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Biomedical subjects

A R Brasier

Publications and source records attributed to A R Brasier.

53 records · Page 3Linked to original sources
PubMed1990

A family of constitutive C/EBP-like DNA binding proteins attenuate the IL-1 alpha induced, NF kappa B mediated trans-activation of the angiotensinogen gene acute-phase response element.

The gene encoding angiotensinogen, the glycoprotein precursor of the potent vasopressor peptide angiotensin II, is transcriptionally activated in hepatocytes during the acute-phase response through interactions of mutually cooperative glucocorticoid receptors and proteins that bind to an acute-phase response element (APRE) 5'-AGTTGGGATTTCCCAACC-3'. The APRE binds a family of constitutive proteins (BPcs) and a cytokine inducible protein (BPi) that is indistinguishable from nuclear factor kappa B (NF kappa B). The interactions of purified proteins with the APRE were studied by in vitro binding and in vivo transcriptional trans-activation assays. BPc is a family of heat-stable DNA binding proteins, the different sized members of which are capable of forming heterodimers. BPcs are recognized by anti-C/EBP antiserum and produce a footprint similar to bacterially expressed C/EBP on the APRE. BPi has a 4- to 5-fold greater affinity for the APRE than the BPcs, and contacts guanosine residues distinct from those contacted by the BPcs, demonstrating that these two classes of proteins contain functionally distinct DNA binding domains. Assays of APRE-luciferase reporter plasmids transfected into HepG2 cells show that a mutated APRE that binds only BPi functions as an IL-1 alpha inducible enhancer, whereas a mutated APRE that binds only BPc does not. The APRE mutant that binds the C/EBP-like BPcs to the exclusion of BPi functions as an uninducible basal enhancer both in the native context of the angiotensinogen gene and when multimerized and placed upstream of a minimal angiotensinogen promoter. The wild-type APRE that binds both BPi and BPc is less inducible by IL-1 alpha than the mutated APRE that binds only BPi. Gel shift competition assays demonstrate in vitro that the mechanism of transcriptional regulation by the APRE involves a competition between BPc and the inducible BPi for binding to the APRE. IL-1 alpha stimulation of hepatocytes leads to nuclear translocation of the NF kappa B-like BPi which competes with the constitutive C/EBP-like BPcs for overlapping binding sites on the APRE and thereby replaces weak transcriptional activators with a stronger one.

Angiotensinogen↗
PubMed1990

An inducible 50-kilodalton NF kappa B-like protein and a constitutive protein both bind the acute-phase response element of the angiotensinogen gene.

The rat angiotensinogen gene is induced in the course of the hepatic acute-phase response. We demonstrate that monocyte conditioned medium can stimulate transcription of a stably introduced reporter construct driven by 615 base pairs of the angiotensinogen 5'-flanking sequence, as well as the endogenous gene, in Reuber H35 cells. Point mutations of a cis-acting element, located 545 base pairs from the transcription start site and sharing sequence identity with known nuclear factor kappa B (NF kappa B)-binding sites, led to loss of cytokine inducibility. When cloned upstream of a minimal promoter, this cis-acting element imparted transcriptional inducibility by monocyte conditioned medium, interleukin-1, and tumor necrosis factor on a luciferase reporter gene in HepG2 cells. Two distinct proteins bound this element in vitro: a heat-stable, constitutively present, hepatic nuclear protein that gave rise to a DNase I-protected footprint covering the functionally defined element; and a binding protein of different mobility, induced by monocyte conditioned medium, which also recognized the NF kappa B-binding site of the murine kappa light-chain enhancer. UV cross-linking showed this inducible protein to have an apparent molecular mass of 50 kilodaltons, similar to that described for NF kappa B and distinct from the constitutively present protein that was shown by Southwestern (DNA-protein) blot to have a molecular mass of 32 kilodaltons. Methylation interference analysis showed that the induced species made contact points with guanine residues in the NF kappa B consensus sequence typical of NF kappa B. Induction of this binding activity did not require new protein synthesis, and 12-O-tetradecanoylphorbol-13-acetate could mimic the induction by cytokines. We thus provide direct evidence for involvement of NF kappa B or a similar factor in the hepatic acute-phase response and discuss the potential role of the presence of a constitutive nuclear factor binding the same cis element.

Acute-Phase Reaction↗
PubMed1990

The permissive role of glucocorticoids on interleukin-1 stimulation of angiotensinogen gene transcription is mediated by an interaction between inducible enhancers.

The acute-phase activation of the rat angiotensinogen (rAT) gene in liver cells is a transcriptional event mediated through an interleukin-1-inducible, NF kappa B-binding, cis-acting element (the acute-phase response element [APRE]). Using a cell culture model for the acute-phase response, we showed that the increase in angiotensionogen mRNA in H35 rat hepatoma cells requires costimulation with glucocorticoids and cytokines. Stably transfected rAT promoter-luciferase reporter genes were also activated by cytokines only in the presence of glucocorticoids. This permissive role of glucocorticoids is dependent on the expression of functional glucocorticoid receptors, because in HepG2 cells naturally deficient in such receptors, rAT gene-luciferase reporter constructs responded to interleukin-1 only when cotransfected with an expression vector for the glucocorticoid receptor. Point mutations in the two rAT gene glucocorticoid response elements located adjacent to the APRE led to loss of interleukin-1 inducibility. Induction of luciferase activity in transfected cells occurred even in the presence of cycloheximide, demonstrating that this synergistic response did not depend on new protein synthesis. Thus, a direct interaction between the interleukin-1-inducible NF kappa B-binding APRE and glucocorticoid response elements, located in cis, underlies the acute-phase activation of the rAT gene.

Angiotensinogen↗
PubMed1990

Synergistic enhansons located within an acute phase responsive enhancer modulate glucocorticoid induction of angiotensinogen gene transcription.

The hepatic transcription of the angiotensinogen gene is regulated by both glucocorticoids and cytokines generated as products of the acute phase reaction. We have identified a multimodular enhancer in the 5'-flanking region of the rat angiotensinogen gene that mediates these responses and consists of an acute phase response element (APRE) flanked on both sides by adjacent glucocorticoid response element consensus motifs (GREs). Induction of transcription by the cytokine interleukin-1 (IL-1) is glucocorticoid dependent and mediated through the APRE. The APRE binds in a mutually exclusive manner a cytokine/phorbol ester-inducible protein (BPi), indistinguishable from nuclear factor kB, and a family of constitutive liver proteins (BPcs) related to the heat-stable transcription factor C/EBP. Using mutated 5'-flanking sequences of the angiotensinogen gene fused to a firefly luciferase reporter gene transfected into hepatoblastoma (HepG2) cells, we have mapped enhanson sequences required for the transcriptional response to glucocorticoids. Two functionally distinct GREs are identified by deletion and site-directed mutagenesis, both of which mediate glucocorticoid-stimulated transcription in vivo. Glucocorticoid-induced transcription mediated by the angiotensinogen gene enhancer is, furthermore, dependent on the occupancy of the APRE by either the BPi or a member of the BPc family because a mutant APRE that binds neither BPi nor BPc exhibits an attenuated glucocorticoid responsiveness. Mutant APREs that permit exclusive binding of either BPi or BPc synergistically transmit the glucocorticoid response mediated by one or the other of the adjacent GREs. Thus, the induction of angiotensinogen gene transcription involves interaction between the glucocorticoid receptor and either one of the APRE-binding proteins: either the cytokine-inducible NFkB or the constitutive family of C/EBP-like proteins, bound to adjacent enhansons in a mutually synergistic enhancer complex.

Acute-Phase Proteins↗
PubMed1989

Multiple cis-acting DNA regulatory elements mediate hepatic angiotensinogen gene expression.

Angiotensinogen is the glycoprotein precursor of angiotensin II, an octapeptide hormone important for the regulation of blood pressure and volume homeostasis. The gene encoding angiotensinogen is expressed in liver and several other tissues, providing a model gene for understanding the role of cis-acting DNA control elements and trans-acting factors in tissue-type specific gene expression. To identify DNA control elements in the rat angiotensinogen gene we prepared an array of fusion genes consisting of either 5' or 3'-deleted sequences of the 5'-flanking region of the gene linked to a firefly luciferase reporter gene and analyzed the relative cellular specificity of expression of these genes after their introduction into hepato-carcinoma cells (Hep G2) that do express and placental cells (JEG-3) that do not express the endogenous angiotensinogen gene. Six transcriptionally active elements were found within 688 base pairs of 5'-flanking DNA. The interactions of DNA binding proteins with these elements was demonstrated by their specific protection to digestion with DNase I in the presence of liver cell extracts. The orientation and spatial requirements for transcription of two of the elements were analyzed further by the construction and expression of synthetic oligonucleotide cassettes incorporating the sequences of these elements when linked to a homologous (angiotensinogen) or a heterologous Simian virus 40 promoter and enhancer. One element located between 60 and 108 base pairs from the start of gene transcription functioned either as a silencer or an enhancer of transcription (SOAP box element), depending upon the distance from the angiotensinogen and viral gene promoters. Moreover, the SOAP box element demonstrated enhancer activity in JEG-3 cells when linked to the Simian virus 40 early promoter. An oligonucleotide mutation of the SOAP box element interfered with protein binding in a gel mobility shift assay and this mutant was transcriptionally inactive in both homologous and heterologous promoters. These observations indicate that expression of the angiotensinogen gene in liver cells is coordinately regulated by multiple cis-acting elements that interact with DNA binding proteins.

Angiotensinogen↗
PubMed1988

Hungry bone syndrome: clinical and biochemical predictors of its occurrence after parathyroid surgery.

The hospital course of 218 consecutive patients with primary hyperparathyroidism admitted over a three-year period for parathyroidectomy at the Massachusetts General Hospital was reviewed to determine the incidence and identify the risk factors for the development of the hungry bone syndrome. Twenty-five patients with the hungry bone syndrome were identified (12.6 percent). Compared to patients with uncomplicated metabolic responses to parathyroid surgery, these patients were older by a mean of 10 years; they had higher preoperative serum levels of calcium, alkaline phosphatase, N-terminal parathyroid hormone, and blood urea nitrogen; and their resected parathyroid adenomata were larger. The mean duration of hospitalization averaged three days longer in the group with hungry bone disease. Stepwise multivariate analysis of preoperative variables enabled the development of a discriminant function for prediction of postoperative hypocalcemia and hypophosphatemia. Identified predictive variables were volume of resected parathyroid adenoma, blood urea nitrogen, alkaline phosphatase, and age. When validated on an independent patient population, these readily obtainable preoperative clinical and laboratory parameters will allow identification of a subgroup of patients who are at greater risk for the development of the hungry bone syndrome following parathyroid surgery.

Adenoma↗
PubMed1988

Recovery of parathyroid hormone secretion after parathyroid adenomectomy.

Using a sensitive two-site immunoradiometric assay which detects only intact human PTH-(1-84), we studied the kinetics of PTH secretion in 19 patients undergoing unilateral neck exploration and removal of a parathyroid adenoma. Preoperative serum PTH values averaged 116 ng/L (normal, 12-65 ng/L). In 8 patients in whom intraoperative sampling was performed, clearance of PTH-(1-84) was rapid, with virtual disappearance of PTH by 120 min after clamping the vascular pedicle to the adenoma. Analysis of the rate of disappearance of PTH-(1-84) indicated an exponential decay with a half-life of 21 min. Thirteen of 19 patients had serum PTH values less than 1 ng/L within 8 h after parathyroidectomy. Recovery of PTH secretion from the suppressed nonsurgically manipulated parathyroid tissue occurred during the nadir of postoperative hypocalcemia. Serum PTH was greater than 10 ng/L in 16 of 19 patients 30 h after removal of the PTH adenoma. Therefore, the functional recovery of atrophic parathyroid tissue is more rapid than that of other endocrine tissues studied to date.

Adenoma↗
PubMed1987

Myopericarditis as an initial presentation of meningococcemia. Unusual manifestation of infection with serotype W135.

Acute meningococcemia is a dramatic clinical syndrome from infection with the gram-negative diplococcus, Neisseria meningitidis. Although pericarditis may complicate the course of meningococcemia, it is distinctly unusual as a presenting sign. A case of disseminated meningococcemia presenting as acute myopericarditis is reported. The serotype isolated, type W135, was a sporadic cause of N. meningitidis in the Boston area. Although the patient had meningitis, bacteremia, and myopericarditis, his course was uncomplicated with early institution of antibiotic therapy.

Humans↗
PubMed1986

Novel expression of the angiotensinogen gene in a rat pancreatic islet cell line. Transcriptional regulation by glucocorticoids.

Angiotensinogen gene expression has a broad tissue specificity. Whereas angiotensinogen mRNA is undetectable in normal rat pancreas, we have identified angiotensinogen mRNA in all tumors and cell lines derived from a rat islet cell line, RIN-r. A subclone with the highest angiotensinogen mRNA levels, 1056A, secreted N-glycosylated angiotensinogen. Angiotensinogen mRNA of 1056A cells was approximately 200 nucleotides longer than that of liver, and this was shown to be due to an extension of the 3'-untranslated region. Dexamethasone increased angiotensinogen mRNA levels approximately 9-fold above control, and this increase was linear over 110 h, indicating a half-life of greater than 55 h for angiotensinogen mRNA during dexamethasone induction. This effect of dexamethasone was inhibited by the glucocorticoid antagonist RU 38486. Dexamethasone increased angiotensinogen gene transcription approximately 5-fold in a nuclear run-on assay. These results demonstrate that dexamethasone induction of angiotensinogen mRNA levels in 1056A cells is due, at least in part, to a transcriptional response and that 1056A cells will be useful for the study of angiotensinogen gene regulation and the identification of glucocorticoid regulatory sequences.

Angiotensinogen↗
PubMed1986

Glucocorticoid and thyroid hormone regulation of angiotensinogen gene expression in a pancreatic islet cell line.

The renin-angiotensin system is an important regulator of blood pressure and volume homeostasis in mammals. Angiotensinogen, a precursor of the octapeptide angiotensin II and an effector of the renin-angiotensin system, is synthesized in numerous rat tissues. Angiotensinogen is expressed in an islet cell line (RIN 1056A) derived from a rat pancreatic tumor. Angiotensinogen mRNA detected by Northern analysis is abundant in the cell line and is approximately 200 bases longer than the mRNA isolated from rat liver, due to both a longer poly(A) tract and the use of a second polyadenylation site. Dexamethasone is a potent inducer of angiotensinogen mRNA, producing a progressive accumulation from 3 to 96 hr in culture (9-fold above control levels). The dexamethasone effect is competitively inhibited by the glucocorticoid antagonist RU486, and transcription rate assays using isolated nuclei indicate that the effect is primarily at the transcriptional level.

Angiotensinogen↗
PubMed1983

A guanine nucleotide-sensitive adenylate cyclase in the yeast Saccharomyces cerevisiae.

Adenylate cyclase in particulate extracts of Saccharomyces cerevisiae utilized either MnATP or MgATP as substrate. A mutation in the CYR1 gene, which codes for the catalytic unit of yeast adenylate cyclase (Matsumoto, K., Uno, I., and Ishikawa, T. (1983) Cell 32, 417-423), eliminated utilization of both MgATP and MnATP, indicating that a single enzyme was responsible for both activities. GTP and guanylyl-5'-imidodiphosphate stimulated yeast adenylate cyclase, while a GDP analog, guanosine-5'-O-(2-thiodiphosphate), competitively inhibited this stimulation. Thermal inactivation studies distinguished putative guanine-nucleotide regulatory protein (N) from the catalytic unit (C) of yeast adenylate cyclase. Yeast N, which conferred guanine nucleotide regulation and the ability to utilize MgATP on yeast C, was quickly inactivated by incubation of particulate extracts at 30 degrees C. In contrast, yeast C, which apparently utilized MnATP as substrate in the absence of a functional N protein, resisted inactivation at 30 degrees C. These observations suggested that physically distinct protein components mediated the catalytic activity of yeast adenylate cyclase and its regulation by guanine nucleotides. These findings indicate a striking homology between the adenylate cyclase systems of S. cerevisiae and those of vertebrate cells.

Adenylyl Cyclases↗
PubMed1982

Partial purification of presynaptic plasma membrane by immunoadsorption.

During transmitter release, synaptic vesicle membrane is specifically inserted into the nerve terminal plasma membrane only at specialized sites or "active zones." In an attempt to obtain a membrane fraction enriched in active zones, we have utilized the electric organ of the marine ray. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an antiserum to purified electric organ synaptic vesicles (anti-SV). The success of this immunoadsorption procedure was demonstrated by increased specific activities of bead-bound nerve terminal cytoplasmic markers and decreased specific activities of markers for contaminating membranes. To obtain a presynaptic plasma membrane (PSPM) fraction, we lysed the bead-bound synaptosomes by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with iodine before immunoadsorption, 10% of this label remained bead-bound after lysis, compared with 2% of the total protein, indicating an approximately fivefold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased approximately 30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic vesicle membrane since two-dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numerous nonvesicle components. Secondly, antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.

Acetylcholine↗
PubMed1982

Nerve terminal components from normal and denervated Narcine electric organ.

Exocytosis at the nerve terminal will only be fully understood when purified synaptic vesicles can be made to fuse with presynaptic plasma membrane in vitro. While the purification of synaptic vesicles from electric organ is now straightforward, isolation of the presynaptic plasma membrane presents a greater difficulty because of lack of specific markers. We have utilized pure synaptic vesicles in a novel way to overcome this difficulty. Antibodies raised to pure synaptic vesicles can be used to detect the presence of vesicle antigens. Thus, for example, we can show that synaptosome preparations isolated from electric organ by conventional procedures have about 5% of their protein in synaptic vesicles. The synaptic vesicle antigens and choline acetyltransferase both disappear from the synaptosome fraction after denervation of the electric organ, verifying that they are nerve terminal specific. Some of the synaptic vesicle antigens can be detected on the outside of the intact synaptosomes by binding rabbit anti-synaptic vesicle antibodies. Such antibody-coated synaptosomes will absorb specifically to goat anti-rabbit IgG attached to polyacrylamide beads. Lysis of such bead-bound synaptosomes leaves a plasma membrane fraction firmly attached to the beads. By raising antibodies to this membrane fraction an antiserum has been generated that binds to antigenic determinants in the electric organ. Some of these antibodies bind to the frog neuromuscular junction. The antigens recognized by this antiserum are, like synaptic vesicle antigens, lost on denervation. Unlike synaptic vesicle antigens, however, the antigens recognized by the anti-plasma membrane serum are present on the outside of resting frog nerve terminals.

Acetylcholinesterase↗
PubMedDate not supplied

Aldosterone synthase gene regulation by angiotensin.

Excessive aldosterone secretion in some hypertensive patients may result from abnormal aldosterone synthase (AS) gene regulation in response to changes in dietary sodium intake. We have utilized NCI-H295 cells, which exhibit stable angiotensin-induced aldosterone secretion, for transient transfections with murine AS/human growth hormone reporter constructs. An angiotensin response element increasing AS gene transcription during angiotensin stimulation appears to reside within the initial 425 nt of the murine AS promoter. We also noted the possible presence of a negatively-acting cis element between nt -425 and -1500. These studies provide an initial step toward characterizing molecular mechanisms by which angiotensin regulates AS gene transcription.

Adrenal Cortex Neoplasms↗
PubMedDate not supplied

Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines.

The enzymatic activity of firefly luciferase provides a sensitive, rapid means to assay transcriptional activity of regulated activation sequences of DNA when fused to the protein coding sequence of the luciferase gene. We have developed several modifications of the luciferase assay and have characterized certain parameters of the assay, resulting in optimization of conditions for the preparation and storage of cell lysates and establishment of substrate concentrations. These findings should be useful to investigators interested in applying the luciferase assay to studies of the transcriptional activity of test DNAs incorporating the luciferase reporter.

Animals↗