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Biomedical subjects

M Courtney

Publications and source records attributed to M Courtney.

At least 73 records · Page 4Linked to original sources
PubMed1989

Evaluation of the inhibition by heparin and hirudin of coagulation activation during r-tPA-induced thrombolysis.

Thrombin bound to a fibrin clot remains active and poorly accessible to heparin-AT III complex. During fibrinolysis, thrombin is released as thrombin-FDP complex and is inactivated by heparin-AT III. However, as successive fibrin layers are removed, inaccessible molecules of thrombin are exposed at the surface of the residual clot, possibly contributing to the occurrence during thrombolytic therapy of coagulation that is poorly controlled by heparin. We have investigated the accessibility of fibrin-bound thrombin to hirudin. The results clearly show that two recombinant hirudin variants neutralize thrombin both in solution and fibrin bound. Furthermore, we have found that in in vitro models, hirudin present in the surrounding medium of a clot under lysis is more efficient than heparin in preventing the activation of coagulation. This observation suggests that hirudin may be effective in the prevention of the rethrombotic process frequently encountered during thrombolytic therapy.

Adsorption↗
PubMed1989

Serum alpha 1-antitrypsin deficiency associated with the common S-type (Glu264----Val) mutation results from intracellular degradation of alpha 1-antitrypsin prior to secretion.

The S-type alpha 1-antitrypsin (alpha 1AT) deficiency allele differs from the normal M1(Val213) allele by a single amino acid substitution (Glu264----Val). To evaluate the molecular pathophysiology responsible for the reduced serum levels of alpha 1AT associated with the S-type allele, alpha 1AT gene expression was examined in blood monocytes, cells which normally produce alpha 1AT, as well as murine fibroblasts modified by retroviral gene transfer to express the S-type and normal M-type human alpha 1AT genes. Northern analysis and S1 protection analysis demonstrated that monocytes of M and S homozygotes both express 1.8-kilobase alpha 1AT mRNA transcripts in comparable levels and similar in structure. Pulse-chase labeling studies demonstrated that both M and S monocytes synthesized and secreted a 52-kDa protein, but the S monocytes secreted significantly less. The cellular lysates of both M and S monocytes contained a newly synthesized 50-kDa precursor form of alpha 1AT, but the S monocytes contained reduced amounts. Pulse-chase labeling in the presence of tunicamycin, an inhibitor of core oligosaccharide addition, demonstrated that S monocytes exhibited a selective inhibition of secretion of 45-kDa nonglycosylated alpha 1AT not observed in M monocytes. Consistent with these observations, murine fibroblasts modified by retroviral gene transfer to contain an integrated human S-type alpha 1AT cDNA demonstrated reduced secretion of alpha 1AT compared with fibroblasts containing an integrated human M-type alpha 1AT cDNA and also reproduced the abnormality of alpha 1AT biosynthesis observed with S-type monocytes. Furthermore, in the presence of leupeptin, an inhibitor of cellular proteinases, the S-type modified fibroblasts demonstrated a selective augmentation of human alpha 1AT secretion not observed for the M-type. Together, these observations are consistent with the concept that the single A----T mutation of the S-type alpha 1AT gene results in reduced cellular secretion of alpha 1AT because the newly synthesized S-type alpha 1AT protein is degraded intracellularly prior to secretion.

Alleles↗
PubMed1989

Purification and biochemical characterization of recombinant hirudin produced by Saccharomyces cerevisiae.

Recombinant hirudin was produced by the yeast Saccharomyces cerevisiae using the alpha-pheromone prepro sequence to direct its secretion into the culture medium. The secreted hirudin was isolated to greater than or equal to 95% purity as measured by 205-nm absorbance integration from a reverse-phase chromatogram. One major activity peak corresponding to the complete, correctly processed molecule and two minor activity peaks corresponding to C-terminally truncated forms were identified. The primary structure of the major peak, determined by N-terminal sequencing of tryptic peptides, was that predicted from the cDNA sequence, and the molecular mass analyzed by fast atom bombardment mass spectrometry (FAB-MS) was 6892.6 (calculated 6892.5). UV spectral analysis suggested that, in contrast to the natural molecule, recombinant hirudin produced by S. cerevisiae is not sulfated.

Amino Acid Sequence↗
PubMed1989

Production and evaluation of recombinant hirudin.

Hirudin, a 65 amino acid polypeptide form the medicinal leech, is an extremely efficient and specific thrombin inhibitor whose therapeutic potential has been demonstrated in a number of animal models. We have developed protocols for the production of recombinant hirudin by secretion from S. cerevisiae and carried out a full biologic evaluation of the purified product. These studies showed that natural and recombinant hirudin was similar in structure and in biologic function in vitro. Moreover, the recombinant protein displayed strong antithrombotic activity in several experimental thrombosis models in vivo, confirming the molecule's promise in the therapy of thrombotic disorders.

Amino Acid Sequence↗
PubMed1989

Point mutations modifying the thrombin inhibition kinetics and antithrombotic activity in vivo of recombinant hirudin.

The hirudin variant HV2 was modified by in vitro site-specific mutagenesis of HV2 cDNA to generate HV2(Asn-47----Lys), HV2(Asn-47----Arg) and HV2(Lys-35----Thr, Asn-47----Lys). Residues 35 and 47 are positioned respectively within the finger and prothrombin-like domains of hirudin, both of which have been suggested as thrombin binding sites. The modified polypeptides were synthesized in Saccharomyces cerevisiae using a secretion vector and purified from culture supernatants. By analysis of the human alpha-thrombin:hirudin inhibition reaction in steady-state conditions it was shown that the dissociation constants for HV2(Lys-47) and HV2(Arg-47) were 5- to 14-fold lower than for unmodified HV2, whereas mutation of Lys-35 did not significantly alter the inhibition kinetics. Furthermore, HV2(Lys-47), whose sequence is identical to a natural hirudin variant, displayed enhanced anti-thrombotic activity in vivo, having a 100-fold lower ED50 compared to HV2 in the rabbit Wessler venous thrombosis model. These results support a role for the prothrombin-like domain in thrombin binding and, moreover, demonstrate that in vivo antithrombotic efficiency correlates with the dissociation constant of the inhibition reaction.

Amino Acid Sequence↗
PubMed1988

Repair of the secretion defect in the Z form of alpha 1-antitrypsin by addition of a second mutation.

Homozygous inheritance of the Z-type mutant form of the alpha 1-antitrypsin (alpha 1AT) gene results in the most common form of alpha 1AT deficiency, a human hereditary disease associated with a high risk for the development of emphysema and an increased incidence of neonatal hepatitis. The alpha 1AT-synthesizing cells of individuals with the Z gene have normal alpha 1AT messenger RNA levels, but alpha 1AT secretion is markedly reduced secondary to accumulation of newly synthesized alpha 1AT in the rough endoplasmic reticulum. Crystallographic analysis of alpha 1AT predicts that in normal alpha 1AT, a negatively charged Glu342 is adjacent to positively charged Lys290. Thus the Glu342----Lys342 Z mutation caused the loss of a normal salt bridge, resulting in the intracellular aggregation of the Z molecule. The prediction was made that a second mutation in the alpha 1AT genet that changed the positively charged Lys290 to a negatively charged Glu290 would correct the secretion defect. When the second mutation was added to the Z-type complementary DNA, the resulting gene directed the synthesis and secretion of amounts of alpha 1AT similar to that directed by the normal alpha 1AT complementary DNA in an in vitro eukaryotic expression system. This suggests the possibility that a human hereditary disease can be corrected by inserting an additional mutation in the same gene.

Animals↗
PubMed1988

Characterization of the gene and protein of the alpha 1-antitrypsin "deficiency" allele Mprocida.

The "deficiency" group of alpha 1-antitrypsin (alpha 1AT) alleles is characterized by alpha 1AT genes that code for alpha 1AT present in serum but in amounts insufficient to protect the lower respiratory tract from progressive destruction by its burden of neutrophil elastase. Mprocida, a rare alpha 1AT allele associated with alpha 1AT serum levels less than 10 mg/dl (normal 150-350 mg/dl), codes for an alpha 1AT molecule that focuses on immobilized pH gradient isoelectric gels slightly cathodal to the common normal M1 (Val213) protein. On a per molecule basis, Mprocida has a mildly reduced function as an inhibitor, with an association rate constant for human neutrophil elastase of 7.0 +/- 0.1 x 10(6) M-1 s-1 (normal M1 (Val213) 9.3 +/- 0.8 x 10(6), p less than 0.01). The Mprocida molecule behaves normally in vivo with a half-life similar to normal M1 alpha 1AT molecules. Restriction endonuclease mapping demonstrates that the cloned Mprocida gene was grossly intact. Sequencing of all the exons, exon-intron junctions, and the major promoter region demonstrated Mprocida to be identical to the M1 (Val213) gene except for a single base substitution in exon II coding for amino acid 41 of the mature protein (M1 (Val213) Leu41 CTG----Mprocida Pro41 CCG). Usefully, the coding sequence of the alpha 1AT residues 40-41 is recognized by the restriction endonuclease PvuII so that using a probe corresponding to this region of exon II, the Mprocida mutation can be rapidly identified by Southern analysis. Evaluation of the crystallographic structure of alpha 1AT suggests the Leu41 to Pro41 mutation may disrupt alpha-helix A in the region of Pro21-Ser45, suggesting the possibility that the alpha 1AT Mprocida molecule is unstable and degraded intracellularly prior to secretion.

Alleles↗
PubMed1988

Isolation of a human gene with protein sequence similarity to human and murine int-1 and the Drosophila segment polarity mutant wingless.

An expressed gene sequence which was identified by the isolation of a methylation free CpG island from human chromosome 7 has been cloned from a human lung cDNA library. The deduced protein sequence contains 360 amino acids and has several features of a secreted protein; it is cysteine rich with a signal peptide sequence and two potential asn-linked glycosylation sites. The protein sequence shows marked similarity with human and murine int-1 and their Drosophila homolog wingless (Dint-1). This human int-1 related protein, int-1 and Dint-1 have diverse patterns of expression, but the inferred structural similarities suggest that some of the functional characteristics of these proteins may be shared.

Amino Acid Sequence↗
PubMed1988

Use of human surfactant low molecular weight apoproteins in the reconstitution of surfactant biologic activity.

Two low molecular weight (LMW) apoproteins were isolated from human pulmonary surfactant. SDS polyacrylamide gel analysis showed one protein (SP 18) to have an apparent molecular weight of 18,000 when unreduced and 9,000 D after reduction. The second protein (SP 9) migrated at approximately 9,000 D in the presence or absence of reducing agents. Both proteins contain a high number of hydrophobic amino acids. The NH2-terminal sequence of SP 18 was determined to be: NH2-phe-pro-ile-pro-leu-pro-tyr-. A cDNA clone isolated from a human adult lung cDNA library contained a long open reading frame encoding at an internal position the human SP 18 amino-terminal sequence. Mixtures of phospholipids (PL) and SP 9 and SP 18 were assessed for their capacity to reduce surface tensions on a pulsating bubble surfactometer. The addition of 1% apoprotein resulted in a reduction of surface tension after 15 s from 42.9 dyn/cm for PL alone to 16.7 and 6.3 dyn/cm for preparations containing SP 9 and SP 18, respectively. In vivo assessment of reconstituted surfactant activity was performed in fetal rabbits. Reconstituted surfactant consisting of PL + 0.5% SP 18 instilled intratracheally at delivery resulted in a marked increase in lung compliance, while the incorporation of 0.5% SP 9 yielded a moderate increase. These data show the ability to produce biologically active surfactant by the addition of isolated LMW apoproteins to defined PL.

Amino Acid Sequence↗
PubMed1988

Lingual vibrotactile screening: an investigation of normal sensitivity based on multiple threshold determinations.

This report describes a standardized training and sensory screening procedure which was used to obtain 7 separate lingual vibrotactile thresholds for each of 5 normal subjects during a 30-day interval. Baseline observations of lingual sensitivity were highly consistent and occurred within a narrow range of contractor displacements. The authors conclude that the screening method provides a quick and reliable clinical tool for general diagnostic use and discuss directions for continued research applications.

Adult↗
PubMed1988

Emphysema associated with complete absence of alpha 1- antitrypsin in serum and the homozygous inheritance [corrected] of a stop codon in an alpha 1-antitrypsin-coding exon.

Homozygous inheritance of the null bellingham alpha 1-antitrypsin (alpha 1AT) gene is associated with early-onset emphysema, resulting from the lack of alpha 1AT to protect the lung from neutrophil elastase. Cloning and sequencing of the null bellingham gene demonstrated that the promoter region, coding exons, and all exon-intron junctions were normal except for a single base substitution in exon III, causing the normal lys217 (AAG) to become a stop codon (TAG). Evaluation of genomic DNA of family members by using oligonucleotides directed toward this region demonstrated that the index case had inherited this mutation in a homozygous fashion. Although the consequences to the individual (i.e., emphysema) are identical to those associated with the common homozygous Z mutation, the homozygous null bellingham form of alpha 1AT deficiency has a very different genetic basis.

Adult↗
PubMed1987

alpha 1-Antitrypsin nullGranite Falls, a nonexpressing alpha 1-antitrypsin gene associated with a frameshift to stop mutation in a coding exon.

alpha 1-Antitrypsin (alpha 1-AT) deficiency is a hereditary disorder associated with serum alpha 1-AT levels less than 35% of normal. There are two categories of alpha 1-AT genes that cause this state: the deficient alleles, in which alpha 1-AT is present in serum but in low levels, and the null alleles, in which no alpha 1-AT in serum can be attributed to the gene. The present study defines the molecular basis for the alpha 1-AT gene nullGranite Falls, identified and cloned from genomic DNA of an individual with severe alpha 1-AT deficiency and emphysema resulting from the heterozygous inheritance of the nullGranite Falls and Z alpha 1-AT genes. Sequencing of the 5'-flanking region, all five coding exons, and all exon-intron junctions of nullGranite Falls demonstrated it was identical with the common normal M1(Ala213) alpha 1-AT gene, except for two bases: a single deletion in the codon for amino acid Tyr160 of the mature protein and a single base substitution 168 base pairs 5' to exon I. Although no role for the promoter region mutation could be assigned, the coding exon deletion [Tyr(TAC)----(TA-)] resulted in a frameshift causing a stop coding to be formed approximately 44% from the N terminus of the precursor protein. Using oligonucleotide probes to evaluate the family of the index case demonstrated the deletion----frameshift/stop mutation was inherited in an autosomal co-dominant fashion. Thus, although the molecular basis for alpha 1-AT deficiency of the alpha 1-AT null haplotype such as nullGranite Falls is very different from the molecular basis of the more common deficient haplotypes such as Z, the phenotypic consequences of the two genes are similar; i.e. severe alpha 1-AT deficiency and an association of a high risk for the development of emphysema.

Adult↗
PubMed1987

Characterization of the M1(Ala213) type of alpha 1-antitrypsin, a newly recognized, common "normal" alpha 1-antitrypsin haplotype.

alpha 1-Antitrypsin (alpha 1AT) is a highly pleomorphic 52-kDa serum glycoprotein that functions as the major inhibitor of neutrophil elastase. Of these, the most common normal alpha 1AT haplotypes identified by isoelectric focusing (IEF) of serum are those of the M family, including M1, M2, and M3. In the course of studying the alpha 1AT type Z gene, we identified a restriction endonuclease BstEII polymorphism in the M1 gene that predicted the existence of a previously unidentified, but relatively common, haplotype of M, referred to as M1(Ala213) [Nukiwa, T., Satoh, K., Brantly, M. L., Ogushi, F., Fells, G. A., Courtney, M., & Crystal, R. G. (1986) J. Biol. Chem. 261, 15989-15994]. In this study we have cloned both alpha 1AT genes from an individual heterozygous for the M1(Ala213) and M1(Val213) haplotypes. Sequencing of the coding exons of both demonstrated that they are identical except for the Ala-Val difference at residue 213. The codominant transmission of the M1(Ala213) gene was demonstrated in a family study. Evaluation of 39 genomic samples of Caucasians with the IEF haplotype M1 demonstrated haplotype frequencies of 68% for M1(Val213) and 32% for M1(Ala213). alpha 1AT serum levels of individuals inheriting the M1(Ala213) gene in a homozygous fashion were in the same range as those for homozygous M1(Val213) as was the rate of association of the M1(Ala213) protein with neutrophil elastase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Clonal gene therapy: transplanted mouse fibroblast clones express human alpha 1-antitrypsin gene in vivo.

A retroviral vector was used to insert human alpha 1-antitrypsin (alpha 1AT) complementary DNA into the genome of mouse fibroblasts to create a clonal population of mouse fibroblasts secreting human alpha 1AT. After demonstrating that this clone of fibroblasts produced alpha 1AT after more than 100 population doublings in the absence of selection pressure, the clone was transplanted into the peritoneal cavities of nude mice. When the animals were evaluated 4 weeks later, human alpha 1AT was detected in both sera and the epithelial surface of the lungs. The transplanted clone of fibroblasts could be recovered from the peritoneal cavities of those mice and demonstrated to still be producing human alpha 1AT. Thus, even after removal of selective pressure, a single clone of retroviral vector-infected cells that expressed an exogenous gene in vitro, continued to do so in vivo, and when recovered, continued to produce the product of the exogenous gene.

Animals↗
PubMed1987

Purification and characterization of human bronchial proteinase inhibitor.

We describe a purification procedure for the human bronchial proteinase inhibitor which involves trichloroacetic acid precipitation of sputum followed by ion-exchange and gel filtration chromatography. The inhibitor shows a major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but exhibits microheterogeneity on high-resolution chromatography. It has a molecular mass of 15.5-16 kDa as determined by electrophoresis and gel filtration and is 90% active against leukocyte elastase. The amino acid sequence of the N-terminal portion of the inhibitor was determined and was found to be identical (through 29 amino acids) to that recently reported for the human seminal plasma proteinase inhibitor I (Seemuller et al. (1986) FEBS Lett. 199, 43-48).

Amino Acid Sequence↗
PubMed1987

Production of glycosylated physiologically "normal" human alpha 1-antitrypsin by mouse fibroblasts modified by insertion of a human alpha 1-antitrypsin cDNA using a retroviral vector.

Alpha 1-Antitrypsin (alpha 1AT) deficiency is a hereditary disorder characterized by reduced serum levels of alpha 1AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment alpha 1AT levels in this disorder with physiologically normal human alpha 1AT, we have integrated a full-length normal human alpha 1AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the alpha 1AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi 2 and infected NIH 3T3. The clones produced three mRNA transcripts (5.8, 4.8, and 2.4 kilobases) containing human alpha 1AT sequences, secreted an alpha 1AT molecule recognized by an anti-human alpha 1AT antibody, with the same molecular mass (52 kDa) as normal human alpha 1AT and that complexed with and inhibited human neutrophil elastase. The psi 2 produced alpha 1AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal alpha 1AT purified from human plasma and markedly longer than that of nonglycosylated human alpha 1AT cDNA-directed yeast-produced alpha 1AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human alpha 1AT cDNA into non-alpha 1AT-producing cells, resulting in the synthesis and secretion of physiologically "normal" human alpha 1AT.

Animals↗
PubMed1987

Protection by recombinant alpha 1-antitrypsin Ala357 Arg358 against arterial hypotension induced by factor XII fragment.

The specificity of serpin superfamily protease inhibitors such as alpha 1-antitrypsin or C1 inhibitor is determined by the amino acid residues of the inhibitor reactive center. To obtain an inhibitor that would be specific for the plasma kallikrein-kinin system enzymes, we have constructed an antitrypsin mutant having Arg at the reactive center P1 residue (position 358) and Ala at residue P2 (position 357). These modifications were made because C1 inhibitor, the major natural inhibitor of kallikrein and Factor XIIa, contains Arg at P1 and Ala at P2. In vitro, the novel inhibitor, alpha 1-antitrypsin Ala357 Arg358, was more efficient than C1 inhibitor for inhibiting kallikrein. Furthermore, Wistar rats pretreated with alpha 1-antitrypsin Ala357 Arg358 were partially protected from the circulatory collapse caused by the administration of beta-Factor XIIa.

Animals↗