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Biomedical subjects

G Brewer

Publications and source records attributed to G Brewer.

At least 55 records · Page 3Linked to original sources
PubMed1996

Regulation of the mRNA-binding protein AUF1 by activation of the beta-adrenergic receptor signal transduction pathway.

In both cell culture based model systems and in the failing human heart, beta-adrenergic receptors ( beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3'-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (approximately 50%) in the abundance of beta1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of beta2-AR mRNA was first described, exposure to beta-AR agonist resulted in a significant increase in AUF1 mRNA and protein (approximately 100%). Conversely, agonist exposure significantly decreased (approximately 40%) beta2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3'-untranslated region of the human beta1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to beta1-AR 3'-untranslated region mRNA.

Base Sequence↗
PubMed1996

Bovine hereditary zinc deficiency: lethal trait A 46.

Bovine hereditary zinc deficiency, also referred to as Adema disease, is an autosomal recessive disorder which results in inadequate amounts of zinc being absorbed from the gastrointestinal tract and leads to a number of clinical abnormalities. Using semen from a homozygous affected bull and obligate heterozygote cows in embryo transfer studies, 7 offspring were obtained. These included 5 affected calves and 1 heterozygous carrier; the seventh calf died within 48 hours of birth undiagnosed. One unaffected, unrelated bull calf was raised as a control. All the calves were raised and maintained under similar management conditions designed to minimize secondary complications that would obscure the clinical and biochemical observations of a zinc deficient state. The first clinical manifestation of zinc deficiency was diarrhea, followed by skin lesions, poliosis, and a decreased ability to sustain a suckle reflex. Trace mineral analysis of plasma blood samples revealed that plasma zinc concentrations of all the calves were normal at birth; however, they gradually declined in affected calves over the course of 3-8 weeks postpartum to below 0.5 ppm. Biochemical analysis of serum samples showed alkaline phosphatase activity consistently paralleled changes in the plasma zinc concentrations. The oral administration of zinc acetate caused a reversal of all clinical, biochemical, and histologic abnormalities in affected calves. The study of these affected calves allows further insight into the biological role of zinc as well as provides an animal model for the continued investigation of the human homologue acrodermatitis enteropathica.

Acrodermatitis↗
PubMed1995

Characterization of adenosine-uridine-rich RNA binding factors.

The adenosine-uridine (AU)-rich sequences within the 3' untranslated region (UTR) of many short-lived mRNAs are important in their rapid degradation. We present evidence that human embryonic lung fibroblasts (W138) contain five major proteins of 70, 45, 40, 38, 32.5 kd, which specifically bind the AU-rich region of human granulocyte-macrophage colony-stimulating factor (GM-CSF) 3'UTR containing 7 x AUUUA motifs. The 40 and 38 kd proteins also bound the 3x and 5 x AUUUA cassettes and even more strongly bound to the AUUUUUUUA motif. All five of these proteins showed more abundant localization in the nucleus than the cytoplasm. The 32.5 kd protein was the major cytoplasmic AU-binding protein. Incubation with actinomycin D resulted in a marked increase in binding activity of 45, 40, 38, and 32.5 kd proteins in the cytoplasm, accompanied by decreased binding activity of the 32.5 kd protein in the nucleus. Antibody against heterogeneous nuclear ribonucleoprotein C (hnRNP C) immunoprecipitated the 40 and 38 kd proteins, and antibody against the AU-rich element RNA-binding protein (AUF1) immunoprecipitated the 45, 40, and 38 kd proteins. The present results not only demonstrated that hnRNP C are AU-binding proteins which are present in the cytoplasm as well as the nucleus, but another group of AU-binding proteins (AUF1 [45, 40, 38 kd], and 32.5 kd), which are not hnRNP, have characteristics related to those of hnRNPs. Taken together with our previous results (Akashi et al., 1994, Blood, 83:3182-3187), AU-binding factors including hnRNP C and AUF1, which bind more than 3 x AUUUA motifs, may be involved in rapid degradation of these transcripts. No significant quantitative changes of these proteins in their binding activity to AU-rich sequences occurred in response to several stimuli that stabilize GM-CSF mRNA, indicating that binding of these proteins to their cognate RNA is not responsible for the stabilization of these transcripts.

Adenosine↗
PubMed1995

Cortisol up-regulates corticotropin releasing factor gene expression in the fetal ovine brainstem at 0.70 gestation.

Glucocorticoids are important for the development of the central nervous system. In the ovine fetus, increased levels of plasma cortisol at term provide a stimulus to initiate parturition. CRF is central to this event in that it is one of the main modulators of the hypothalamic-pituitary-adrenal (HPA) axis. The purpose of the present study was to determine the effect of physiological increases in fetal plasma cortisol levels on corticotropin-releasing factor (CRF) gene expression in the developing ovine brain. Fetal plasma cortisol levels were chronically elevated at 0.70 gestation (100 days) to physiological levels found at 0.90 gestation (130 days; term 145 +/- 2 days) when glucocorticoid-induced maturational changes are known to occur in the HPA axis. The 3' end of the ovine CRF gene encodes 4 putative polyadenylation (poly(A)) signals that may post-transcriptionally regulate gene expression through stability, translation and localization of the mRNA in a temporal and spatial manner. To determine whether CRF mRNA levels or poly(A) site usage are differentially regulated by cortisol in a region-specific manner, we used an RNase protection assay with an antisense CRF RNA probe from the 3' coding and untranslated regions of the gene to quantify changes in mRNA levels in the hypothalamus (Hypo), hippocampal-amygdala complex (H and A), frontal cerebral cortex (FCC) and brainstem. Our novel finding was a 3.5-fold increase in CRF mRNA levels in the medulla oblongata of fetuses from the cortisol group compared to those from the saline group (P = 0.001). CRF mRNA levels in the Hypo, H and A and FCC did not change significantly in fetuses from the cortisol group.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1994

Characterization of cDNAs encoding the murine A+U-rich RNA-binding protein AUF1.

A+U-rich elements (ARE) serve to control the degradation of some proto-oncogene and lymphokine mRNAs. The protein, AUF1, which consists of two polypeptides of 37 and 40 kDa (p37 and p40, respectively) when purified from cytosol, has been implicated in ARE-directed mRNA turnover due to its binding to ARE. Molecular cloning of a cDNA (p37AUF1) corresponding to human p37 predicted a polypeptide containing two non-identical RNA recognition motifs (RRM) and a C-terminal Gln-rich domain [Zhang et al. Mol. Cell. Biol. 13 (1993) 7652-7665]. Two cDNAs, designated muAUF1-3 and muAUF1-7, were isolated from a murine fetal cDNA library, using as a probe, a fragment of the p37AUF1 cDNA encoding RRM1 and approximately half of RRM2. The muAUF1-3 open reading frame (ORF) was very homologous to human p37AUF1 with the greatest homology between the corresponding RRMs and the C-terminal Gln-rich motif. Clone muAUF1-7 was highly homologous to muAUF1-3, but was truncated within the region encoding the RNP-1 box in RRM2. Clone muAUF1-3 encoded 19 amino acids in RRM1 not encoded by either muAUF1-7 or human p37AUF1. Such alterations in sequence could modify the RNA-binding properties of these proteins and have concomitant effects on ARE-directed posttranscriptional processes.

Adenine↗
PubMed1994

Corticotropin releasing factor mRNA and peptide levels are differentially regulated in the developing ovine brain.

The regulation of CRF mRNA and protein in the developing ovine brain has been studied to assess the hypothesis that CRF is differentially regulated in the hypothalamus (Hypo), hippocampal-amygdala complex (H & A), frontal cerebral cortex (FCC) and brainstem (BS). We used a quantitative RNase protection assay and radioimmunoassay to determine mRNA and peptide concentrations, respectively, from the last third of gestation until term (i.e., from 95 to 142 days gestation (dg); term approximately 145 days). The major findings from this study are: (1) Hypothalamic CRF mRNA was increased by 2-fold in 140-142 dg fetuses compared to 128-138 and 95-123 dg fetuses; P = 0.016. (2) In the hypothalamus of 140-142 dg fetuses, there was a 2.5-fold increase in CRF mRNA derived from polyadenylation at poly(A) sites 2, 3 or 4; P = 0.005. (3) In 128-138 dg fetuses, CRF mRNA in the frontal cortex was 2-fold higher than in the other brain regions during this time period; P = 0.008. (4) CRF peptide concentrations in the Hypo were 2.5-fold higher in 140-142 dg fetuses compared to 95-106 and 128-138 dg fetuses; P = 0.007. (5) CRF peptide concentrations in the frontal cortex were 5.5-fold higher in 140-142 dg fetuses compared to fetuses at 95-106 dg; P = 0.004. (6) CRF peptide concentrations in the H & A were 5-fold higher in 140-142 dg fetuses compared to 95-106 dg fetuses; P = 0.029. The results from the present study demonstrate for the first time that CRF mRNA and peptide are differentially regulated in a region-specific manner during development.

Amygdala↗
PubMed1994

Effect of vesicular stomatitis virus matrix protein on host-directed translation in vivo.

Vesicular stomatitis virus infection causes a rapid and potent inhibition of both host transcription and translation. Recently, the viral matrix (M) protein was shown to inhibit host-directed transcription in vivo in the absence of any other viral component (B. L. Black and D. S. Lyles, J. Virol. 66:4058-4064, 1992). The goal of this study was to determine the effect of M protein on host-directed translation. In vitro-transcribed mRNAs encoding M protein and chloramphenicol acetyltransferase (CAT) were cotransfected into BHK cells to determine the effect of M protein expression on translation of CAT mRNA. The results presented here show that M protein did not inhibit host-directed translation of CAT mRNA. On the contrary, this study gave the unexpected result that M protein actually stimulated host-directed translation under the same conditions in which it potently inhibited host-directed transcription. Under these conditions, the combined effect on host gene expression was a greater-than-20-fold inhibition. Furthermore, the enhancement of host translation mediated by M protein was genetically correlated with M protein's ability to inhibit host transcription. Thus, the results of this study establish that M protein does not inhibit host protein synthesis under the same conditions in which it potently inhibits host transcription and suggest that the inhibition of transcription and that of translation by vesicular stomatitis virus require separate viral gene products.

Animals↗
PubMed1993

Endocardial pacing, cardioversion and defibrillation using a braided endocardial lead system.

The clinical efficacy and safety of a second-generation braided endocardial pacing, cardioversion and defibrillation lead system was evaluated in 25 patients with ventricular tachycardia (VT) or ventricular fibrillation (VF). The lead system consisted of two 8Fr active fixation endocardial leads each with pacing and defibrillation electrodes and a thoracic patch electrode. Monophasic and biphasic shocks were delivered using a triple-electrode configuration with a right ventricular common cathode and right atrial and thoracic patch anodes. VT and VF were electrically induced. Rapid VT (rate > or = 180 beats/min) and VF were initially terminated by 20 J (550 V) shocks and slow VT (rate < 180 beats/min) by 10 J (400 V) shocks. One hundred fourteen episodes (rapid VT/VF 73, slow VT 41) were treated with 128 shocks (monophasic 80, biphasic 48). Mean ventricular pacing threshold was 0.7 +/- 0.5 ms before and 0.9 +/- 0.5 ms after endocardial shock delivery (p > 0.2). Mean ventricular electrogram amplitude in sinus rhythm was 11.9 +/- 5.7 mV before and 11.4 +/- 5.1 mV after shock delivery (p > 0.2). Simultaneous monophasic endocardial shocks terminated 53% of VF episodes at < or = 20 J. Simultaneous biphasic shocks terminated 94% of all VF episodes at < or = 20 J (p < 0.03). Efficacy of > or = 10 J shocks for rapid VT/VF was greater for biphasic (92%) versus monophasic (74%) shocks (p < 0.05) at lower average shock energy (15 +/- 7 J vs 19 +/- 7 J, respectively, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1993

Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1.

The degradation of some proto-oncogene and lymphokine mRNAs is controlled in part by an AU-rich element (ARE) in the 3' untranslated region. It was shown previously (G. Brewer, Mol. Cell. Biol. 11:2460-2466, 1991) that two polypeptides (37 and 40 kDa) copurified with fractions of a 130,000 x g postribosomal supernatant (S130) from K562 cells that selectively accelerated degradation of c-myc mRNA in a cell-free decay system. These polypeptides bound specifically to the c-myc and granulocyte-macrophage colony-stimulating factor 3' UTRs, suggesting they are in part responsible for selective mRNA degradation. In the present work, we have purified the RNA-binding component of this mRNA degradation activity, which we refer to as AUF1. Using antisera specific for these polypeptides, we demonstrate that the 37- and 40-kDa polypeptides are immunologically cross-reactive and that both polypeptides are phosphorylated and can be found in a complex(s) with other polypeptides. Immunologically related polypeptides are found in both the nucleus and the cytoplasm. The antibodies were also used to clone a cDNA for the 37-kDa polypeptide. This cDNA contains an open reading frame predicted to produce a protein with several features, including two RNA recognition motifs and domains that potentially mediate protein-protein interactions. These results provide further support for a role of this protein in mediating ARE-directed mRNA degradation.

Amino Acid Sequence↗
PubMed1993

Developmental changes in renin gene expression in ovine kidney cortex.

The ontogeny of renin mRNA and renin content from renal cortical slices was studied in two groups of ovine fetuses at 92-94 days (0.64 gestation) and at 138-142 days (0.96 gestation), newborn lambs (0.4-2 days old), and adult sheep. Renal renin mRNA was identified by hybridization with a 32P-labeled full length rat renin cDNA. Renal renin content was measured as nanograms of angiotensin I generated per hour (active renin). There was a significant age effect on renin mRNA levels (F = 10.0, P < 0.001); values increase significantly between 0.64 and 0.95 g (P < 0.005), remain elevated in the newborns (P < 0.05), and subsequently decline in adulthood (P < 0.005). Likewise, renal renin content was significantly higher in late gestation fetuses and newborn lambs than in early gestation and adults (F = 8.3, P < 0.003). The renal renin content was strongly correlated with renin mRNA levels (R = 0.88, P < 0.0001). These results suggest that 1) the renin gene is developmentally regulated in the ovine kidney and 2) the renal content of active renin in basal conditions is regulated, at least in part, by events at the transcriptional level.

Animals↗
PubMed1993

Homeostasis of zinc in marginal human zinc deficiency: role of absorption and endogenous excretion of zinc.

Although biochemical and immunologic aspects of mild or marginal human zinc deficiency have been well characterized, there is a paucity of data regarding the effects of prolonged marginal zinc deficiency on zinc homeostasis. It appears that human beings are able to maintain zinc homeostasis by increasing efficiency of zinc absorption and decreasing endogenous excretion of zinc when they are subjected to short-term dietary zinc restriction. However, a mild deficiency of zinc in human beings under usual circumstances is an outcome of chronic exposure to diets low in zinc lasting for many months and years. Therefore, it is important to determine whether or not the adapted zinc homeostasis during the short duration of dietary zinc deprivation is also maintained during a prolonged period of dietary zinc restriction. We assessed the efficiency of zinc absorption as well as endogenous zinc excretion during a 6-month period of dietary zinc restriction (63.1 mumol/day) in human volunteers by using a stable zinc (70Zn). Prolonged marginal zinc deficiency did not impair the functional role of endogenous zinc excretion in zinc homeostasis, but efficiency of zinc absorption was not sustained and decreased in the majority of our volunteers when the zinc-restricted diet was continued for 6 months. Such altered mechanisms of zinc homeostasis suggest that chronic human zinc deficiency warrants reassessment of dietary zinc requirement to preserve normal zinc status in the subsets of population that are vulnerable to zinc deficiency.

Adult↗
PubMed1991

Oxygen affinity of hemoglobin and peripheral nerve degeneration in experimental diabetes.

Peripheral neuropathy remains a major complication of diabetes. Numerous etiological theories of metabolic and/or vascular disturbances have been suggested including decreased endoneurial oxygen tension with presumed tissue hypoxia. Increases in the affinity of hemoglobin for oxygen (Hb-O2 affinity) may also produce tissue hypoxia and such Hb-O2 affinity changes have been implicated in the pathogenesis of diabetic microangiopathy. In order to test whether affinity hypoxia might contribute to the development of diabetic peripheral neuropathy, we have utilized a rat model of high and normal Hb-O2 affinity produced by backcrossing animals with increased and decreased levels of 2,3-diphosphoglycerate (DPG). Diabetes was induced in ten high and ten low DPG animals with a tail vein injection of 55 mg/kg streptozotocin (STZ). Five animals in each group were treated with 2.4 U protamine zinc insulin (PZI)/day while the remaining animals were untreated. All rats were killed after 30 days, sections of tibial and sural nerve were rapidly removed and processed for teased fiber analysis. A minimum of 125 axons were assessed per nerve for E degeneration (myelin ovoids) using the classification developed by Dyck et al. Untreated animals, regardless of DPG levels, demonstrated 0% neuropathy. In contrast, all insulin-treated animals showed degeneration (0.4-17%) that inversely correlated with the DPG level (r = -0.59, P less than 0.04). The results of this study suggest that the level of RBC DPG (and presumably the Hb-O2 affinity) with its attendant effect on tissue oxygen release may play a role in the development of peripheral neuropathy in STZ-induced diabetic rats treated with insulin.

2,3-Diphosphoglycerate↗
PubMed1991

An A + U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro.

Transient expression of some proto-oncogenes, cytokines, and transcription factors occurs as a cellular response to growth factors, 12-O-tetradecanoylphorbol-13-acetate, antigen stimulation, or inflammation. Expression of these genes is mediated in part by the rapid turnover of their mRNAs. A + U-rich elements in the 3' untranslated regions of these mRNAs serve as one recognition signal targeting the mRNAs for rapid degradation. I report the identification of a cytosolic factor that both binds to the proto-oncogene c-myc A + U-rich element and specifically destabilizes c-myc mRNA in a cell-free mRNA decay system which reconstitutes mRNA decay processes found in cells. Proteinase K treatment of the factor abolishes its c-myc mRNA degradation activity without affecting its RNA-binding capacity. Thus, RNA substrate binding and degradation appear to be separable functions. These findings should aid in understanding how the cell selectively targets mRNAs for rapid turnover.

Adenine↗
PubMed1990

Drug-eluting collar--a new approach to reducing threshold.

This study evaluated the clinical performance of leads with a drug-eluting (less than 0.5 mg of dexamethasone sodium phosphate) collar (DEC) placed adjacent to a 4-mm2 Pt/Ir coated electrode (Telectronics model 030-368). Ten trailing tined ventricular silicone DEC leads and ten control leads (model 030-359) were implanted transvenously in patients of comparable age, sex, and indication for pacing. Early threshold rise, chronic thresholds (Vario), and lead impedances were monitored by pacer telemetry. The DEC lead had substantially lower thresholds in the first few weeks (P less than 0.0005) as well as chronically (P less than 0.025). The study has shown that a drug dose of less than 0.5 mg, released from a drug-eluting collar (DEC), is effective in reducing thresholds.

Aged↗
PubMed1989

Regulation of c-myc mRNA stability in vitro by a labile destabilizer with an essential nucleic acid component.

The turnover rates of some mRNAs vary by an order of magnitude or more when cells change their growth pattern or differentiate. To identify regulatory factors that might be responsible for this variability, we investigated how cytosolic fractions affect mRNA decay in an in vitro system. A 130,000 X g supernatant (S130) from the cytosol of exponentially growing erythroleukemia cells contains a destabilizer that accelerates the decay of polysome-bound c-myc mRNA by eightfold or more compared with reactions lacking S130. The destabilizer is deficient in or absent from the S130 of cycloheximide-treated cells, indicating that it is labile or is repressed when translation is blocked. It is not a generic RNase, because it does not affect the turnover of delta-globin, gamma-globin, or histone mRNA and does not destabilize a major portion of polysomal polyadenylated mRNA. The destabilizer accelerates the turnover of the c-myc mRNA 3' region, as well as subsequent 3'-to-5' degradation of the mRNA body. It is inactivated in vitro by mild heating and by micrococcal nuclease, suggesting that it contains a nucleic acid component. c-myb mRNA is also destabilized in S130-supplemented in vitro reactions. These results imply that the stability of some mRNAs is regulated by cytosolic factors that are not associated with polysomes.

Cytosol↗
PubMed1989

Exonuclease activity that degrades histone mRNA is stable when DNA or protein synthesis is inhibited.

The induction and repression of histone synthesis during the cell cycle are regulated, in part, by modulating histone mRNA stability. When DNA synthesis stops, histone mRNA seems to be destabilized, perhaps via an autoregulatory circuit triggered by cytoplasmic histones. We have used an in vitro mRNA decay system to determine whether differential histone mRNA turnover is linked to changes in the basal activity of cytoplasmic mRNA-degrading enzymes. The basal level of the polysome-associated exonuclease enzyme or enzymes that degrade histone mRNA was similar in untreated cells and in cells exposed to DNA or protein synthesis inhibitors. Histone mRNA decay was accelerated in reactions supplemented with histones and soluble cytoplasmic factor(s) (S130), but S130s from control and inhibitor-treated cells were indistinguishable in these assays. The data indicate that basal exonuclease activity is stable or constitutive. The putative factor(s) required for autoregulating histone mRNA decay also do not change appreciably when DNA or protein synthesis is inhibited. The implications of these results with regard to the autoregulation of histone mRNA turnover are discussed.

Cycloheximide↗