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Biomedical subjects

G Murphy

Publications and source records attributed to G Murphy.

At least 235 records · Page 13Linked to original sources
PubMed1994

Physical mapping of the rat MHC class II genes shows a high level of interspecies conservation.

We report here a pulsed-field gel electrophoresis map of the rat major histocompatibility complex (MHC) class II region. Using probes for the recently discovered Tap-1 and Tap-2 genes and the different MHC class II genes, we found the gene order in the rat MHC (RT1) region to be RT1.H-Tap-1-Tap-2-Bb-Ba-Db-Da. Moreover, the distance between the Tap-1 and the RT1.Da genes is approximately 150 kb. This, together with recent mapping of the RT1 class II region, demonstrates an extensive colinearity in the MHC region of different species.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Mutation of the active site glutamic acid of human gelatinase A: effects on latency, catalysis, and the binding of tissue inhibitor of metalloproteinases-1.

Human gelatinase A, a member of the matrix metalloproteinase family, is secreted from cells as the M(r) 72,000 latent precursor, progelatinase A. The autolytic removal of an N-terminal propeptide generates the M(r) 66,000 active form. Mutants of recombinant progelatinase A, altered such that the proposed active site glutamic acid residue (E375) was replaced by either an aspartic acid (proE375-->D), an alanine (proE375-->A) or a glutamine (proE375-->Q), were purified from medium conditioned by transfected NS0 mouse myeloma cells. Like wild-type progelatinase A, the mutant proenzymes were inactive and could bind tissue inhibitor of metalloproteinases (TIMP)-2 but not TIMP-1 to their C-terminal domains. Their rates of autolytic processing induced by the organomercurial (4-aminophenyl) mercuric acetate, however, were markedly slower and, of the three M(r) 66,000 forms so produced, only E375-->D displayed any proteolytic activity against either a synthetic substrate (kcat/Km = 10% that of the wild-type enzyme) or denatured type I collagen (specific activity = 0.9% that of the wild-type enzyme). ProE375-->A and proE375-->Q could be more rapidly processed to their M(r) 66,000 forms by incubation with a deletion mutant of gelatinase A that has full catalytic activity but lacks the C-terminal domain [delta (418-631) gelatinase A]. These two M(r) 66,000 forms displayed low activity on a gelatin zymogram (approximately 0.01% that of the wild-type enzyme) but, like E375-->D were able to bind TIMP-1 with an affinity equal to that of the activated wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Analysis of the role of the COOH-terminal domain in the activation, proteolytic activity, and tissue inhibitor of metalloproteinase interactions of gelatinase B.

Recombinant human progelatinase B and a COOH terminally truncated version, pro-delta426-688 gelatinase B have been prepared from a myeloma cell expression system. Both proenzymes could be processed to active forms by stromelysin-1 to give an NH2 terminus of Phe88, or by treatment with 4-aminophenylmercuric acetate resulting in an NH2-terminal Met75. The kinetics of activation using either treatment was not affected by removal of the enzyme COOH-terminal domain. The specific activities of both gelatinase B and delta426-688 gelatinase B, activated using either method, were found to be similar using either a quenched fluorescent peptide or gelatin as the substrate. Fibroblast monolayers were shown to mediate processing of both progelatinases at similar rates in the presence of either plasminogen or prostromelysin-1. Active wild-type gelatinase B was inhibited by tissue inhibitor of metalloproteinase (TIMP) -1 at a much faster rate than TIMP-2. COOH-terminal truncation of either enzyme or inhibitor gave a marked reduction in the rate constant for TIMP-1 inhibition but had no effect on the rate of TIMP-2 binding. It can be concluded that the COOH-terminal domain of progelatinase B is not involved in autolytic or cellular activation and does not affect the catalytic activity of the enzyme. However, COOH-terminal domain interactions between active gelatinase B and TIMP-1 significantly enhance the rate of complex formation.

Amino Acid Sequence↗
PubMed1994

Assessment of the role of the fibronectin-like domain of gelatinase A by analysis of a deletion mutant.

The properties of a deletion mutant delta V191-Q364 of gelatinase A, which represents the removal of the fibronectin-like type II repeats defined by exons 5-7, were compared with those of full-length gelatinase A. Both enzymes underwent self-activation over a similar time course in the presence of 4-aminophenylmercuric acetate. The fully active enzymes had similar kcat/Km values for the cleavage of an octapeptide substrate, but the deletion mutant had 50% of the activity of wild type gelatinase A against beta-casein and 10% of the activity against gelatin. The cleavage pattern for gelatin was similar for both enzymes but differed for type IV collagen. Comparison of the rates of association of the tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 and their N-terminal domains to both forms of gelatinase indicated that the fibronectin-like domain plays little role in TIMP binding. The deletion mutant failed to bind to collagen, while the wild type gelatinase bound tightly, indicating that the fibronectin-like domain is the sole site of collagen binding. Both gelatinases could be activated by concanavalin A-activated fibroblasts, suggesting that the fibronectin-like domain is not required for the membrane-mediated activation process.

Animals↗
PubMed1994

Proteolysis of human native and oxidised alpha 1-proteinase inhibitor by matrilysin and stromelysin.

Matrilysin is shown to rapidly inactivate alpha 1PI, an inhibitor of elastase, by cleaving the Pro357-Met358 peptide bond of its reactive centre. The rate of inactivation of alpha 1PI by matrilysin is four times higher than stromelysin. Matrilysin cleaves oxidised alpha 1PI at the Phe352-Leu353 bond, whilst stromelysin cleaves oxidised alpha 1PI at the Met358-Ser359 bond. We conclude that matrilysin is a potent serpinase which could play a role in inflammatory tissue damage by proteolytically inactivating alpha 1PI.

Amino Acid Sequence↗
PubMed1994

Different domain interactions are involved in the binding of tissue inhibitors of metalloproteinases to stromelysin-1 and gelatinase A.

The matrix metalloproteinases gelatinase A and stromelysin-1 have definable N-terminal (catalytic) and C-terminal domains. In order to analyze their interactions with the N- and C-terminal domains of the tissue inhibitors of metalloproteinases TIMP-1 and -2, mutants of both the enzymes and the inhibitors were prepared in which the C-terminal domains had been deleted. Since the Ki values for TIMP inhibition of the matrix metalloproteinases are in the picomolar range, it was not possible to measure these accurately within the sensitivity of available activity assays. Rate constants for the association of the wild-type proteins were therefore determined and systematically compared with those for the deletion mutants. It was found that TIMP-1 binds more rapidly than TIMP-2 to stromelysin-1 and that the C-terminal domain of the enzyme does not affect the rate of association of enzyme and inhibitor. This is in contrast to gelatinase A, where the C-terminal domain has been shown to play an important role in increasing the rate of complex formation with the TIMPs (Willenbrock et al., 1993). The TIMPs are also comprised of an N- and C-terminal domain. By deletion mutagenesis, we found that the C-terminal domain of both TIMPs contributed less to the rate of complex formation with stromelysin-1 than to that with gelatinase A. Hybrids of the N- and C-terminal domains of gelatinase A and stromelysin-1 were prepared and used to analyze further the differences in domain interactions with the TIMPs. They demonstrated that the interactions between the C-terminal domains of enzyme and inhibitor can occur irrespective of the nature of the N-terminal domain. We can conclude that the TIMPs have two major binding regions which associate in different ways with the domains of the enzymes gelatinase A and stromelysin-1. The N-terminal domains of the TIMPs bind to the enzyme catalytic domains to inhibit activity. The TIMP C-terminal domain acts to increase the association rate constant by binding to the N-terminal domain of stromelysin or the C-terminal domain of gelatinase A.

Amino Acid Sequence↗
PubMed1994

Sotalol controlled-release systems for arrhythmias: in vitro characterization, in vivo drug disposition, and electrophysiologic effects.

An array of controlled-release formulations of sotalol were investigated for epicardial drug delivery in short-term and chronic long-term treatment models with dogs. A nondegradable matrix formulation of sotalol made with polyurethane was studied by use of short-term treatment model with dogs, and the electrophysiologic effects were compared with those resulting from an intravenous dose of 2 mg/kg of body weight. Epicardial sotalol-polyurethane matrices were also used in 7-day canine implant studies. A sotalol-silicone rubber matrix was used in 60-day epicardial canine implant studies. A biodegradable poly(dl-lactide-co-glycolide) (PLGA) microsphere formulation of sotalol was also studied as a pericardial injection in another series involving 30-day dog experiments. The short-term treatment electrophysiologic effects observed with the epicardial (left ventricular) implantation of a sotalol-polyurethane matrix formulation were comparable to those observed with intravenous sotalol. However, the total dose delivered by the matrix over a 2-h experimental period was 25 times smaller than the intravenous dose (0.077 versus 2 mg/kg). Furthermore, coronary venous sotalol levels after sotalol-polyurethane matrix implantation were in the therapeutic range (1812.4 +/- 415.1 ng/mL), whereas simultaneous peripheral venous levels were more than 1 order of magnitude lower (149.8 +/- 14.1 ng/mL). An intravenous bolus administration of sotalol (2 mg/kg) resulted in coronary venous levels (1537.1 +/- 44.6 ng/mL) that were very close to simultaneous peripheral venous levels (1428.6 +/- 63.9 ng/mL).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Activation markers on T cells infiltrating melanoma metastases after therapy with dinitrophenyl-conjugated vaccine.

Treatment of metastatic melanoma patients with an autologous vaccine modified by the hapten, dinitrophenyl (DNP), produces a striking immunological effect: the induction of clinically evident inflammatory responses in metastatic tumors. Histological examination shows these tumors to be infiltrated with T lymphocytes. We studied the expression of activation markers on those cells and compared them with matched peripheral blood lymphocytes (PBL) and with lymphocytes extracted from metastases before treatment with DNP-conjugated vaccine. The median fraction of cells that were T cells in post-vaccine tumors was 41%, as compared with 9% in pre-treatment tumors, and those T cells were predominantly CD8+ (mean CD8/CD4 ratio = 5.0). A high proportion of both pre- and post-treatment infiltrating T cells expressed HLA-DR (mean +/- SE = 48% +/- 4%), CD69 (56% +/- 7%), and ganglioside GD3 (68% +/- 5%). This distinguished them from matched PBL in which expression of those markers was significantly lower (HLA-DR = 10% +/- 2%; CD69 = 2% +/- 0.4%; GD3 = 49% +/- 4%). These changes were not accompanied by increased cell-surface expression of interleukin-2 (IL-2) receptors, either CD25 or p75, which were expressed by 1%-2% and 12% of tumor-infiltrating lymphocytes (TIL), respectively. The pattern of activation marker expression that we identified appears to be characteristic of tissue T cells with the memory phenotype. The low expression of IL-2 receptors could indicate functional impairment of TIL in situ, perhaps because of inhibitory molecules produced by melanoma cells.

Biomarkers↗
PubMed1994

T-cell recognition of an allogeneic RT1-Dbu class II MHC peptide.

The allo-antibody response of several rat strains to an unconjugated synthetic 20 amino acid peptide derived from the alpha helical region of the RT1-Du beta chain was tested. The LEW (RT1l) and WAG (RT1u) strains produced little or no antibody; the PVG (RT1c) and DA (RT1av1) strains produced moderate amounts of antibody; while the BN (RT1n) strain produced strong primary and secondary antibody responses. This suggested that the BN strain was able to process and present the RT1-Dbu peptide on its class II molecules. In vitro proliferation studies demonstrated that LEW T cells did not respond to the peptide, whereas BN T cells responded strongly, and that the response in the BN strain was found only in the CD4+ T-cell subset. However, immunisation of BN rats with the RT1-Dbu peptide failed to cause any acceleration of rejection of WAG skin or kidney grafts. Moreover, BN rats primed with WAG skin and kidney grafts did not produce T cells reactive to the RT1-Dbu synthetic peptide. This suggests that the T-cell response of the BN strain to the synthetic major histocompatibility complex peptide was not relevant to the indirect T-cell allo-recognition response to naturally processed RT1-Du beta chains.

Amino Acid Sequence↗
PubMed1994

Pharmacokinetics and pharmacodynamics of MK-383, a selective non-peptide platelet glycoprotein-IIb/IIIa receptor antagonist, in healthy men.

MK-383 (L-tyrosine, N-(n-butylsulfonyl)-O-[4-butyl(4-piperidinyl)], monohydrochloride monohydrate) is a potent and specific platelet fibrinogen receptor antagonist that may be useful in preventing processes that lead to occlusive thrombus formation in the lumen of the blood vessel. Two placebo-controlled phase I trials were completed in 56 healthy volunteers to investigate the safety, tolerability, pharmacokinetics, and pharmacodynamics of MK-383 administered as 1- and 4-hour infusions in the presence and absence of aspirin. When administered to healthy male subjects by constant infusions up to 0.4 microgram/kg/min over 1 hour or up to 0.2 microgram/min over 4 hours, it provided a well-tolerated reversible means of inhibiting platelet function. At infusion rates of 0.25 and 0.15 microgram/kg/min for 1 and 4 hours, respectively, MK-383 extended baseline bleeding time by 2.0- to 2.5-fold and inhibited adenosine diphosphate (ADP)-induced platelet aggregation by at least 80%. The pharmacokinetics of MK-383 include a mean plasma clearance of 329 ml/min, steady-state volume of distribution of 76 L, and half-life of 1.6 hours. The percentage of dose excreted in the urine was 37%. Correlations between MK-383 plasma concentration (C) and inhibition of platelet aggregation were examined by fitting with a sigmoid maximum-effect model. The plasma concentration yielding 50% inhibition (C50) for MK-383 in healthy volunteers is approximately 13 ng/ml, with a Hill coefficient > 5. Based on a naive pooled analysis, an exponential empirical model best describes the MK-383 C-extension of template bleeding time (BTE) relationship. The model indicates that the MK-383 plasma concentration necessary to double BTE is approximately 30 ng/ml (i.e., 2.5-fold greater than the C50 for ADP-induced inhibition of platelet aggregation). The pharmacokinetics of MK-383 was unaffected by pretreatment with 325 mg aspirin 1 day before and 1 hour before infusion. Conversely, aspirin pretreatment reduced C50 and increased bleeding time extension, suggesting that aspirin may have an additive effect with respect to inhibition of platelet function. Based on the putative role of the fibrinogen receptor in thrombotic processes and an acceptable human pharmacokinetic-pharmacodynamic profile, MK-383 should be evaluated in patients with unstable angina.

Analysis of Variance↗
PubMed1994

Folding and stability of the active N-terminal domain of tissue inhibitor of metalloproteinases-1 and -2.

The truncated forms of tissue inhibitor of metalloproteinase-1 and -2 (delta TIMP-1 and -2), comprising the N-terminal active domain, are ideal molecules for structural analysis by intrinsic fluorescence as each contains a single conserved tryptophan residue. In this paper we describe studies on their conformational stability, unfolding/refolding kinetics and the environment of the unique tryptophan as judged by its fluorescence properties in the native state and exposure to an external quencher, acrylamide. Two forms of delta TIMP-2 were studied: delta TIMP-2 T21 derived from the full-length cDNA clone isolated from a mixed-tumour library, and delta TIMP-2 A21 containing the highly conserved V18IRAK22 sequence. In all three delta TIMP proteins the tryptophan environments in the native state appeared to be similar, but substantial differences were seen in their conformational stabilities and refolding kinetics. delta TIMP-1 was approximately twice as stable as delta TIMP-2 T21 and 1.4-fold more stable than delta TIMP-2 A21. This stability difference between delta TIMP-1 and delta TIMP-2 was shown to be independent of N-linked glycosylation. delta TIMP-1 and delta TIMP-2 A21 both showed simple two-state refolding kinetics, whereas delta TIMP-2 T21 refolding was more complex and biphasic in character. These differences between delta TIMP-2 T21 and A21 suggest that residue 21 is a structurally important site in the TIMP protein. All three truncated molecules can be considered as stable independent folding domains ideally suited for further structural analysis.

Base Sequence↗
PubMed1994

Structure-function relationships in the tissue inhibitors of metalloproteinases.

The tissue inhibitors of metalloproteinases (TIMPs) are proteins that specifically inhibit the matrix metalloproteinases. They consist of two distinct structural and functional domains. In order to elucidate the role of these domains, we have prepared mutants of TIMP-1 and TIMP-2 that lack a C-terminal domain. The N-terminal domain alone is an efficient inhibitor of all the matrix metalloproteinases through interaction with the enzyme catalytic domain. The C-terminal domain has at least two separate enzyme binding sites, one for gelatinase A and the other for stromelysin-1. The rate of inhibition of either enzyme is increased by interaction with the TIMP C-terminal domain. As no conformational change is observed, we propose that the rate enhancement is due to an anchoring effect in which binding of the TIMP C-terminal domain aligns the TIMP N-terminal domain with the enzyme active site. Site-directed mutagenesis of TIMP-1 has demonstrated that the N-terminal amino acids, His7 and Gln9, are important for inhibition.

Amino Acid Sequence↗
PubMed1994

The effects of selective inhibitors of matrix metalloproteinases (MMPs) on bone resorption and the identification of MMPs and TIMP-1 in isolated osteoclasts.

We have compared the effects of a general matrix metalloproteinase (MMP) inhibitor (CT435) with those of a concentration-dependent specific gelatinase inhibitor (CT543; Ki < 20 nM) on bone resorption in vitro. The test systems consisted of measuring: (i) the release of 45Ca2+ from prelabelled mouse calvarial explants; (ii) the release of 45Ca2+ from prelabelled osteoid-free calvarial explants co-cultured with purified chicken osteoclasts; and (iii) lacunar resorption by isolated rat osteoclasts cultured on ivory slices. Both CT435 and CT543 dose-dependently inhibited the release of 45Ca2+ from neonatal calvarial bones stimulated by either parathyroid hormone or 1,25-dihydroxyvitamin D3. Moreover, CT543 produced a 40% inhibition at a concentration (10(-8) M) selective for the inhibition of human gelatinases A and B. CT435 (10(-5) M) and CT543 (10(-5) M) partially inhibited the release of 45Ca2+ from osteoid-free calvarial explants by chicken osteoclasts with a maximum of approximately 25% for unstimulated cultures, and approximately 36% for cultures stimulated by interleukin-1 alpha (IL-1 alpha; 10(-10) M). Neither inhibitor prevented lacunar resorption on ivory by unstimulated rat osteoclasts, but the compounds produced a partial reduction in both the number and total surface area of lacunae in IL-1 alpha-stimulated cultures, with maximal action at 10(-5) M. Neither of the inhibitors affected protein or DNA synthesis, nor the IL-1 alpha-stimulated secretion of the lysosomal enzyme beta-glucuronidase. Immunocytochemistry demonstrated that isolated rabbit osteoclasts constitutively expressed gelatinase A and synthesized gelatinase B, collagenase and stromelysin, as well as the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) following IL-1 alpha stimulation. These experiments have shown that in addition to collagenase, gelatinases A and B are likely to play a significant role in bone resorption. They further suggest that MMPs produced by osteoclasts are released into the sub-osteoclastic resorption zone where they participate in bone collagen degradation.

Animals↗