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.

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.

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.

The binding site for Xenopus glucocorticoid receptor accessory factor and a single adjacent half-GRE form an independent glucocorticoid response unit.

In Xenopus laevis, transcription of the gamma-fibrinogen subunit gene is activated by glucocorticoids. Hormone induction is regulated by three glucocorticoid response element (GRE) half-sites and an additional DNA sequence which binds a novel hepatocyte nuclear protein, Xenopus glucocorticoid receptor accessory factor (XGRAF). The XGRAF binding site (GAGTTAA) is located directly upstream of the most distal half-GRE. The proximity of the binding sites for XGRAF and the glucocorticoid receptor (GR) led to the hypothesis that these two sites form a glucocorticoid response unit (GRU). By transfecting DNA into primary hepatocytes, we showed that this GRU confers hormone responsiveness in the absence of other half-GREs. The XGRAF binding site enhances function of the half-GRE without itself responding to glucocorticoids. The GRU retains efficacy in other locations relative to the gamma-fibrinogen gene promoter, further increases transcription when present in multiple copies, and activates a heterologous promoter. Despite the contiguity of the XGRAF binding site and half-GRE, the two sites can be occupied simultaneously in vitro. The binding characteristics correlate with function since mutations that disrupt concurrent XGRAF and GR binding also impair transcription. This novel GRU represents a new regulatory mechanism that may be applicable to other glucocorticoid responsive genes that lack a full GRE.

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.

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.

Analysis of the DNA-binding site for Xenopus glucocorticoid receptor accessory factor. Critical nucleotides for binding specificity in vitro and for amplification of steroid-induced fibrinogen gene transcription.

In addition to the glucocorticoid receptor, DNA-binding proteins called accessory factors play a role in hormone activation of many glucocorticoid-responsive genes. Hormonal regulation of the gamma-fibrinogen subunit gene from the frog Xenopus laevis requires a novel DNA sequence that binds a liver nuclear protein called Xenopus glucocorticoid receptor accessory factor (XGRAF). Here we demonstrate that the recognition site for XGRAF encompasses GAGTTAA at positions -175 to -169 relative to the start site of transcription. This sequence is not closely related to the binding sites for known transcription factors. The two guanosines make close contact with XGRAF, as shown by the methylation interference assay. Single-point mutagenesis of every nucleotide in the 9-base pair region from positions -177 to -169 showed an excellent correlation between ability to bind XGRAF in vitro and ability to amplify hormone-induced transcription from DNA transfected into Xenopus primary hepatocytes. Conversely, XGRAF had little or no effect on basal transcription of the gamma-fibrinogen gene. Maximal hormonal induction also requires three half-glucocorticoid response elements (half-GREs) homologous to the downstream half of the consensus GRE. Interestingly, the XGRAF-binding site is immediately adjacent to the most important half-GRE. This close proximity suggests a new mechanism for activation of a gene lacking a conventional full GRE.

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.

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.

Physiological correlates of simulated wheelchair racing in trained quadriplegics.

This study examined the physiological responses during a 7.5-km simulated wheelchair race (SR) performed on rollers by 8 male quadriplegic marathon racers and analyzed the factors associated with SR time. Cardiac output (Q) was estimated during the SR using carbon dioxide rebreathing, from which stroke volume (SV) and (alpha-v)O2 diff were calculated. Subjects raced at 90 and 93% of peak oxygen uptake (VO2) and peak heart rate, respectively. SR time was inversely related (p < 0.05) to peak VO2, and VO2, Q, and SV during the SR, but not (alpha-v)O2 diff, age, and lesion level. Multiple regression analysis included only absolute SR VO2 in the equation to predict SR time: Y = -29.7X + 65.9; SE = 5.8. SR VO2 was significantly (p < 0.05) related to Q and SV but not to (alpha-v)O2 diff. These descriptive data suggest that SR performance in trained male quadriplegics might be limited by central, as opposed to peripheral, factors that determine VO2.

Reliability of the maximal aerobic power and ventilatory threshold in adults with cerebral palsy.

This study examines the test-retest reliability of the ventilatory threshold (VT) and the maximum oxygen uptake (VO2max) in adults with cerebral palsy (CP). Nine subjects completed two continuous incremental tests on either a wheelchair ergometer (WE, n = 5) or a cycle ergometer (CE, n = 4) to volitional fatigue. Metabolic and cardiorespiratory responses were continuously monitored during the tests with an automated metabolic cart interfaced with an electrocardiogram. Two experienced evaluators identified the VT using standardized respiratory gas exchange criteria. The reliability coefficient for the VO2max during the two trials was significant (r = 0.83, p < .05). However, there was no significant relationship (p > .05) between the two trials for the oxygen uptake (VO2) at the VT identified by the two evaluators (r = 0.45 and 0.43). The correlations for this variable between the two evaluators were highly significant on each trial (r = 0.99 and 1.00, p < .01). No significant differences were observed between the two trials for the VO2 at the VT and the VO2max. These results suggest that when monitoring the cardiorespiratory fitness of adults with CP, it may be more appropriate to evaluate the VO2max rather than the VT, because the former variable can be reliably determined in these individuals whereas the latter lacks consistency.

Primary thrombocythaemia: a composite approach to diagnosis.

Traditional diagnostic criteria for primary thrombocythaemia (PT) remain essentially negative, aiming to exclude other myeloproliferative disorders and causes of reactive thrombocytosis (RT). It would be useful to have positive markers. We have examined several parameters to see how well they discriminate between PT and RT. Three groups of patients were studied: new, untreated PT (17), treated PT (12) and RT (17). Data consisted of: ESR, plasma fibrinogen, factor VIIIC, von Willebrand factor antigen (vWF:Ag), PDW, platelet nucleotide ratio (ATP:ADP) serum erythropoietin (Epo), ristocetin cofactor (vWF:RiCoF), multimeric structure of vWF, interleukin-6, evidence of clinical ischaemia and erythroid colony formation. Erythroid colonies were assayed in a serum-free system with the addition of Epo, IL3 or alpha-IFN to produce a discriminant function (DF) successfully used in the diagnosis of primary polycythaemia in an earlier study. Acute phase reactants (ESR, fibrinogen, VIIIC, vWF:Ag) and IL6 were the best discriminants, while PDW and serum Epo were less so. ATP:ADP and clinical ischaemia were nondiscriminatory in this study. Reduction in vWF:RiCof and in high molecular weight multimers were clearly associated with PT. Endogenous erythroid colonies were nondiscriminatory, but half the PT group and only one patient in the RT group obtained a DF suggestive of myeloproliferative disorder. Judicious use of a battery of tests may provide support for diagnosis of PT in difficult cases.

Physiological responses during wheelchair racing in quadriplegics and paraplegics.

The purposes of this study were: (1) to compare the physiological responses during simulated wheelchair racing (SR) between male quadriplegics and paraplegics, (2) to test the validity of the SR against a track race (TR) and (3) to examine the relationship between the peak oxygen uptake (peak VO2) and wheeling velocity (WV) during the SR and TR. Seven quadriplegics (C5-8 lesions) and six paraplegics (T5-L4 lesions) completed (1) an incremental wheelchair velocity test, (2) a SR (1.6 km for quadriplegics and 3.2 km for paraplegics), and (3) an indoor TR of the same distance. The subjects performed the incremental velocity test and SR in their personal wheelchairs mounted on a roller system interfaced with customized software programmed to provide velocity and distance feedback. Physiological responses were monitored using an automated metabolic cart and electrocardiogram. Blood lactate concentration [La] was determined from finger prick samples. Peak VO2 and peak heart rate (peak HR) were significantly higher in the paraplegics compared to quadriplegics: 1.90 +/- 0.54 vs 1.07 +/- 0.35 l/min, and 188 +/- 11 beats/min vs 117 +/- 12 beats/min respectively. The paraplegics exercised at significantly (p < 0.05) higher percentages of peak VO2 and peak HR during the SR compared to quadriplegics (95% vs 76% and 95% vs 86%, respectively). No significant relationships (p < 0.05) were observed between the peak VO2 and WV during the SR and TR in either group.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional regulation of the Xenopus laevis B beta fibrinogen subunit gene by glucocorticoids and hepatocyte nuclear factor 1: analysis by transfection into primary liver cells.

The blood-clotting protein fibrinogen is composed of three subunits, designated A alpha, B beta, and gamma, which are encoded by a family of related genes. As part of the acute-phase response, expression of the fibrinogen genes is coordinately regulated in the liver by glucocorticoids. To understand the factors underlying this hormonal response, we have examined control of transcription from fibrinogen gene fragments transfected into hepatocytes from the frog Xenopus laevis. This analysis is the first in any species to define transcriptional regulatory elements for the fibrinogen genes by transfection into primary liver cells, rather than liver-derived cell lines. A transfection vector was constructed containing the Xenopus B beta gene transcription start site and 1293 bp of the 5' flanking sequence linked to the firefly luciferase gene. When this construct was transfected into primary liver parenchymal cells, luciferase expression was induced approximately 14-fold by glucocorticoids, an increase similar to the transcriptional stimulation of the endogenous B beta subunit gene. DNA fragments with as little as 284 bases of upstream sequence retained full hormone responsiveness. This region contains a sequence resembling the canonical glucocorticoid response element (GRE) at bases -148 to -162. Deletions or specific point mutations eliminating this putative GRE led to complete loss of glucocorticoid inducibility. Physical association of the steroid hormone receptor with this functional GRE was demonstrated with a truncated form of the rat glucocorticoid receptor containing the DNA-binding domain. A second possible GRE at positions -526 to -540 was not hormone-responsive, in either the presence or the absence of the more proximal GRE. The regulatory region also has a sequence similar to the binding site for a liver-specific transcription factor, hepatocyte nuclear factor 1 (HNF-1), at positions -120 to -132. Specific point mutations in the HNF-1-binding site, in a construct containing a wild-type GRE, reduced promoter activity by a factor of 10, while stimulation by glucocorticoids was retained. Binding studies confirmed specific interaction between this site and the transcription factor HNF-1 alpha from mouse. Thus, we have identified a GRE sufficient to account for full glucocorticoid inducibility and an HNF-1 site close to the promoter that are major determinants of transcriptional control of the Xenopus fibrinogen B beta subunit gene in cells from normal liver tissue.

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.

Anaerobic threshold in wheelchair athletes with cerebral palsy: validity and reliability.

This study examined the validity and reliability of the anaerobic threshold (AT) using blood lactate (ATLa) and respiratory gas exchange (ATg) criteria during cycle ergometry (CE) and wheelchair ergometry (WE) in athletes with spastic cerebral palsy (CP). Eleven subjects attempted a discontinuous incremental test protocol, two minutes work interspersed with one minute rest, twice each on the CE and WE. Only five out of the 11 subjects were able to complete the CE tests, whereas all the subjects were able to complete the WE test. Inadequate hip flexion due to muscle spasticity was the primary limiting factor during the CE tests. Although the maximal aerobic power using this protocol was reliable during WE (r = 0.89, p < .05), the validity and reliability of the AT identified by two independent evaluators using these two techniques was questionable. Evaluator 1 was able to identify ATLa and ATg in seven out of the 11 cases, whereas evaluator 2 was successful in five and seven cases, respectively. It is unclear from these results whether the poor validity and reliability of the AT was due to the discontinuous test protocol used, or whether it was due to inconsistencies in the rate of lactate diffusion from the muscle into the blood due to variations in muscle spasticity during the test.

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.

A major estrogen-regulated protein secreted from the liver of Xenopus laevis is a member of the serpin superfamily. Nucleotide sequence of cDNA and hormonal induction of mRNA.

Estrogen treatment of Xenopus frogs causes four mRNAs to become highly abundant in the liver. Three of these mRNAs have been previously identified as coding for vitellogenin, ferritin, and serum retinol binding protein. We show here that the fourth abundant liver messenger RNA comprises about 1500 nucleotides and codes for a 45-kDa secreted protein, designated Ep45. A clone complementary to Ep45 mRNA was isolated, and its identity was confirmed by hybridization selection of mRNA that translated in vitro into the Ep45 precursor. Nucleotide sequence analysis of the nearly full length cDNA revealed a total length of 1454 base pairs consisting of: 36 nucleotides of the 5' noncoding region, 1308 base pairs encoding an open reading frame of 436 amino acids, and 110 nucleotides of the 3' untranslated region. Ep45 mRNA may originate from as many as four closely spaced transcription start sites, which are 15 to 21 bases upstream of the first nucleotide of the cDNA clone. The Xenopus laevis genome appears to contain a single Ep45 gene. The deduced amino acid sequence indicates that Ep45 has features typical of a secreted protein, including a signal peptide of 16 amino acids and three potential sites for N-linked glycosylation, and is related to the serine protease inhibitors, a large family of proteins with very diverse physiological functions. Ep45 mRNA was absent in the liver of normal male frogs and increased at least 100-fold in response to estradiol-17 beta. Thus, both Ep45 and vitellogenin mRNAs are switched from undetectable to very high levels, a pattern of expression not found for any other mRNAs in Xenopus liver.

Maximal aerobic power in cerebral palsied wheelchair athletes: validity and reliability.

This study examined the validity and reliability of maximal aerobic power (VO2max) during wheelchair ergometry (WE) in wheelchair athletes with cerebral palsy. Six class 3 and class 4 male athletes with cerebral palsy completed two graded exercise tests to volitional fatigue on a wheelchair ergometer mounted on frictionless rollers. Four athletes were also able to complete two tests of bicycle ergometry (BE). Although the reliability coefficients for the VO2max during the two exercise modes were high (.89 and .93 for the WE and BE tests, respectively), the validity coefficients for this variable (ie, the correlations between WE and BE) were poor (.31 and -.24 for trials 1 and 2, respectively). Examination of the individual data indicated that athletes who used wheelchairs as their primary mode of ambulation had higher VO2max values during WE; whereas, those who used canes or no aids for daily ambulation had higher values on BE. Because of the specificity of the, VO2max response, it is recommended that the primary mode of ambulation be considered when deciding on the testing mode for evaluating the cardiorespiratory fitness of athletes with cerebral palsy.