Long-period orbits in the Stark spectrum of lithium.
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Journal Article↗
Biomedical subjects
M Courtney
Publications and source records attributed to M Courtney.
Alaproclate acts as a potent, reversible and noncompetitive antagonist of the NMDA receptor coupled ion flow.
We studied the effect of alaproclate [2-(4-chlorophenyl)-1,1-dimethyl 2-aminopropanoate] on the N-methyl-D-aspartate (NMDA)-induced changes in membrane potential and intracellular free Ca++ in cerebellar granule cells by using the fluorescent indicators DiBaC4(3) and fura-2, respectively. The NMDA-induced responses were blocked by the 5-hydroxytryptamine reuptake blocker alaproclate in a noncompetitive manner with an IC50 value of 0.3 microM. The effect of alaproclate was stereoselective because the S-(-)-enantiomer was more potent than the R-(+)-enantiomer. The inhibitory response was rapidly reversed if alaproclate was removed by perfusion. The same was the case with the reversible noncompetitive NMDA receptor antagonists dextromethorphan, dextrorphan, amitriptyline and desipramine. The inhibition caused by the noncompetitive antagonist dizolcipine could not be reversed by perfusion. The glycine-sensitivity of the NMDA response was unaffected by alaproclate, and high concentrations of glycine were unable to reverse the inhibition of alaproclate. Alaproclate also did not affect the sensitivity of the responses to Mg++. The results suggest that alaproclate, which has previously been in clinical trials for depressive illness, acts as a reversible noncompetitive antagonist of the NMDA receptor.
Alanine↗
New class of universal correlations in the spectra of hydrogen in a magnetic field.
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Journal Article↗
Teachers' ratings of disruptive behaviors: the influence of halo effects.
This study evaluated the accuracy of teachers' ratings and examined whether these ratings are influenced by halo effects. One hundred thirty-nine elementary school teachers viewed videotapes of what they believed were children in regular fourth-grade classrooms. In fact, the children were actors who followed prepared scripts that depicted a child engaging in behaviors characteristic of an attention-deficit hyperactivity disorder (ADHD), an oppositional defiant disorder or a normal youngster. The findings provide support for a bias that was unidirectional in nature. Specifically, teachers rated hyperactive behaviors accurately when the child behaved like an ADHD youngster. However, ratings of hyperactivity and of ADHD symptomatic behaviors were spuriously inflated when behaviors associated with oppositional defiant disorder occurred. In contrast, teachers rated oppositional and conduct problem behaviors accurately, regardless of the presence of hyperactive behaviors. The implications of these findings regarding diagnostic practices and rating scale formats are discussed.
Adult↗
In memoriam. Jean-Pierre Lecocq, 1947-1992.
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Belgium↗
A hybrid protein of urokinase growth-factor domain and plasminogen-activator inhibitor type 2 inhibits urokinase activity and binds to the urokinase receptor.
The binding of urokinase-type plasminogen activator (uPA) to its specific cell-surface receptor (uPAR) localises the proteolytic cascade initiated by uPA to the pericellular environment. Inhibition of uPA activity or prevention of uPA binding to uPAR might have a beneficial effect on disease states wherein this activity is deregulated, e.g. cancer and some inflammatory diseases. To this end, a bifunctional hybrid molecule consisting of the uPAR-binding growth-factor domain of uPA (amino acids 1-47; GFuPA) at the N-terminus of plasminogen-activator inhibitor type 2 (PAI-2) was produced in Saccharomyces cerevisiae. The purified protein inhibited uPA with kinetics similar to placental or recombinant PAI-2 and was also found to bind to U937 cells and to FL amnion cells. GFuPA-PAI-2 competed with uPA, the N-terminal fragment of uPA and a proteolytic fragment of uPA (amino acids 4-43) in cell binding experiments, indicating that the molecule bound to the cells via uPAR. Hence, both the uPA-inhibitory and uPAR-binding domains of the hybrid molecule were functional, demonstrating the feasibility of the novel concept of introducing an unrelated, functional domain onto a member of the serine-protease-inhibitor superfamily.
Amino Acid Sequence↗
Processing and characterization of recombinant von Willebrand factor expressed in different cell types using a vaccinia virus vector.
The cloning of the cDNA encoding von Willebrand factor (vWF) has revealed that it is synthesized as a large precursor (pre-pro-vWF) molecule and it is now clear that the prosequence or vWAgII is responsible for the intracellular multimerization of vWF. We have cloned the complete vWF cDNA and expressed it using a recombinant vaccinia virus as vector. We have characterized the structure and function of the recombinant vWF (rvWF) secreted from five different cell types: baby hamster kidney (BHK), Chinese hamster ovary (CHO), human fibroblasts (143B), mouse fibroblasts (L) and primary embryonic chicken cells. Forty-eight hours after infection, the quantity of vWF antigen found in the cell supernatant varied from 3 to 12 U/dl depending on the cell type. By SDS-agarose gel electrophoresis, the percentage of high molecular weight forms of vWF varied from 39 to 49% relative to normal plasma for BHK, CHO, 143B and chicken cells but was less than 10% for L cells. In all cell types, the two anodic subbands of each multimer were missing. The two cathodic subbands were easily detected only in BHK and L cells. By SDS-PAGE of reduced samples, pro-vWF was present in similar quantity to the fully processed vWF subunit in L cells, present in moderate amounts in BHK and CHO and in very low amounts in 143B and chicken cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Expression of recombinant platelet-derived endothelial cell growth factor in the yeast Saccharomyces cerevisiae.
A platelet-derived endothelial cell growth factor cDNA has been cloned, sequenced and expressed using the Saccharomyces cerevisiae PRB1 promoter. Soluble recombinant platelet-derived endothelial cell growth factor constituted 0.5-1.0% of total soluble protein. Yeast soluble protein extracts containing recombinant platelet-derived endothelial cell growth factor stimulate the growth of calf pulmonary artery endothelial cells in vitro.
Cell Division↗
Purification and characterisation of plasminogen activator inhibitor 2 produced in Saccharomyces cerevisiae.
Expression of plasminogen activator inhibitor 2 (PAI-2) under the control of the protease B gene promoter in a mutant strain of Saccharomyces cerevisiae, DS569, resulted in its accumulation intracellularly at up to 20% of the soluble cell protein. Provision of an N-terminal signal sequence resulted in the secretion of a hyperglycosylated molecule. The intracellularly produced PAI-2 was purified by copper-chelate and anion-exchange chromatography to greater than 95% pure and was fully active. The recombinant PAI-2 formed SDS-stable complexes with urokinase and tissue-type plasminogen activator and inhibited the proteases with similar reaction kinetics to placental PAI-2 (second-order rate constant for uPA, 2.4 x 10(6) M-1 s-1, and for two-chain tPA, 0.7 x 10(5) M-1 s-1). As is the case for placental PAI-2, the N-terminus of the yeast-derived recombinant PAI-2 was blocked. The high productivity and consequent ease of purification mean that S. cerevisiae provides an excellent source of recombinant PAI-2 for investigation of its therapeutic potential in the treatment of neoplastic and inflammatory diseases.
Base Sequence↗
Sulphation of hirudin in BHK cells.
Hirudin, a thrombin inhibitor of the leech, was expressed in BHK cells; the alpha 1-antitrypsin signal peptide was used to direct secretion into the culture medium. The recombinant hirudin so produced inhibited thrombin and was shown by labelling experiments with [35S]sulphate to have been posttranslationally modified.
Amino Acid Sequence↗
Expression of human alpha 1-antitrypsin using a recombinant adenovirus vector.
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Adenoviridae↗
Reactivity of alpha 1-antitrypsin mutants against proteolytic enzymes of the kallikrein-kinin, complement, and fibrinolytic systems.
Increased extracellular proteolysis because of unregulated activation of blood coagulation, complement, and fibrinolysis is observed in thrombosis, shock, and inflammation. In the present study, we have examined whether the plasma kallikrein-kinin system, the classical pathway of complement, and the fibrinolytic system could be inhibited by alpha 1-antitrypsin reactive site mutants. Wild-type alpha 1-antitrypsin contains a Met residue at P1 (position 358), the central position of the reactive center. It did not inhibit plasma kallikrein, beta-factor XIIa, plasmin, tissue-type plasminogen activator (t-PA), or urokinase. In contrast, these serine proteases were inhibited by alpha 1-antitrypsin Arg358. For the inhibition of C1s, a double mutant having Arg358 and a Pro----Ala mutation at P2 (position 357) was required. This double modification was made because C1-inhibitor, the natural inhibitor of C1s, has Arg and Ala residues at positions P1 and P2. Plasminogen activator inhibitor 1, the natural inhibitor of t-PA, also has Arg and Ala residues at positions P1 and P2. In a purified system, alpha 1-antitrypsin Ala357-Arg358 was 150-fold less efficient against C1s than C1-inhibitor and 27,000-fold less efficient against t-PA than plasminogen activator inhibitor-1. In plasma, 2.3 microM alpha 1-antitrypsin Ala357-Arg358 reduced by 65% the formation of a complex between kallikrein and C1-inhibitor following activation of the intrinsic pathway of blood coagulation by kaolin. Furthermore, after supplementation by 2.0 microM alpha 1-antitrypsin Ala357-Arg358, zymographic analysis showed that the majority of the free t-PA of normal plasma formed a bimolecular complex with the double mutant. In contrast, 3.4 microM alpha 1-antitrypsin Ala357-Arg358 did not prevent the activation of the classical pathway of complement observed when normal serum is supplemented with anti-C1-inhibitor F(ab')2 fragment. These results demonstrate that alpha 1-antitrypsin Ala357-Arg358 has therapeutic potential for disorders with unregulated activation of the intrinsic pathway of blood coagulation and the fibrinolytic system; however, the double mutant is not an efficient inhibitor for the classical pathway of complement.
Amino Acid Sequence↗
Inhibition by recombinant hirudins of experimental venous thrombosis and disseminated intravascular coagulation induced by tissue factor in rats.
Antithrombotic potency of recombinant hirudins rHV2, rHV2-Lys47 and rHV2-Arg47 was studied in a model of experimental thrombosis induced by tissue factor in the rat. Venous thrombosis was induced by i.v. injection of 25 mg/kg tissue factor followed by stasis of the inferior vena cava. In this model natural recombinant hirudins, rHV2 and rHV2-Lys47 injected 5 min before thrombo-plastin totally inhibited thrombosis in the same micrograms range as heparin or natural hirudin extracted from leeches. However, the mutant variant rHV2-Arg47 gave a maximal 60% inhibition of thrombosis. Variants rHV2-Lys47 (30 micrograms/kg) and rHV2-Arg47 (157 micrograms/kg) injected 5 min before thromboplastin prevented by 90 to 100% the drop in platelet count observed during the disseminated intravascular coagulation induced by thromboplastin injection. Recombinant hirudins were less anticoagulant than heparin as measured by an APTT on rat plasma. After rat tail transection, rHV2-Lys47 caused a 2-fold smaller prolongation of the bleeding time than an equivalent antithrombotic dose of heparin. Plasmatic elimination of rHV2-Lys47 from rat plasma after i.v. injection had a fast distribution phase with a half-life of 3 min during which 90% of injected rHV2-Lys47 was lost and was followed by a slower elimination phase. Thus recombinant hirudin rHV2-Lys47 appears as a promising potent antithrombotic agent for the prevention of thrombin-dependent venous thrombosis and disseminated intravascular coagulation.
Animals↗
Inhibition of activated protein C by recombinant alpha 1-antitrypsin variants with substitution of arginine or leucine for methionine358.
alpha 1-Antitrypsin (alpha 1-AT) was recently identified as a major physiologic plasma inhibitor of activated protein C. The reaction with activated protein C of recombinant alpha 1-AT containing amino acid substitutions at the reactive center was studied. The substitution of Arg358 for Met, as observed in a patient with a severe bleeding disorder with the mutant alpha 1-AT Pittsburgh, increased the association rate constant for activated protein C from 1.1 x 10(1) to 4.9 x 10(4) M-1 s-1. The association rate constant of activated protein C with protein C inhibitor, a native plasma serpin that contains Arg354 at the reactive site, is 6 x 10(3) M-1 s-1 in the absence of heparin. Plasma containing 4 microM [Arg358]alpha 1-AT inhibited activated protein C activity by greater than 95% in 15 s, and the inhibited activated protein C was shown by immunoblotting to exist as activated protein C-inhibitor complexes. In controls 50% loss of activated protein C activity in normal plasma occurred in 19 min. Double-substituted [Pro357,Met358]alpha 1-AT----[Ala357,Arg358]alpha 1-AT had similar reactivity toward activated protein C as the single-substituted [Arg358]alpha 1-AT. Thus, replacement of the reactive center Met358 of alpha 1-AT by Arg358, analogous to Arg354 of protein C inhibitor, results in an activated protein C inhibitor that is more potent than either of the native inhibitors. Comparison of the association rate constant of the [Arg358]alpha 1-AT for activated protein C to that for thrombin (4 x 10(4) versus 3 x 10(5) M-1 s-1) suggests that thrombin would be more effectively inhibited than activated protein C, thereby giving an explanation for bleeding rather than thrombosis in the alpha 1-AT Pittsburgh patient.
Arginine↗
Fast-LC determination of 1-methyl-1H-tetrazole-5-thiol in human plasma with column-switching sample clean-up.
A simple, fast and sensitive method for the quantitative determination of 1-methyl-1H-tetrazole-5-thiol using HPLC is reported. Samples are deproteinated by plasma water filtration and injected into a HPLC system capable of column switching and backflushing the analytical column. The limit of quantitation was determined to be 70 ng ml-1 and the limit of detection 22 ng ml-1. Between-day precision (expressed as percent coefficient of variation) of standard curve slopes was +/- 3.5% with a range of within-day percent coefficient of variations from 0.28 to 1.4%. Recovery and precision of spiked control samples was 96 +/- 7% over a concentration range of 630-6300 ng ml-1.
Calibration↗
Heterologous protein expression by transimmortalized differentiated liver cell lines derived from transgenic mice (hepatomas/alpha 1 antitrypsin/ONC mouse).
A number of therapeutic plasma proteins are synthesized by human hepatocytes. Since many of these proteins undergo liver-specific post-translational modifications which are required for full biological activity, it may therefore be necessary to develop hepatocyte-based expression systems for their production. Using transgenic mice we have developed a transimmortalisation technique for the isolation of differentiated hepatic cell lines, already engineered to secrete human alpha 1 antitrypsin (alpha 1 AT), a plasma protein which is produced mainly in liver cells. This was achieved by co-expression of the mouse c-myc proto-oncogene and a genomic copy of the human alpha 1 AT gene, both under the control of the human alpha 1 AT promoter. Transgenic mice carrying this construct developed hepatomas producing human alpha 1 AT. Under defined culture conditions, cell lines secreting active alpha 1 AT were derived from these tumours. These cells maintain a differentiated hepatic phenotype and continue to secrete human alpha 1 AT for at least 40 generations.
Animals↗
Maternal and fetal catecholamines and uterine incision-to-delivery interval during elective cesarean.
The fetal sympathoadrenal system is activated during periods of intrauterine stress such as inadequate uterine perfusion. During cesarean, the period of interruption of utero-placental blood flow is extended as the time interval from uterine incision to delivery increases. An increasing uterine incision-to-delivery interval with spinal or general anesthesia has been associated with a poorer neonatal outcome. This association has not been demonstrated previously in patients undergoing cesarean delivery under epidural anesthesia. We investigated the correlation between prolonged uterine incision-to-delivery intervals, fetal catecholamine concentrations, and fetal blood gas values at delivery in 25 parturients undergoing cesarean under epidural anesthesia and in 28 under spinal anesthesia. Infants delivered after prolonged uterine incision-to-delivery intervals had significantly lower pH values in both the epidural and spinal groups. With longer uterine incision-to-delivery intervals, umbilical arterial norepinephrine concentrations were increased significantly. Umbilical arterial pH values were significantly lower in infants with higher umbilical arterial catecholamine concentrations. The importance of minimizing the uterine incision-to-delivery interval, regardless of the type of anesthetic selected, is demonstrated.
Anesthesia, Epidural↗
Production in Escherichia coli of a biologically active subfragment of von Willebrand factor corresponding to the platelet glycoprotein Ib, collagen and heparin binding domains.
A full-length cDNA for vWF has been cloned from a human lung cDNA library and a fragment of this cDNA has been modified to allow its expression in E. coli. This fragment, which corresponds to Val 449-Asn 730 of vWF and includes the GPIb-binding domain and binding sites for collagen and heparin, was subcloned into an expression vector containing an inducible lambda PL promoter. On induction, the expressed recombinant vWF subfragment migrated with a mol wt of around 38,000 after SDS-PAGE. It was identified as a vWF fragment by Western blotting using either a polyclonal or a monoclonal antibody which inhibits the binding of vWF to GPIb. Following solubilization in urea, the bacterial extract inhibited ristocetin-induced platelet aggregation and bound to ristocetin-treated platelets, to collagen and to heparin.
Binding Sites↗