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Biomedical subjects

G Murphy

Publications and source records attributed to G Murphy.

At least 145 records · Page 8Linked to original sources
PubMed1998

TNF-alpha converting enzyme (TACE) is inhibited by TIMP-3.

TNF-alpha converting enzyme (TACE; ADAM-17) is a membrane-bound disintegrin metalloproteinase that processes the membrane-associated cytokine proTNF-alpha to a soluble form. Because of its putative involvement in inflammatory diseases, TACE represents a significant target for the design of specific synthetic inhibitors as therapeutic agents. In order to study its inhibition by tissue inhibitors of metalloproteinases (TIMPs) and synthetic inhibitors of metalloproteinases, the catalytic domain of mouse TACE (rTACE) was overexpressed as a soluble Ig fusion protein from NS0 cells. rTACE was found to be well inhibited by peptide hydroxamate inhibitors as well as by TIMP-3 but not by TIMP-1, -2 and -4. These results suggest that TIMP-3, unlike the other TIMPs, may be important in the modulation of pathological events in which TNF-alpha secretion is involved.

ADAM Proteins↗
PubMed1998

Collagenase 2 (MMP-8) expression in murine tissue-remodeling processes. Analysis of its potential role in postpartum involution of the uterus.

Neutrophil collagenase or collagenase 2 (MMP-8) is unique among the family of matrix metalloproteinases (MMPs) because of its exclusive pattern of expression in inflammatory conditions. At present, no evidence of the occurrence of this enzyme in tissues other than human has been reported. In this work, we have cloned the murine homologue of human collagenase 2. The isolated cDNA contains an open reading frame coding for a polypeptide of 465 amino acids, which is 74% identical to its human counterpart. The mouse collagenase 2 exhibits the domain structure characteristic of several MMPs, including a signal sequence, a prodomain with the cysteine residue essential for enzyme latency, an activation locus with the Zinc-binding site, and a COOH-terminal fragment with sequence similarity to hemopexin. It also contains the three conserved residues (Tyr-209, Asp-230, and Gly-232) located around the Zinc-binding site and are distinctive of the collagenase subfamily. Northern blot analysis of RNAs isolated from a variety of mouse tissues revealed that collagenase 2 is expressed at late stages during mouse embryogenesis, coinciding with the appearance of hematopoietic cells. In addition, collagenase 2 was highly expressed in the postpartum uterus starting at 1 day postpartum and extending up to 5 days. Enzymatic analysis revealed that matrilysin, another MMP overexpressed in uterine tissue, is able to activate murine procollagenase 2. These data suggest that both enzymes could form an activation cascade resulting in the generation of the collagenolytic activity required during the process of massive connective tissue resumption occurring in the involuting uterus.

Amino Acid Sequence↗
PubMed1998

Kinetic analysis of the mechanism of interaction of full-length TIMP-2 and gelatinase A: evidence for the existence of a low-affinity intermediate.

We have undertaken a detailed analysis of the mechanism of inhibition of matrix metalloproteinase-2 (gelatinase A) by tissue inhibitor of metalloproteinase-2 (TIMP-2). Quenched fluorescent substrates have been used to analyze the rate of inhibition of gelatinase A by TIMP-2 over a wide range of TIMP-2 concentrations. When the values of the observed rate constant for the inhibition are plotted against TIMP-2 concentration, saturation is observed at high concentrations, providing evidence for formation of an intermediate in the pathway. Rate constants for the formation and dissociation of the intermediate are 5.9 x 10(6) M-1 s-1 and 6.3 s-1 respectively, giving a Ki for the initial step of approximately 1 microM. The rate constant for the association of the final complex is 33 s-1. By studying the dissociation of 125I-labeled TIMP-2 from a gelatinase A-TIMP-2 complex using ligand exchange experiments, we obtained a rate constant for the dissociation of the final stable complex of 2 x 10(-)8 s-1. This gives a value for the overall dissociation constant of approximately 0.6 fM.

Binding, Competitive↗
PubMed1998

Induction of matrix metalloproteinase activation cascades based on membrane-type 1 matrix metalloproteinase: associated activation of gelatinase A, gelatinase B and collagenase 3.

SW1353 chondrosarcoma cells cultured in the presence of interleukin-1, concanavalin A or PMA secreted procollagenase 3 (matrix metalloproteinase-13). The enzyme was detected in the culture medium by Western blotting using a specific polyclonal antibody raised against recombinant human procollagenase 3. Oncostatin M enhanced the interleukin-1-induced production of procollagenase 3, whereas interleukin-4 decreased procollagenase 3 synthesis. The enzyme was latent except when the cells had been treated with concanavalin A, when a processed form of 48 kDa, which corresponds to the active form, was found in the culture medium and collagenolytic activity was detected by degradation of 14C-labelled type I collagen. The concanavalin A-induced activation of procollagenase 3 coincided with the processing of progelatinase A (matrix metalloproteinase-2) by the cells, as measured by gelatin zymography. In addition, progelatinase B (matrix metalloproteinase-9) was activated when gelatinase A and collagenase 3 were in their active forms. Concanavalin A treatment of SW1353 cells increased the amount of membrane-type-1 matrix metalloproteinase protein in the cell membranes, suggesting that this membrane-bound enzyme participates in an activation cascade involving collagenase 3 and the gelatinases. This cascade was effectively inhibited by tissue inhibitors of metalloproteinases-2 and -3. Tissue inhibitor of metalloproteinases-1, which is a much weaker inhibitor of membrane-type 1 matrix metalloproteinase than tissue inhibitors of metalloproteinases-2 and -3 [Will, Atkinson, Butler, Smith and Murphy (1996) J. Biol. Chem. 271, 17119-17123], was a weaker inhibitor of the activation cascade.

Blotting, Western↗
PubMed1998

Inhibition of matrix metalloproteinase 2 maturation and HT1080 invasiveness by a synthetic furin inhibitor.

The close correlation observed between matrix metalloproteinase 2 (MMP-2) activation and metastatic progression in various tumors suggests that MMP-2 is a 'master switch' triggering tumor spread. Recently, membrane type 1 MMP (MT1-MMP) was identified as a potential physiological activator of MMP-2. Like all other MMPs, MT1-MMP possesses a pro-domain which must be removed for the enzyme to acquire its catalytic potential. The presence of a typical recognition motif (RXKR) for the furin-like convertases at the end of its pro-domain suggests a potential role for these proteinases in MT1-MMP processing. In order to evaluate the implication of furin in pro-MT1-MMP processing, we treated HT1080 cells with a synthetic furin inhibitor and monitored their ability to activate pro-MMP-2 as well as their invasive potential. Our results demonstrated that the furin inhibitor decreased pro-MT1-MMP processing as well as pro-MMP-2 activation and cell invasiveness. Therefore, our data bring further evidence that furin is a key factor in the maturation of MMPs associated with the invasive and metastatic potential of tumor cells.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana.

The plant Arabidopsis thaliana (Arabidopsis) has become an important model species for the study of many aspects of plant biology. The relatively small size of the nuclear genome and the availability of extensive physical maps of the five chromosomes provide a feasible basis for initiating sequencing of the five chromosomes. The YAC (yeast artificial chromosome)-based physical map of chromosome 4 was used to construct a sequence-ready map of cosmid and BAC (bacterial artificial chromosome) clones covering a 1.9-megabase (Mb) contiguous region, and the sequence of this region is reported here. Analysis of the sequence revealed an average gene density of one gene every 4.8 kilobases (kb), and 54% of the predicted genes had significant similarity to known genes. Other interesting features were found, such as the sequence of a disease-resistance gene locus, the distribution of retroelements, the frequent occurrence of clustered gene families, and the sequence of several classes of genes not previously encountered in plants.

Arabidopsis↗
PubMed1998

MT1-MMP on the cell surface causes focal degradation of gelatin films.

The membrane-type matrix metalloproteinases (MT-MMPs) are a subclass of the matrix metalloproteinase (MMP) family which uniquely possess a C-terminal transmembrane domain and are initiators of an activation cascade for progelatinase A (MMP-2). Recent studies have shown that they can also efficiently directly degrade a number of matrix macromolecules. We now show that cells expressing MT1-MMP on their cell surfaces cause subjacent proteolysis of a gelatin film and that this proteolysis is inhibited by TIMP-2 but not by TIMP-1. These data indicate that expression of MT1-MMP on the cell surface may lead to both progelatinase A activation and extracellular matrix degradation.

Animals↗
PubMed1998

The TIMP2 membrane type 1 metalloproteinase "receptor" regulates the concentration and efficient activation of progelatinase A. A kinetic study.

We have used C-terminal domain mutants to further define the role of interactions of progelatinase A and membrane type 1 matrix metalloproteinase (MT1 MMP) in the binding of TIMP2 and in the cell-associated activation of progelatinase A. Soluble constructs of MT1 MMP were used to demonstrate that binding with TIMP2 occurs primarily through N-terminal domain interactions, leaving the C-terminal domain free for interactions with progelatinase A. The rate of autolytic activation of progelatinase A initiated by MT1 MMP cleavage could be potentiated by concentration of the proenzyme by binding to heparin. Residues 568-631 of the progelatinase A C-terminal domain are important in formation of the heparin binding site, since replacement of this region with the corresponding stromelysin-1 sequence abolished binding to heparin and the potentiation of activation. The same region of gelatinase A was required for binding of latent and active enzyme to TIMP2, but residues 418-474 were not important. A similar pattern was seen using cell membrane-associated MT1 MMP; residues 568-631 were required for binding and activation of progelatinase A, whereas residues 418-474 were not. Neither region was required for activation in solution. The addition of TIMP2 to HT1080 membrane preparations expressing MT1 MMP, but depleted of endogenous TIMP2, resulted in potentiation of progelatinase A activation. This effect was dependent upon TIMP2 binding to MT1 MMP rather than at an independent membrane site. Together, the data suggest that TIMP2 forms a receptor with MT1 MMP that regulates the concentration and efficient generation of functionally active gelatinase A.

Animals↗
PubMed1998

Human myeloma cells shed the interleukin-6 receptor: inhibition by tissue inhibitor of metalloproteinase-3 and a hydroxamate-based metalloproteinase inhibitor.

Interleukin-6 (IL-6) is the major growth factor for human myeloma cells, exerting its effect through the IL-6 receptor (IL-6R). A soluble form of IL-6R (sIL-6R) has been identified, which increases the sensitivity of myeloma cells to IL-6. In patients with multiple myeloma (MM), serum concentrations of sIL-6R are elevated and associated with poor prognosis. The present study was undertaken to determine whether proteolytic cleavage of IL-6R could contribute to sIL-6R release from human myeloma cells, and also to identify the class of proteinase responsible for this event. Human myeloma cell lines were shown to express IL-6R upon their surface and also to release sIL-6R into culture supernatants. In addition, phorbol 12-myristate 13-acetate (PMA) stimulated a loss of IL-6R from the cell surface, with a corresponding increase in the concentration of sIL-6R in the supernatant. Inhibitors of serine and cysteine proteinases, and tissue inhibitor of metalloproteinase (TIMP) -1 and TIMP-2, were shown to have no effect on the magnitude of sIL-6R release. In contrast, TIMP-3 and a hydroxamate-based metalloproteinase inhibitor (BB-94), inhibited both constitutive and PMA-induced release of sIL-6R. Myeloma cells freshly isolated from the bone marrow of a patient with MM were also shown to express IL-6R upon their surface, and to shed this receptor in response to PMA. These data demonstrate that increased proteolytic cleavage of IL-6R, mediated by a non-matrix-type metalloproteinase, is likely to contribute to the elevated concentrations of sIL-6R found in the serum of patients with MM. Inhibition of sIL-6R release by hydroxamate-based metalloproteinase inhibitors may represent a novel therapeutic approach to the treatment of MM.

Bone Marrow Cells↗
PubMed1998

Distinct T-cell subtypes induced with whole cell and acellular pertussis vaccines in children.

Recent clinical trials have demonstrated that new generation acellular pertussis vaccines can confer protection against whooping cough. However, the mechanism of protective immunity against Bordetella pertussis infection induced by vaccination remains to be defined. We have examined cellular immune responses in children immunized with a range of acellular and whole cell pertussis vaccines. Immunization of children with a potent whole-cell vaccine induced B. pertussis-specific T cells that secreted interferon-gamma (IFN-gamma), but not interleukin-5 (IL-5). In contrast, T cells from children immunized with acellular pertussis vaccines secreted IFN-gamma and/or IL-5 following stimulation with B. pertussis antigens in vitro. These observations suggest that protective immunity conferred by whole-cell vaccines, like natural immunity, is mediated by type 1 T cells, whereas the mechanism of immune protection generated with acellular vaccines may be more heterogeneous, involving T cells that secreted type 1 and type 2 cytokines.

Antigens, Bacterial↗
PubMed1998

Two-month treatment of obese subjects with the oral growth hormone (GH) secretagogue MK-677 increases GH secretion, fat-free mass, and energy expenditure.

Obesity is associated with blunted GH secretion, unfavorable body composition, and increased cardiovascular mortality. The objective of this study was to investigate the effects of oral treatment with the GH secretagogue MK-677 on GH secretion and body composition in otherwise healthy obese males. The study was randomized, double blind, parallel, and placebo controlled. Twenty-four obese males, aged 18-50 yr, with body mass indexes greater than 30 kg/m2 and waist/hip ratios greater than 0.95, were treated with MK-677 25 mg (n = 12) or placebo (n = 12) daily for 8 weeks. Serum insulin-like growth factor I (IGF-I) increased approximately 40% with MK-677 treatment (P < 0.001 vs. placebo). Serum IGF-binding protein-3 was also significantly increased (P < or = 0.001 vs. placebo). GH and PRL (peak and area under the curve values) were significantly increased after the initial dose of MK-677. Significant increases, with the exception of peak PRL, persisted at 2 and 8 weeks of treatment. The increases in GH and PRL after the initial dose were significantly greater than the increase seen after multiple doses. Serum and urinary concentrations of cortisol were not increased at 2 and 8 weeks (P = NS, vs. placebo). Fat-free mass increased significantly in the MK-677 treatment group when determined with dual energy x-ray absorptiometry (P < 0.01) or using a four-compartment model (P < 0.05). Total and visceral fat were not significantly changed with active therapy. The basal metabolic rate was significantly increased at 2 weeks of MK-677 treatment (P = 0.01) but not at 8 weeks (P = 0.1). Fasting concentrations of glucose and insulin were unchanged, whereas an oral glucose tolerance test showed impairment of glucose homeostasis at 2 and 8 weeks. We conclude that 2-month treatment with MK-677 in healthy obese males caused a sustained increase in serum levels of GH, IGF-I, and IGF-binding protein-3. The effects on cortisol secretion were transient. Changes in body composition and energy expenditure were of an anabolic nature, with a sustained increase in fat-free mass and a transient increase in basal metabolic rate. Further studies are needed to evaluate whether a higher dose of MK-677 or a more prolonged treatment period can promote a reduction in body fat.

Adult↗
PubMed1998

The activation of ProMMP-2 (gelatinase A) by HT1080 fibrosarcoma cells is promoted by culture on a fibronectin substrate and is concomitant with an increase in processing of MT1-MMP (MMP-14) to a 45 kDa form.

We have assessed the effect of fibronectin and laminin-1 on the expression of molecules involved in the activation pathway of MMP-2, a key proteinase in tissue remodelling. HT1080 fibrosarcoma cells cultured on fibronectin were shown to activate endogenous MMP-2, to a level comparable with that elicited by treatment with phorbol ester. In contrast, the MMP-2 expressed by HT1080 cells cultured on laminin-1 was mainly in the pro- (inactive form). Culture of the cells on peptide fragments of fibronectin derived from the central cell binding domain also promoted MMP-2 activation, indicating that signals via fibronectin binding to integrin receptors may be involved. HT1080 cells cultured on immobilised antibodies to the alpha5 and beta1 integrin subunits secreted levels of active MMP-2 similar to those observed for full length fibronectin, whereas cells cultured on an antibody to the alpha6 integrin subunit secreted mainly proMMP-2. The data demonstrate that the activation of MMP-2 by HT1080 cells is regulated by the nature of the extracellular matrix, and that signals via the alpha5beta1 integrin receptor may be involved in the fibronectin induced up-regulation of MMP-2 activation. We then assessed the effect of fibronectin on the components of the putative MT1-MMP/TIMP-2 'receptor' complex implicated in MMP-2 activation. Levels of TIMP-2 protein expressed by HT1080 cells did not vary detectably between cells cultured on fibronectin or laminin-1. However, the expression of MT1-MMP protein was up-regulated when the cells were cultured on fibronectin, which could be attributed to an increase in levels of a truncated 45 kDa form. Parallel studies using gelatin zymography demonstrated that the up-regulation of the production of the 45 kDa band was concomitant with MMP-2 activation. Inhibitor studies revealed that the truncation of MT1-MMP to a 45 kDa form is MMP mediated, although not inhibited by TIMP-1. In vitro, the 45 kDa form could be generated by cleavage of membrane-bound native MT1-MMP with several recombinant MMPs, including both active MT1-MMP and MMP-2. The implication that either MMP-2 or MT1-MMP can process MT1-MMP to 45 kDa, raises the possibility that truncation of MT1-MMP represents a self-regulatory end-point in the activation pathway of MMP-2.

Cell Adhesion↗
PubMed1998

Effects of collagenase-cleavage of type I collagen on alpha2beta1 integrin-mediated cell adhesion.

In this paper we show that collagenase-3 cleavage of type I collagen has a marked effect on alpha2beta1 integrin-mediated interactions with the collagen fragments generated. Isolated alpha2beta1 integrin and alpha2 integrin A-domain were found to bind to both native collagen and native 3/4 fragment and, to a lesser degree, native 1/4 fragment. Whole integrin and integrin A-domain binding were lost after heat denaturation of the collagen fragments. At physiological temperature, cell adhesion to triple-helical 3/4 fragment via alpha2beta1 integrin was still possible; however, no alpha2beta1 integrin-mediated adhesion to the 1/4 fragment was observed. Unwinding of the collagen fragment triple helices by heating to physiological temperatures prior to adsorption to plastic tissue culture plates resulted in total abrogation of HT1080 cell attachment to either fragment. These results provide significant evidence in support of a role for matrix-metalloproteinase cleavage of the extracellular matrix in modifying cell-matrix interactions.

Animals↗
PubMed1998

Treatment with the oral growth hormone secretagogue MK-677 increases markers of bone formation and bone resorption in obese young males.

The effect of 2 months of treatment with the oral growth hormone (GH) secretagogue MK-677 on markers of bone metabolism was determined in healthy obese male subjects. This was a randomized, double-blind, parallel, placebo-controlled study. Twenty-four healthy obese males, 19-49 years of age, with body mass index > 30 kg/m2 were treated with MK-677 (25 mg/day; n = 12) or placebo (n = 12) for 8 weeks. MK-677 increased markers of bone formation; a 23% increase in the carboxy-terminal propeptide of type I procollagen levels and a 28% increase in procollagen III peptide levels were seen with as little as 2 weeks of MK-677 treatment (p < 0.01 and p = 0.001 vs. placebo, respectively) while a 15% increase in serum levels of osteocalcin was not detected until 8 weeks of treatment (p < 0.01 vs. placebo). Markers of bone resorption were induced within 2 weeks of treatment with MK-677; serum levels of the carboxy-terminal cross-linked telopeptide of type I collagen were increased 26% at 8 weeks (p = 0.001 vs. placebo), and urine hydroxyproline/creatinine and calcium/creatinine ratios at 8 weeks were increased by 23% (p < 0.05 vs. placebo) and 46% (p < 0.05 vs placebo), respectively, MK-677 increased serum insulin-like growth factor binding protein-5 (IGFBP-5) by 43-44% after 2-8 weeks of treatment (p < 0.01 vs. placebo). Serum IGFBP-4 was increased by 25% after 2 weeks of treatment (p < 0.001 vs. placebo) but no significant change from baseline was observed after 8 weeks of treatment. Plasma interleukin-6 was not significantly changed by active treatment. In conclusion, short-term treatment of healthy obese male volunteers with the GH secretagogue MK-677 increases markers of both bone resorption and formation. Large increases in serum levels of IGF-1 and IGFBP-5 and a transient increase in serum IGFBP-4 were found. Future long-term studies are needed to investigate if prolonged treatment with MK-677 increases bone mass.

Administration, Oral↗