon estradiol as is transcription from the major start points. The transcripts are polyadenylated, but are not detectable in polysomes. Run-on assays indicated that the low concentration of the upstream initiated transcripts is due both to low transcription levels and to low transcript stability. The sequence around the upstream start points does not show strong homologies with consensus sequences of promoters for eukaryotic protein encoding genes. Nevertheless, the upstream sequences are transcribed in vivo by RNA polymerase II.

Splicing pathways of the chicken apo very low density lipoprotein II (pre)messenger RNA.

The precursor-mRNA transcribed from the chicken apo very low density lipoprotein II gene was identified. This gene which is under full estrogen control and only expressed in the liver, possesses three introns. Splicing intermediates were characterized by hybridization with intron-specific probes, and by electron microscopy of R-loops. The introns appear to be excised in a non-obligatory order, but at different rates.

Overexpression of alcohol oxidase in Pichia pastoris.

The protein import capacity of peroxisomes in methylotrophic yeasts was studied using Pichia pastoris containing one or two extra copies of the gene encoding the peroxisomal protein alcohol oxidase. The alcohol oxidase overproduced in this strain was only partially imported and assembled into the active, octameric form of the protein. The excess remained in the cytosol as protein aggregates composed of monomers. These results are discussed in view of the possible application of peroxisomes as storage compartments for heterologous proteins.

The N-terminus of amine oxidase of Hansenula polymorpha contains a peroxisomal targeting signal.

Here we describe the identification of the targeting sequence of peroxisomal amine oxidase (AMO) of H. polymorpha. Deletion analysis revealed that essential targeting information is located within the extreme N-terminal 16 amino acids. Moreover, this sequence can direct a reporter protein to the peroxisomal matrix of H. polymorpha. The N-terminal 16 amino acids of AMO contain a sequence with strong homology to the conserved PTS2 sequence. Therefore, AMO is considered to be a PTS2 protein.