Xenopus fibrinogen: characterization of the mRNAs for the three subunits.

We purified and characterized the mRNAs coding for each of the three subunits of Xenopus fibrinogen. Purification was accomplished by electrophoretic separation of liver polyadenylated RNA in a fully denaturing gel, followed by recovery of the RNA from the gel via transfer to an ion-exchange membrane. This procedure yielded fractions which were highly enriched for the mRNAs for each of the fibrinogen chains. The fibrinogen mRNAs were identified by two methods: (i) in vitro translation followed by subunit-specific cleavage with the proteases thrombin and batroxobin; and (ii) cross-hybridization with cDNA clones for individual subunits of rat fibrinogen. The results demonstrate that the A alpha and gamma chains of frog fibrinogen are each coded by a single mRNA species. The A alpha mRNA is ca. 3,100 nucleotides in length, which is nearly twice the minimum size required to code for the A alpha precursor polypeptide. The gamma chain mRNA comprises about 1,600 bases and includes only a small untranslated region. In contrast, the B beta subunit is synthesized from two mRNAs, one of which is 2,500 and the other 1,800 nucleotides long. The 2,500-base mRNA includes a large noncoding region, whereas the smaller one is near the minimum required size. The larger B beta mRNA is ca, fivefold more abundant that the smaller species.

Coordinate regulation of fibrinogen subunit messenger RNA levels by glucocorticoids in primary cultures of Xenopus liver parenchymal cells.

Fibrinogen synthesis is specifically induced by a synthetic glucocorticoid, dexamethasone, in primary liver parenchymal cell cultures of the frog Xenopus laevis. Here we demonstrate that this increase in the level of fibrinogen protein production is accompanied by an induction in the three mRNAs coding for the fibrinogen subunits, designated A alpha, B beta, and gamma. The stimulation of fibrinogen mRNA levels appears to be mediated by the glucocorticoid receptor, because 1) the dose-response relationship parallels the reported affinity of dexamethasone for the Xenopus glucocorticoid receptor; and 2) the induction is blocked by RU 486, a potent antiglucocorticoid. All three subunit mRNA levels are induced coordinately by the hormone. The response is characterized by a detectable increase as early as 2-4 h after dexamethasone addition, continuing to a final 10- to 30-fold increase over basal levels by 60 h. The induction is specific for the fibrinogen mRNAs; total cellular RNA content and the levels of other mRNAs are unaffected by the hormone. Dexamethasone-mediated stimulation of A alpha and B beta mRNA production occurs in the absence of protein synthesis, whereas increased production of gamma mRNA is completely blocked under the same conditions. Thus, the A alpha and B beta genes are probably regulated at least in part by direct transcriptional activation by glucocorticoid-receptor complexes. Induction of the gamma gene is dependent on newly synthesized or labile proteins, which could be required for either transcription or posttranscriptional processes. These data suggest that different proteins are involved in regulation of the three fibrinogen genes.

Novel accessory factor-binding site required for glucocorticoid regulation of the gamma-fibrinogen subunit gene from Xenopus laevis.

Glucocorticoids induce gene expression by binding to an intracellular receptor that interacts with genomic DNA and stimulates transcription of specific genes. The consensus DNA-binding site for the glucocorticoid receptor, called a glucocorticoid response element (GRE), is GGTACAnnnTGTTCT. In the classical model, binding of the receptor as a dimer to the two halves of the GRE is required for activation of transcription. For some glucocorticoid-regulated genes, additional DNA-binding proteins called accessory factors are necessary for hormonal responsiveness. We have identified a new factor required for glucocorticoid-induced expression of the gamma-fibrinogen subunit gene from the frog Xenopus laevis. Transfection of cloned DNA fragments into primary Xenopus hepatocytes showed that the DNA between 163 and 187 bp upstream of the transcription initiation site is essential for hormonal activation. A single complex forms when this small region of DNA is incubated in vitro with Xenopus liver nuclear proteins. The protein recognition site has been narrowed to AAGAGTTAA, a sequence not previously described as a transcription factor-binding site. We have named the protein(s) bound to this sequence Xenopus glucocorticoid receptor accessory factor (XGRAF). In addition to the XGRAF-binding site, glucocorticoid regulation of the gamma-fibrinogen gene requires at least three nearby GREs, each of which is a poor match to the consensus GRE. The position of the binding site for XGRAF overlaps the putative upstream half of the most important GRE. Models are presented to show possible ways that the novel accessory factor and the glucocorticoid receptor could act through closely juxtaposed sites on the DNA.

Heterodimerization between the glucocorticoid receptor and the unrelated DNA-binding protein, Xenopus glucocorticoid receptor accessory factor.

The adrenal steroid hormones, glucocorticoids, control many physiological responses to trauma, including elevated synthesis of fibrinogen, a major blood-clotting protein. Glucocorticoid regulation of the gamma-fibrinogen subunit gene in Xenopus laevis is mediated by a binding site for Xenopus glucocorticoid receptor accessory factor (XGRAF) and a contiguous glucocorticoid response element (GRE) half-site. Here, we characterize the protein:DNA complex formed by a cooperative interaction between XGRAF, GR, and the DNA. We demonstrate that the complex contains XGRAF by competition in a gel shift assay. The presence of GR is established by two criteria: 1) size dependence of the XGRAF:GR:DNA complex on the size of the GR component and 2) interference with complex formation by GR antibody. Cooperative binding of XGRAF and GR to the DNA was quantitated, showing that GR favors binding to XGRAF:DNA compared with free DNA by a factor of 30. The cooperative interaction between XGRAF and GR can occur on nicked DNA but is disrupted when 1 bp is inserted between the XGRAF binding site and half-GRE. Significantly, this loss of physical association in vitro correlates with loss of XGRAF amplification of GR activity in transiently transfected primary Xenopus hepatocytes. The simplest explanation for cooperativity between XGRAF and GR is formation of a DNA-bound heterodimer of these two proteins. This mechanism represents a new mode of transcriptional regulation in which GR and a nonreceptor protein form a heterodimer, with both partners contacting their specific DNA sites simultaneously.

Low frequency of elevated prothrombin times in patients with lupus anticoagulants when using a recombinant thromboplastin reagent: implications for dosing and monitoring of oral anticoagulant therapy.

Many patients with lupus anticoagulants (LA) are treated with oral anticoagulation and monitored using the international normalised ratio (INR) derived from the prothrombin time (PT). Recent reports have produced conflicting conclusions about the extent to which LA interferes with PT determination. The degree of anticoagulation may be overestimated in a patient whose LA affects the PT. A number of reports conclude that specific thromboplastin reagents containing recombinant tissue factor are sensitive to the presence of LAs and should not be used to monitor oral anticoagulant therapy in these patients. These studies were performed on orally anticoagulated patients. The present retrospective study on 400 patients with LAs who were not receiving therapeutic anticoagulation was performed to ascertain the frequency of prolonged PT in these patients when using Innovin recombinant thromboplastin. Only 17 (4.3%) out of 400 had prolonged PT in the presence of LA. As this is a low prevalence, and not all patients with LAs will require anticoagulant therapy, it is concluded that baseline INR determination should be used to highlight the need to monitor individual patients with LA-insensitive reagents. As the use of moderate-intensity oral anticoagulation for patients with LAs and previous thrombosis is receiving wider acceptance, an informed approach to anticoagulant monitoring will reduce the possibility of under-anticoagulating patients receiving this therapy.

Coordinate transcriptional regulation of the three fibrinogen subunit genes by glucocorticoids in cultured primary liver cells from Xenopus laevis.

Xenopus laevis primary hepatocytes in culture are induced by glucocorticoid hormones to synthesize and secrete fibrinogen. The increase in production of the protein is preceded by a 10-to 30-fold elevation of the mRNAs coding for the three fibrinogen subunits, A alpha, B beta, and gamma. To analyze the mechanisms underlying this coordinate control of independent genes in a common regulatory network, we show here that the steroid hormone induced simultaneous activation of transcription of the three fibrinogen subunit genes. Using an optimized transcription run-on assay for nuclei from Xenopus primary liver cells, we demonstrate that glucocorticoids rapidly stimulated transcription of the A alpha fibrinogen subunit gene by 15- to 20-fold, the B beta gene by 5- to 10-fold, and the gamma gene by 5- to 15-fold. The three genes exhibited a highly concerted response to the hormone, in which maximal stimulation occurred by 30 min and was maintained for at least 16 h. Blocking new protein synthesis before hormone treatment reduced total transcription by 45% and partially inhibited specific hormonal induction of all three fibrinogen subunit genes. The effect of glucocorticoids on fibrinogen transcription, therefore, was dependent in part on ongoing protein synthesis, suggesting that hormonal stimulation uses already synthesized stable factors, but also requires labile or newly synthesized factors for the full effect.

cDNA and amino-acid sequences and organization of the gene encoding the B beta subunit of fibrinogen from Xenopus laevis.

Fibrinogen, the major blood-clotting protein, is made up of three chains, A alpha, B beta and gamma, which are synthesized and secreted by the liver. In this communication, we describe the complete cDNA sequence, deduced amino acid (aa) sequence and organization of the gene encoding the B beta subunit of fibrinogen from Xenopus laevis (Xl). The cDNA representing the predominant form of the B beta mRNA comprises 2390 nucleotides (nt), with an open reading frame of 1467 nt coding for a 488-aa protein. The percent identity between Xl B beta and that of other animals ranges from 50% for lamprey to 66% for human. The Xl B beta gene consists of nine exons, one more than found in the human gene. The exon/intron boundaries in the frog and human B beta genes are in exactly conserved positions, except for junctions in the highly variable fibrinopeptide-encoding regions. Three of the exon/intron boundaries in the Xl B beta gene are also analogous to ones in A alpha and gamma genes of other species, supporting the notion of a close evolutionary relationship between the genes for all three subunits. This analysis of B beta from an amphibian provides the first complete description of the arrangement of exons and introns in any fibrinogen subunit gene from a non mammal and gives insight into the most highly conserved aspects of fibrinogen protein structure and gene organization.

Inhibitor to factor VIII in mild haemophilia.

Factor VIII inhibitors in mild haemophilia are uncommon and the management of such patients is controversial. The development of a persistently high responding F VIII inhibitor in a mild haemophiliac is reported and the behaviour of the inhibitor discussed in the context of the various therapeutic regimes employed for symptomatic management. When inhibitor titres were low, endogenous F VIII stimulation, by DDAVP, was less immunogenic than the administration of exogenous F VIII concentrates. This inhibitor displayed characteristics of an autoantibody, and was characterised as an immunoglobulin of IgG subtype.

Estrogen induction of a 45 kDa secreted protein coordinately with vitellogenin in Xenopus liver.

In the frog Xenopus laevis, vitellogenin is the major estrogen-induced protein in the liver. We have characterized an additional secreted protein, of 45,000 Da and designated Ep45, which is also responsive to estrogen treatment. Like vitellogenin, Ep45 is not normally found in the plasma nor synthesized by the liver of the male frog. Its synthesis increases 6-fold between days 2 and 8 following a single 2 mg injection of estradiol-17 beta. For comparison, we have also studied a third estrogen-regulated protein, Ep20, with a molecular weight of approximately 20,000. This protein exhibits a different set of characteristics with regard to hormone responsiveness. Ep20 is synthesized in the liver of normal males and therefore is not absolutely hormone-dependent. Its level increases only about 4-fold following estrogen stimulation. The messenger RNAs for both Ep45 and Ep20 have been identified and purified, using a high-resolution RNA fractionation technique. By this procedure, it was possible to demonstrate that following high doses of estrogen the predominant mRNAs in the liver are those coding for vitellogenin, Ep45 and Ep20. Thus estrogen suppression of virtually all other liver proteins appears to act at the messenger RNA level for intracellular as well as secreted proteins.

Isolation and characterization of cDNA clones for the gamma subunit of Xenopus fibrinogen, the product of a coordinately regulated gene family.

Fibrinogen, the major structural protein involved in blood coagulation, is synthesized and secreted by the liver. In the frog Xenopus laevis, fibrinogen production is dramatically induced by glucocorticoids. The hormonal stimulation requires synthesis of three separate subunits, designated A alpha, B beta, and gamma. For investigation of the molecular mechanisms underlying this coordinate induction, we have isolated cDNA clones for the subunits of Xenopus fibrinogen. In this communication we describe the identification of clones for the gamma chain. Initially, a Xenopus liver cDNA library in pBR322 was screened with a rat gamma chain cDNA and a clone representing half of the 1600-base frog gamma mRNA was identified. This clone was shown to be complementary to gamma mRNA by hybrid selection of mRNA that translated in vitro into the gamma polypeptide. A clone about 1460 base pairs in length was then isolated from a Xenopus liver lambda gt10 cDNA library and subcloned into Bluescript SK-. This clone, designated X1 gamma 3, contains the entire 3'-end and lacks 38 bases at the 5'-end of gamma mRNA. The deduced amino acid sequence at the N-terminal is compatible with a signal peptide of 20-23 amino acids, in agreement with the calculated size of the frog gamma chain signal peptide. Following the signal sequence is a region of highly conserved amino acids that participate in disulfide bond formation critical for the maintenance of tertiary structure in mammalian fibrinogen. The gamma cDNA clone was used to measure gamma mRNA in purified Xenopus liver cells treated with glucocorticoids in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of cDNA for the B beta subunit of Xenopus fibrinogen, the product of a coordinately-regulated gene family.

Fibrinogen, the principal blood-clotting protein, is made up of three different subunits synthesized in the liver. In vitro administration of glucocorticoids to liver cells from the frog Xenopus laevis causes a dramatic increase in fibrinogen synthesis. Investigations of molecular mechanisms underlying this hormonal stimulation at the mRNA level require cDNA clones complementary to the mRNAs coding for the three fibrinogen subunits, called A alpha, B beta, and gamma. We describe here the isolation and characterization of cDNA clones for the B beta subunit of Xenopus fibrinogen. cDNA libraries in both plasmid (pBR322) and phage (lambda gt10) cloning vectors were constructed from frog liver mRNA and screened with a rat B beta cDNA. Clones thus isolated hybridized to two Xenopus liver mRNAs 2500 and 1800 bases long, the previously-determined sizes for B beta mRNAs. The identity of the plasmid clone B beta-27 was confirmed by hybridization-selection of complementary mRNA which translated in vitro into the B beta polypeptide, as determined by size and susceptibility to thrombin cleavage. lambda/B beta 10, a clone representing nearly all of the 2500-base B beta mRNA, was isolated from the phage cDNA library. The 3'-end of this clone includes a polyadenylation signal about 20 residues upstream of a stretch of 34 adenosine residues, which probably represents the 3'-poly(A) tail of the messenger RNA. lambda/B beta 10 lacks only 20 nucleotides of full-length B beta mRNA at the 5'-end and there is one major start site of transcription. The 2500-base B beta mRNA has a 700-base extension at the 3'-end that is not present in the 1800-base mRNA. The Xenopus laevis genome contains two or three genes for the B beta fibrinogen subunit. Using the cDNA clone as a probe, B beta mRNA was shown to be induced at least 20-fold by glucocorticoid treatment of purified parenchymal cells of Xenopus liver maintained in primary culture.

Bleeding diathesis coincident with chronic myelomonocytic leukaemia.

Two important haematological problems were found in an otherwise healthy 78 year old man: chronic myelomonocytic leukaemia; and a complex, acquired, hyperfibrinolytic bleeding disorder characterized by prolonged coagulation times, deficiency of coagulation factors V, X, and XI, anti-thrombin III and proteins C and S, with high concentrations of circulating tissue plasminogen activator, and low concentrations of plasminogen activator inhibitor. There may be a causal relation between the two conditions, with the peripheral blood monocytes mediating the hyperfibrinolytic process by the abnormal production of tissue plasminogen activator, though no previous description of a similar association has been reported.

Energy-dependent UV light-induced disruption of (-)sulpiride antagonism of dopamine.

The dopamine D2 receptor antagonist sulpiride decreases the spontaneous locomotor activity of Planaria in an enantiomeric-selective and dose-dependent manner. We now report that (-)sulpiride (0.1 microM)-induced decrease of planarian locomotor activity is significantly (P<0.05) attenuated by low-energy (366 nm) ultraviolet (UV) light and to a greater extent by high-energy (254 nm) UV light. The phenomenon offers a novel approach for studying dopamine D2 receptor transduction processes in a simple in vivo model.

Quantitative assessment of dopamine D2 antagonist activity using invertebrate (Planaria) locomotion as a functional endpoint.

INTRODUCTION: Dopaminergic ligands, including drugs of abuse, modulate the locomotor activity of planarians and induce characteristic abnormal patterns of motility at high doses. It has been presumed that the effect is related to dopamine receptors based on ligand specificity and effects on second messenger levels. However, to date, the measured changes have been mostly qualitative in nature and it is not completely clear that the effect is related to stereospecific receptor mechanisms. METHODS: The present study addressed these issues by devising a convenient and sensitive metric (locomotor velocity, pLMV) and applied the method to test Planaria enantiomer-sensitivity to a dopamine D2-receptor antagonist. RESULTS: pLMV was remarkably constant over the observation period and established a stable baseline against which to study and quantitate pharmacologic intervention. Further, S(-)-sulpiride at low doses (10(-10) to 10(-8) M) attenuated pLMV in a dose-dependent manner, but R(+)-sulpiride was only 1/25th as potent. DISCUSSION: The new methodology thus provides a method for quantifying actions of D2 ligands in a simple in vivo system.

Age-related deficits in prospective memory: the influence of task complexity.

Younger and older subjects were asked to perform an action whenever target words occurred during a short-term memory task. The difficulty of this prospective memory task was manipulated by varying the delay preceding the occurrence of a target event and by varying the number of different target events. Age-related performance differences emerged when there were several different target events but not when there was one target event presented several times. Age-related performance differences, when they occurred, were associated with poorer retrospective memory for the target events. The results were interpreted in terms of a componential analysis of prospective memory, which assumes both similarities and differences between prospective and retrospective memory.

Acquired haemophilia A: errors in the diagnosis.

The distinction between a specific factor inactivator and a non-specific inhibitor is important when confronted by a patient with a history of bleeding and abnormal in-vitro coagulation tests. We report on two patients who presented with bleeding and a prolonged activated partial thromboplastin time. Initial factor assays suggested combined deficiency of factors VIII and IX as a result of the presence of inactivators. The use of dilution studies, chromogenic assays, a novel in-house enzyme-linked-immunosorbent-assay-based technique and phospholipid neutralization, demonstrated that Case 1 had a genuine factor VIII inactivator resulting in factor VIII levels of less than 1 IU/dl but no factor IX deficiency. Case 2 had normal levels of factor VIII on further testing and no specific inactivator to either factor VIII or IX but a potent antiphospholipid antibody which had interfered with the phospholipid-dependent in-vitro assays. Care must be taken in the interpretation of laboratory assays in the presence of antiphospholipid antibodies to ensure that the correct diagnosis is made and inappropriate treatment avoided.

Xenopus fibrinogen synthesis and secretion. Analysis of precursor polypeptides and their post-translational modification.

Xenopus fibrinogen is distinct from that of mammals in that the B beta subunit of the secreted protein has a higher apparent molecular weight than either the A alpha or the gamma subunit. A precursor polypeptide for each subunit was identified among the products translated in vitro from liver mRNA. Pre-A alpha is larger than pre-B beta and pre-gamma is the smallest of the three. Purified liver parenchymal cells cultured in the presence of tunicamycin secrete fibrinogen polypeptides which lack carbohydrate moieties. Our analysis of these forms of Xenopus fibrinogen demonstrated the presence of a signal peptide on each of the precursor polypeptides, the loss of a COOH-terminal peptide from the pre-A alpha chain, and the presence of one carbohydrate moiety on the mature gamma chain and two carbohydrate moieties on the mature B beta chain. The B fibrinopeptide on the NH2-terminus of the B beta chain is unusually large. The number of amino acids in this peptide is approximately twice that characteristic of mammalian B fibrinopeptides and it is glycosylated. On the basis of these data, together with information in the literature, we propose that when vertebrates first arose the B fibrinopeptide was a large glycosylated peptide. It retained this basic structure during the evolution of all subsequent vertebrates, with the exception of mammals. In contrast, the A fibrinopeptide increased in length early in vertebrate evolution. The alpha portion of the A alpha subunit of fibrinogen appears to have originally been a polypeptide about the same length as the beta chain. Concomitant with the evolution of mammals, the alpha polypeptide significantly increased in length.

Efficient recovery of functionally intact mRNA from agarose gels via transfer to an ion-exchange membrane.

A simple method is described for the efficient recovery of intact mRNA from high resolution agarose gels. Fractionation of RNA is accomplished by gel electrophoresis under denaturing conditions using methylmercuric hydroxide. The RNA in the gel is then transferred electrophoretically to a diethylaminoethyl (DEAE)-membrane. After reversing the methylmercuric modification of the RNA, the membrane is sliced into narrow sections and the RNA is eluted at 65 degrees with a high ionic strength buffer containing 6M guanidine hydrochloride. RNA isolated by this procedure is suitable for subsequent enzymatic reactions, including in vitro translation and reverse transcription. The major advantages offered by this procedure are: 1) The membrane-bound RNA is a replica of the high resolution fractionation pattern achieved in the gel. 2) The immobilization and concentration of RNA and the removal of gel matrix contaminants are all accomplished in one step. 3) Small quantities of RNA are efficiently recovered and are suitable for subsequent biochemical manipulations. The method is of general utility for any biological system. We have applied its use to the fractionation, recovery, and analysis of mRNA from Xenopus liver and have identified cDNA clones complementary to albumin mRNA.

Modulation of transcription from chromatin assembled in vitro.

A small plasmid DNA was assembled into chromatin in vitro by incubation in an extract prepared frog eggs of Xenopus laevis. The plasmid DNA contrained the regulatory region of the Escherichia coli lac operon, the transcription of which is under positive regulation by catabolite activator protein (CAP) and negative regulation by lac repressor. After incubation in the egg extract the plasmid DNA acquired approximately 60% of the predicted maximum number of nucleosomes. Chromatin was treated with protein and DNA cross-linking agents prior to transcriptin in order to demonstrate that regions of the DNA organized into nucleosomes served as templates for transcription. Cross-linking abolished transcription of chromatin but had no effect on transcription of the DNA, suggesting that transcription of untreated chromatin was not solely attributable to nucleosome-free regions. In support of this conclusion, the average size of the RNA transcribed from chromatin was approximately 1000 bases, which was approximately 5 times longer than the average distance between nucleosomes. Transcription of in vitro assembled plasmid chromatin by E. coli RNA polymerase was stimulated by catabolite activator protein. The CAP-mediated stimulation of transcription was detectable as an increase in total transcription that was specific to chromatin made from a plasmid containing the lac regulatory DNA sequences. The specific increase in the amount of RNA whose synthesis was initiated within the lac region was demonstrated by hybridization of transcription products to complementary DNA fragments bound to nitrocellulose filters. Preliminary investigation of the action of lac repressor suggested that it also modulated transcription from the chromatin template.