Two divergently transcribed genes of Dictyostelium discoideum are cyclic AMP-inducible and coregulated during development.

The cysteine proteinase 1 (CP1) gene of Dictyostelium discoideum encodes a developmentally regulated sulfhydryl proteinase. We characterized the DNA sequences upstream of the CP1 gene and found a second developmentally regulated gene, which we term DG17. The translational open reading frame of the DG17 gene encoded a 458-amino-acid cysteine- and lysine-rich protein of unknown function. In several regions, the cysteine and lysine residues were arranged in a manner characteristic of the zinc-binding domains found in proteins which interact with nucleic acids. During normal development, the DG17 and CP1 genes are coordinately activated late in aggregation. The addition of exogenous cyclic AMP (cAMP) induced the premature expression of both mRNAs. By measuring the rate of specific mRNA synthesis in isolated nuclei, we showed that cAMP acted at the transcriptional level to activate both genes. The two genes were separated by 910 nucleotides and were divergently transcribed. The intergenic region was predominantly composed of A + T residues except for four short G-rich regions. These sequences coincided with the positions of four nuclease-hypersensitive sites, which appear during aggregation when the DG17 and CP1 genes are transcribed (J. Pavlovic, E. Banz, and R. W. Parish, Nucleic Acids Res. 14:8703-8722, 1986). Two of the G-rich regions formed the core of two almost identical 80-nucleotide repeats located 220 and 320 nucleotides upstream of the CP1 gene. Using the Dictyostelium transformation system, we showed that a restriction fragment containing the intergenic region was capable of directing bidirectional transcription in a cAMP-dependent manner.

A sequence-specific RNA-binding protein complements apobec-1 To edit apolipoprotein B mRNA.

The editing of apolipoprotein B (apo-B) mRNA involves the site-specific deamination of cytidine to uracil. The specificity of editing is conferred by an 11-nucleotide mooring sequence located downstream from the editing site. Apobec-1, the catalytic subunit of the editing enzyme, requires additional proteins to edit apo-B mRNA in vitro, but the function of these additional factors, known as complementing activity, is not known. Using RNA affinity chromatography, we show that the complementing activity binds to a 280-nucleotide apo-B RNA in the absence of apobec-1. The activity did not bind to the antisense strand or to an RNA with three mutations in the mooring sequence. The eluate from the wild-type RNA column contained a 65-kDa protein that UV cross-linked to apo-B mRNA but not to the triple-mutant RNA. This protein was not detected in the eluates from the mutant or the antisense RNA columns. Introduction of the mooring sequence into luciferase RNA induced cross-linking of the 65-kDa protein. A 65-kDa protein that interacted with apobec-1 was also detected by far-Western analysis in the eluate from the wild-type RNA column but not from the mutant RNA column. For purification, proteins were precleared on the mutant RNA column prior to chromatography on the wild-type RNA column. Silver staining of the affinity-purified fraction detected a single prominent protein of 65 kDa. Our results suggest that the complementing activity may function as the RNA-binding subunit of the holoenzyme.

Insight into mammalian selenocysteine insertion: domain structure and ribosome binding properties of Sec insertion sequence binding protein 2.

The cotranslational incorporation of the unusual amino acid selenocysteine (Sec) into both prokaryotic and eukaryotic proteins requires the recoding of a UGA stop codon as one specific for Sec. The recognition of UGA as Sec in mammalian selenoproteins requires a Sec insertion sequence (SECIS) element in the 3' untranslated region as well as the SECIS binding protein SBP2. Here we report a detailed analysis of SBP2 structure and function using truncation and site-directed mutagenesis. We have localized the RNA binding domain to a conserved region shared with several ribosomal proteins and eukaryotic translation termination release factor 1. We also identified a separate and novel functional domain N-terminal to the RNA binding domain which was required for Sec insertion but not for SECIS binding. Conversely, we showed that the RNA binding domain was necessary but not sufficient for Sec insertion and that the conserved glycine residue within this domain was required for SECIS binding. Using glycerol gradient sedimentation, we found that SBP2 was stably associated with the ribosomal fraction of cell lysates and that this interaction was not dependent on its SECIS binding activity. This interaction also occurred with purified components in vitro, and we present data which suggest that the SBP2-ribosome interaction occurs via 28S rRNA. SBP2 may, therefore, have a distinct function in selecting the ribosomes to be used for Sec insertion.

An RNA-binding protein recognizes a mammalian selenocysteine insertion sequence element required for cotranslational incorporation of selenocysteine.

In mammalian selenoprotein mRNAs, the recognition of UGA as selenocysteine requires selenocysteine insertion sequence (SECIS) elements that are contained in a stable stem-loop structure in the 3' untranslated region (UTR). In this study, we investigated the SECIS elements and cellular proteins required for selenocysteine insertion in rat phospholipid hydroperoxide glutathione peroxidase (PhGPx). We developed a translational readthrough assay for selenoprotein biosynthesis by using the gene for luciferase as a reporter. Insertion of a UGA or UAA codon into the coding region of luciferase abolished luciferase activity. However, activity was restored to the UGA mutant, but not to the UAA mutant, upon insertion of the PhGPx 3' UTR. The 3' UTR of rat glutathione peroxidase (GPx) also allowed translational readthrough, whereas the PhGPx and GPx antisense 3' UTRs did not. Deletion of two conserved SECIS elements in the PhGPx 3' UTR (AUGA in the 5' stem or AAAAC in the terminal loop) abolished readthrough activity. UV cross-linking studies identified a 120-kDa protein in rat testis that binds specifically to the sense strands of the PhGPx and GPx 3' UTRs. Direct cross-linking and competition experiments with deletion mutant RNAs demonstrated that binding of the 120-kDa protein requires the AUGA SECIS element but not AAAAC. Point mutations in the AUGA motif that abolished protein binding also prevented readthrough of the UGA codon. Our results suggest that the 120-kDa protein is a significant component of the mechanism of selenocysteine incorporation in mammalian cells.

Induction of RNA editing at heterologous sites by sequences in apolipoprotein B mRNA.

An RNA editing mechanism modifies apolipoprotein B (apo-B) mRNA in the intestine by converting cytosine at nucleotide (nt) 6666 to uracil. To define the sequence requirements for editing, mutant apo-B RNAs were analyzed for the ability to be edited in vitro by enterocyte extracts. Editing was detected by a sensitive and linear primer extension assay. An upstream region (nt 6648 to 6661) which affected the efficiency of editing was identified. RNAs with mutations in this efficiency sequence were edited at 22 to 160% of wild-type levels. Point mutations in a downstream 11-nt mooring sequence (nt 6671 to 6681) abolished editing, confirming previous studies (R. R. Shah, T. J. Knott, J. E. Legros, N. Navaratnam, J. C. Greeve, and J. Scott, J. Biol. Chem. 266:16301-16304, 1991). The optimal distance between the editing site and the mooring sequence is 5 nt, but a C positioned 8 nt upstream is edited even when nt 6666 contains U. The efficiency and mooring sequences were inserted individually and together adjacent to a heterologous C in apo-B mRNA. The mooring sequence alone induced editing of the C at nt 6597 both in vitro and in transfected rat hepatoma cells. Editing at nt 6597 was specific, was independent of editing at nt 6666, and was stimulated to wild-type levels when the efficiency sequence was also inserted. Introduction of the mooring sequence into a heterologous mRNA, luciferase mRNA, induced editing of an upstream cytidine. Although UV cross-linking studies have previously shown that proteins of 60 to 66 kDa cross-link to apo-B mRNA, these proteins did not cross-link to the luciferase translocation mutants.

Molecular cloning of apobec-1 complementation factor, a novel RNA-binding protein involved in the editing of apolipoprotein B mRNA.

The C-to-U editing of apolipoprotein B (apo-B) mRNA is catalyzed by a multiprotein complex that recognizes an 11-nucleotide mooring sequence downstream of the editing site. The catalytic subunit of the editing enzyme, apobec-1, has cytidine deaminase activity but requires additional unidentified proteins to edit apo-B mRNA. We purified a 65-kDa protein that functionally complements apobec-1 and obtained peptide sequence information which was used in molecular cloning experiments. The apobec-1 complementation factor (ACF) cDNA encodes a novel 64.3-kDa protein that contains three nonidentical RNA recognition motifs. ACF and apobec-1 comprise the minimal protein requirements for apo-B mRNA editing in vitro. By UV cross-linking and immunoprecipitation, we show that ACF binds to apo-B mRNA in vitro and in vivo. Cross-linking of ACF is not competed by RNAs with mutations in the mooring sequence. Coimmunoprecipitation experiments identified an ACF-apobec-1 complex in transfected cells. Immunodepletion of ACF from rat liver extracts abolished editing activity. The immunoprecipitated complexes contained a functional holoenzyme. Our results support a model of the editing enzyme in which ACF binds to the mooring sequence in apo-B mRNA and docks apobec-1 to deaminate its target cytidine. The fact that ACF is widely expressed in human tissues that lack apobec-1 and apo-B mRNA suggests that ACF may be involved in other RNA editing or RNA processing events.

Properties of two isolated antigens associated with bovine leukemia virus infection.

We isolated an ether-resistant internal antigen and an ether-sensitive antigen previously described in relation to bovine leukemia virus infection. These two antigens have now been isolated by isoelectric focusing and concanavalin A affinity chromatography, respectively. The ether-resistant antigen exhibited isoelectric heterogeneity with a major peak at pH 7.2 and a minor peak at pH 6.2. Its molecular weight, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was 23,000 (p23), and it gave a sedimentation value of 2.3s. For material containing ether-sensitive antigen, analyzed by SDS-PAGE, protein staining revealed four components with molecular weights of 18,000, 25,000, 45,000, and 55,000. Two of these [45,000 (gp45) and 55,000 (gp55)] were stained by periodic acid-Schiff reagent. Isoelectric point and sedimentation value of the major glycoprotein (gp45) were pH 5.0 and 3.4s, respectively; no immunologic cross-reactivity was found between p23 and glycoprotein antigen.

Concanavalin A and the production of bovine leukemia virus antigen in short-term lymphocyte cultures.

The influence of the mitogen concanavalin A (Con A) on the production of bovine leukemia virus (BLV) antigen in short-term lymphocyte cultures was determined by means of a single radial immunodiffusion test. Con A did not affect viral antigen production in peripheral blood lymphocytes from 60% of both experimentally and naturally infected cattle. Antigen production was stimulated by Con A in lymphocytes from 28% of the cattle, but it was inhibited in lymphocytes from 12%. Similar results were also obtained with lymphocytes from both blood and lymph nodes from 10 cattle with lymphosarcoma and from 10 clinically normal cattle with histologically normal lymph nodes. In sheep and goats, Con A had no effect on lymphocytes from 50%, stimulated BLV production in 43%, and inhibited BLV production in 7%. These results indicated that lymphocytes should be cultured with and without Con A to identify every BLV-infected animal.

B-lymphocytes and T-lymphocytes in three types of bovine lymphosarcoma.

Lymphoid cells of peripheral blood, lymph nodes, and thymus from clinically normal cattle, cattle infected with bovine leukemia virus (BLV), and cattle with lymphosarcoma were characterized for T- and B-cell surface markers. B-cells were detected by the erythrocyte-antibody-complement (EAC) rosette test and the surface immunoglobulin (sig) immunofluorescence assay. Peripheral blood from BLV-infected cattle had a higher than normal percentage of B-cells by both EAC rosette and sig immunofluorescence assays. Lymphoid cells from tumorous lymph nodes of cattle with the adult type of lymphosarcoma had a higher than normal percentage of sig-bearing cells, but in the same cell preparation the EAC rosette-positive cells were fewer than sig-positive cells. T-cells were detected by the erythrocyte rosette test. The percentage of T-cells by this test in lymph nodes of adult type lymphosarcoma was lower than that in normal cattle. A distinctly lower than normal percentage of lymphocytes could be characterized as either B- or T-cells in lymph nodes thymus, and peripheral blood from the calf type and thymic type of lymphosarcoma.

Influence of breast and formula feeding on hepatic concentrations of apolipoprotein and low-density lipoprotein receptor mRNAs.

We tested the hypothesis that breast and formula feeding differentially affect hepatic mRNA concentrations for LDL receptor (LDL-R) and apolipoproteins A-I, B and E in infant baboons during the preweaning period. The mRNA concentrations were measured in liver biopsies obtained prior to weaning at 14 weeks from 43 baboons that were either breast-fed (n = 17) or fed formulas with a high (n = 12) or low (n = 14) polyunsaturated/saturated (P:S) fat ratio. Breast-fed baboons had 99% higher LDL-R mRNA concentrations compared with infants fed formulas, but there were no differences among breast and formula-fed baboons in mRNA concentrations of apolipoproteins A-I, B or E. The fatty acid P:S ratio of the formulas did not affect hepatic LDL-R or apolipoprotein mRNA concentrations. These results suggest that breast-feeding increases LDL-R gene expression even though breast milk is higher in cholesterol and saturated fat compared with formulas.

The interaction between respiratory and autonomic function during sleep-related changes in pharyngeal airway patency.

Sleep-related changes in pharyngeal function result in an increased resistance to airflow and in some people complete pharyngeal occlusion. Clinically, pharyngeal occlusion causes obstructive sleep apnoea syndrome (OSA). This is a prevalent disorder, which is an independent risk factor for the development of systemic hypertension. Several mechanisms contribute to the sleep-related changes in pharyngeal function in both health and disease, including a reduction in respiratory-related muscle activation, and an increase in latency of the pharyngeal reflex to negative intralumenal pressure. Arousal from sleep causes increases in ventilation and autonomic cardiovascular function that far exceed physiological requirements--the so-called 'waking reflex'. In patients with OSA the waking reflex is augmented either by hypoxemia, hypercapnia, or large swings in intrathoracic pressure. How these factors interact to cause the acute surges in heart rate and systemic blood pressure that occur at the termination of an apnoea will be reviewed, together with the longer term consequences of pharyngeal occlusion during sleep.

Baboon lecithin cholesterol acyltransferase (LCAT): cDNA sequences of two alleles, evolution, and gene expression.

Lecithin cholesterol acyltransferase (LCAT) is a key enzyme of cholesterol metabolism that catalyzes esterification of cholesterol for packaging in high-density lipoprotein (HDL) particles. In this study, we cloned and sequenced LCAT cDNA from baboon, a nonhuman primate model of atherosclerosis. LCAT sequences have been highly conserved over approximately 25 million years since the divergence of the baboon and human lineages. The baboon and human sequences are 97% identical at the nucleotide (nt) level and 98% identical at the amino acid (aa) level. Only 18% of the nt substitutions change the aa sequence (nonsynonymous substitutions). The substitutions between baboon and human LCAT do not alter key functional sites including the interfacial substrate active site, asparagine-linked glycosylation sites, or sites at which rare mutations cause human familial LCAT deficiencies. We also sequenced LCAT cDNA for a less common allele that is associated with higher LCAT activities and altered lipoprotein phenotypes. There were no sequence differences between the two alleles, which suggests that genotypic effects are most likely due to allelic differences in gene expression. The tissue specificity of LCAT expression was investigated using an RNase protection assay calibrated with known amounts of synthetic human LCAT RNA. In a survey of baboon tissues, the highest levels of LCAT mRNA were found in the cerebellum and liver and trace amounts in the ileum, spleen and cerebral cortex.

Cognitive organization of impressions: effects of incongruency in complex representations.

Two experiments investigated the organization in memory of expectancy-congruent and expectancy-incongruent information pertaining to multiple trait concepts in an impression-formation task. In Experiment 1, when multiple trait concepts were represented in the information describing the target person, both congruent and incongruent items reflecting the same trait concept were stored together and were directly associated in memory, and both types of items were recalled equally well. In Experiment 2, when only one trait concept was represented in the information, incongruent items were recalled with higher probability than congruent items, and the latter were not directly associated in memory. Results suggest that with increasing categorical complexity of stimulus information, processes are invoked that do not occur in simpler impression-formation contexts. Implications for theoretical models of person memory are discussed.

Quantification of lumbar intradiscal deformation during flexion and extension, by mathematical analysis of magnetic resonance imaging pixel intensity profiles.

STUDY DESIGN: A magnetic resonance imaging study of the internal kinematic response of normal lumbar intervertebral discs to non-weight-bearing flexion and extension. OBJECTIVES: To quantify the pattern of magnetic resonance imaging pixel intensity variation across discs, and noninvasively monitor displacement of the nucleus pulposus during sagittal-plane movements. SUMMARY OF BACKGROUND DATA: Invasive techniques used to study intradiscal movements of the nucleus pulposus have suggested that it moves posteriorly during flexion and anteriorly during extension. A noninvasive study based on magnetic resonance images gave similar results for normal young women. Quantification has been problematic, and the invasive procedures may have altered disc dynamics. METHODS: Ten male subjects (age, 21-38 years) with healthy backs were positioned in a magnetic resonance imaging portal with their lumbar spine stabilized in flexion and extension by supporting pads. For each disc, a T2-weighted image was obtained, as was a computer-generated profile of pixel intensities along a horizontal mid-discal transect. Mathematical curve-fitting regression analysis was used to characterize the shape of the intensity profile and to compute the point of maximum pixel intensity. RESULTS: A single equation fitted the profile for all normal discs. The intensity peak shifted posteriorly during flexion, anteriorly during extension. CONCLUSIONS: Automated mathematical modeling of magnetic resonance imaging pixel data can be used to describe the fundamental shape of the pixel intensity profile across a normal lumbar disc, to determine the precise location of the site of maximum pixel intensity, and to measure the movement of this peak with flexion and extension. This technique may be of value in recognizing incipient degenerative changes in lumbar discs.

Insulin inhibits changes in the phospholipid profiles in sciatic nerves from streptozocin-induced diabetic rats: a phosphorus-31 magnetic resonance study.

Sciatic nerve phospholipids obtained from insulin-treated streptozocin-induced diabetic, non-treated streptozocin-induced diabetic, and healthy, control male Sprague-Dawley rats after eighteen weeks of diabetes were studied by 31P NMR spectrometry. Eleven phospholipids resonances were identified as follows: Phosphatidic acid (Chemical shift, 0.30 ppm), dihydrosphingomyelin (0.13 ppm), ethanolamine plasmalogen (0.07 ppm), phosphatidylethanolamine (0.03 ppm), phosphatidylserine (-0.05 ppm), sphingomyelin (-0.09 ppm), lysophosphatidylcholine (-0.28 ppm), phosphatidylinositol (-0.30 ppm), alkylacylglycerophosphorylcholine (-0.78 ppm), choline plasmalogen (-0.80 ppm), and phosphatidylcholine (-0.84 ppm). Diabetic rats showed that phosphatidylcholine was significantly elevated (p < 0.05), and ethanolamine plasmalogen and choline plasmalogen were significantly lower when compared with both control and insulin treated rats. The choline ratio (choline-containing phospholipids over noncholine phospholipids) was significantly elevated in the diabetic group, when compared with both control and insulin-treated groups. The ethanolamine ratio (ethanolamine-containing phospholipids over nonethanolamine phospholipids) and the ratio of the ethanolamine ratio over the choline ratio, was significantly elevated in the control and the insulin-treated groups when compared with the diabetic group. The presence of phosphatidic acid and the significance in phosphatidylcholine and ethanolamine plasmalogen, suggested that insulin had a role in the phosphatidylcholine metabolism in the rat nerve.

RNA binding proteins and selenocysteine.

Selenocysteine is incorporated into protein by a complex co-translational mechanism that involves both cis and trans acting factors. Among the trans-acting factors are RNA binding proteins that interact with the selenoprotein 3' UTRs at a sequence known as the selenocysteine insertion sequence (SECIS). These factors are generally referred to as SBPs, and in this review we will discuss the history of the SBPs, and give a detailed description of the recently identified SBP2 which is the only SBP known to be required for Sec insertion. The mechanism by which SBP2 may be involved in this process will be discussed.

Selenocysteine incorporation directed from the 3'UTR: characterization of eukaryotic EFsec and mechanistic implications.

The mechanism of selenocysteine incorporation in eukaryotes has been assumed for almost a decade to be inherently different from that in prokaryotes, due to differences in the architecture of selenoprotein mRNAs in the two kingdoms. After extensive efforts in a number of laboratories spanning the same time frame, some of the essential differences between these mechanisms are finally being revealed, through identification of the factors catalyzing cotranslational selenocysteine insertion in eukaryotes. A single factor in prokaryotes recognizes both the selenoprotein mRNA, via sequences in the coding region, and the unique selenocysteyl-tRNA, via both its secondary structure and amino acid. The corresponding functions in eukaryotes are conferred by two distinct but interacting factors, one recognizing the mRNA, via structures in the 3' untranslated region, and the second recognizing the tRNA. Now, with these factors in hand, crucial questions about the mechanistic details and efficiency of this intriguing process can begin to be addressed.

Intragastric pH monitoring.

Buffering of intragastric pH is an accepted treatment modality for prophylaxis against the development of gastric stress ulcers. This method of prophylaxis is commonly based on the pH value acquired by measurement of gastric aspirate. Recent literature suggests pH measurement techniques that involve gastric aspirate specimens have many flaws. The purpose of this study was to compare gastric pH measurements with the use of a nasogastric sensor, meter system, and pH-sensitive test paper. Fifteen hundred paired serial measurements of intragastric pH were obtained on 19 thermally injured patients (16 men and three women, ages 23 to 79 years, total body surface area burn 25% to 80%). A double-lumen tube containing an antimony/graphite pH sensor incorporated into the tip of the tube was inserted with the use of a standard technique. Each tube was in place an average of 5.7 days (range 1 to 15 days). Patients were randomized into two groups. The first group (six patients) received non-acid-buffering prophylaxis therapy. The second group (13 patients) received standard antacid or antacid/H2 histamine-blocking agent combination prophylaxis therapy. Analysis of the 539 paired measurements for the non-acid-buffering revealed a correlation coefficient of r = 0.532. The 961 measurements from the group receiving gastric acid buffering revealed a correlation coefficient of r = 0.569. Paired t test values for the sample showed a significant difference (18.52, p < 0.0000) between measurement techniques.

Bovine leukemia virus-associated antigens in lymphocyte cultures.

Short-term lymphocyte cultures from bovine leukemia virus (BLV)-infected cattle were tested for BLV-associated antigens at various times after incubation. Several immunologic methods were used, including fluorescent antibody tests, immunodiffusion, and radial immunodiffusion. Antigens were not detected in uncultured lymphocytes. The BLV-associated antigens were detected as early as 3 hours, with maximum antigen production occurring at 18 to 24 hours after incubation. These results indicate that culturing of lymphocytes in vitro is necessary for the expression of the virus.

Burn dressings: a critical indicator for patient care classification in burn units.

Nursing services consume approximately 60% of a hospital personnel budget, requiring justification for staffing levels and manpower expenses. The lack of an adequate level of nursing care affects the operation of the entire health care team. The purpose of this descriptive study is to establish the mean tasking time to apply burn dressings. The number of nursing staff required to complete a burn dressing application is dependent on the location of injury and the ability of the patient to cooperate. Establishing the essential mean tasking time for routine post-operative dressing assists in the production of accurate reporting of nursing manpower requirements.