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Biomedical subjects

I M Clark

Publications and source records attributed to I M Clark.

At least 37 records · Page 2Linked to original sources
PubMed1997

Transcriptional activity of the human tissue inhibitor of metalloproteinases 1 (TIMP-1) gene in fibroblasts involves elements in the promoter, exon 1 and intron 1.

The active forms of all of the matrix metalloproteinases (MMPs) are inhibited by a family of specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Inhibition represents a major level of control of MMP activity. A detailed knowledge of the mechanisms controlling TIMP gene expression is therefore important. We have isolated a genomic clone of the human TIMP-1 gene. A 3 kbp XbaI fragment has been sequenced; this fragment contains 1718 bp 5' flanking sequences, exon 1, a 929 bp intron 1 and part of exon 2. Computer analysis reveals 10 consensus sequences for Sp1, six for activating protein 1 (AP-1), six for polyoma enhancer A3 (PEA3), 12 for AP-2 and five CCAAT boxes. The region hybridizing with a murine TIMP-1 promoter fragment has been subcloned and analysed further. RNase protection identifies six transcription start points, making exon 1 up to 48 bp in length. Transient transfection of promoter-chloramphenicol O-acetyltransferase reporter constructs into primary human connective tissue fibroblasts shows that a 904 bp fragment that hybridizes to a murine TIMP-1 promoter fragment contains a functional promoter. Constructs of -738/+95 to -194/+21 are inducible with serum or phorbol ester to a similar extent to the endogenous TIMP-1 gene. These results and further mapping with 5' deletion mutants from the -738/+95 region have demonstrated that an AP-1 site at -92/-86 is essential for basal expression of the gene. Point mutations within this region have further confirmed the role of this site, along with a more minor role for a neighbouring PEA3 site, in basal expression. Deletions from the 3' end also implicate a region across the exon 1/intron 1 boundary and especially +21 to +58 in basal expression. The +21/+58 region contains a putative binding site for the transcription factor leader-binding protein 1 (LBP-1). Gel-shift analysis shows that protein binds specifically to this region, but competition studies suggest that it is unlikely to be LBP-1.

Animals↗
PubMed1997

Spatially distinct expression of two new cytochrome P450s in leaves of Nepeta racemosa: identification of a trichome-specific isoform.

Using a PCR-based approach, two novel cytochrome P450 cDNAs were isolated from a catmint (Nepeta racemosa) leaf cDNA library. The cDNAs (pBSK3C7 and pBSK4C3) were 76.9% identical in their nucleotide sequences, indicating that they are the products of two closely-related genes. A comparison of the sequence of these cDNAs with database sequences indicated that they represent new members of the CYP71 gene family of plant cytochrome P450s. Clone pBSK3C7 contains the full-length coding sequence of a cytochrome P450, whilst pBSK4C3 lacks ca. 6 codons at the 5' end. The cytochromes P450 encoded by these clones were designated CYP71A5 and CYP71A6 (pBSK3C7 and pBSK4C3, respectively). Southern blot analysis indicated that the corresponding genes were present as single copies in the genome of N. racemosa. Northern blot analysis showed that a gene homologous with CYP71A5 was expressed in the related species N. cataria, but no homologue of CYP71A6 was detected in this species. Expression of CYP71A5 in N. racemosa was maximal in flowers, tissues within the apical bud, and young expanded leaves. That of CYP71A6 was maximal in older leaves. Expression of CYP71A5 occurred exclusively in trichomes present on the leaf surfaces, in contrast to that of CYP71A6, which occurred predominantly within the leaf blade tissues.

Amino Acid Sequence↗
PubMed1995

Development of an enzyme-linked immunosorbent assay to measure total TIMP-1 (free TIMP-1 and TIMP-1 in combination with matrix-metalloproteinases) and measurement of TIMP 1 and CRP in serum.

A panel of six monoclonal antibodies (MAbs) was raised against purified human fibroblast tissue inhibitor of metalloproteinase-1 (TIMP-1) and characterised. All possible antibody pairs were tested for their suitability as capture and revealing antibodies in a two-site enzyme-linked immunosorbent assay (ELISA) to measure total TIMP-1 (both free TIMP-1 and TIMP-1 together with matrix metalloproteinases (MMPs)). Using the best combination of MAbs the assay was optimised. The sensitivity of detection of the assay was 1.4 ng/ml, and inter- and intra-assay coefficients of variation were between 10.4-13.7% and 8.8-9.7%, respectively. Dilution series of human cerebrospinal and synovial fluids, plasma and sera paralleled those of the TIMP-1 standard curve indicating that the immunoreactivity detected in these samples was authentic TIMP-1. TIMP-2 shows no detectable cross reactivity in this assay confirming that this ELISA is specific for TIMP-1. The levels of total TIMP-1 and collagenase were measured in conditioned medium from A2058 human melanoma cells cultured in the absence or presence of human recombinant interleukin-1 alpha (hrIL-1 alpha). Total TIMP-1 was also measured in serum samples with known C-reactive protein (CRP) (n = 100) and alpha 1 antichymotrypsin (ACT) (n = 52) concentrations; no correlation was found between TIMP-1 levels and either of these acute phase reactants although the levels of TIMP-1 were raised when compared to normal sera. This ELISA provides a rapid and convenient procedure for the quantitation of total TIMP-1 in human biological fluids and supernatants from cultured cell lines.

Antibodies, Monoclonal↗
PubMed1995

Structure of full-length porcine synovial collagenase reveals a C-terminal domain containing a calcium-linked, four-bladed beta-propeller.

BACKGROUND: The collagenases are members of the family of zinc-dependent enzymes known as the matrix metalloproteinases (MMPs). They are the only proteinases that specifically cleave the collagen triple helix, and are important in a large number of physiological and pathological processes. Structures are known for the N-terminal catalytic' domain of collagenases MMP-1 and MMP-8 and of stromelysin (MMP-3). This catalytic domain alone, which comprises about 150 amino acids, has no activity against collagen. A second domain, of 200 amino acids, is homologous to haemopexin, a haem-binding glycoprotein. RESULTS: The crystal structure of full-length MMP-1 at 2.5 A resolution gives an R-factor of 21.7%. Two domains are connected by an exposed proline-rich linker of 17 amino acids, which is probably flexible and has no secondary structure. The catalytic domain resembles those previously observed, and contains three calcium-binding sites. The haemopexin-like domain contains four units of four-stranded antiparallel beta sheet stabilized on its fourfold axis by a cation, which is probably calcium. The domain constitutes a four-bladed beta-propeller structure in which the blades are scarcely twisted. CONCLUSIONS: The exposed linker accounts for the difficulty in purifying full-length collagenase. The C-terminal domain provides a structural model for haemopexin and its homologues. It controls the specificity of MMPs, affecting both substrate and inhibitor binding, although its role remains obscure. These structural results should aid the design of site-specific mutants which will reveal further details of the specificity mechanism.

Amino Acid Sequence↗
PubMed1995

A fibroblast elongation factor purified from colon carcinoma cells shares sequence identity with TIMP-1.

We have previously reported that human colon cancer cells secrete a factor(s) which induces elongation of colon fibroblasts in vitro. Isolation of this factor led to the identification of a 55kD protein with fibroblast stretching activity. Two internal amino acid sequences identified in this protein (YEI; GFQALGDAADI) share complete homology with tissue inhibitor of metalloproteinase (TIMP-1).

Amino Acid Sequence↗
PubMed1995

Recombinant porcine collagenase: purification and autolysis.

Collagenase is a member of the matrix metalloproteinase family whose members are all capable of degrading extracellular matrix components. The mature form of porcine collagenase has been expressed in Escherichia coli using the pAX5 expression vector. The fusion protein consists of beta-galactosidase at the N-terminus joined to a collagen hinge region and a blood-coagulation factor Xa cleavage site linked to an active form of collagenase. Recombinant collagenase was biologically active in the form of a fusion protein; this was cleaved with factor Xa to yield collagenase with the authentic N terminus (phenylalanine) found in vivo and purified in a single step on a peptide hydroxamic acid affinity column. On purification the recombinant porcine collagenase undergoes autolysis at a number of different bonds in the region connecting the active site domain with the C-terminal hemopexin-like domain. This may represent a loop region of poor secondary structure, making it susceptible to relatively nonspecific cleavage. The N-terminal fragment retains a reduced level of collagenolytic activity, along with that against casein and gelatin.

Amino Acid Sequence↗
PubMed1994

Fragments of human fibroblast collagenase: interaction with metalloproteinase inhibitors and substrates.

On purification, active human fibroblast collagenase breaks down by an autolytic mechanism into two major forms (M(r) 22,000 and M(r) 27,000) and one minor form (M(r) 25,000). The ability of human collagenase to bind to the tissue inhibitor of metalloproteinases (TIMP) and to TIMP-2 resides mainly in the active site area of the 22,000 M(r) N-terminal domain of the molecule, but the 27,000 M(r) C-terminal domain also has a role in stabilizing these interactions. The 22,000 M(r) fragment is able to form a complex with TIMP and TIMP-2 which is stable to gel filtration in a similar manner to the whole molecule, but no such complexes are formed by the 27,000 M(r) fragment. Complex formation with the whole molecule is prevented by EDTA and by 1,10-phenanthroline demonstrating the importance of the active site; additionally TIMP and TIMP-2 will compete with a reversibly bound peptide hydroxamic acid inhibitor for the active site. The inhibition of enzyme activity by TIMP and TIMP-2 is less pronounced in the 22,000 M(r) fragment when compared to the whole molecule and a similar effect is seen with the peptide hydroxamic acid inhibitor and also with alpha 2-macroglobulin, suggesting a role for the C-terminal domain in interacting with these inhibitors. Whole molecule collagenase and the 27,000 M(r) fragment bind to type 1 collagen-Sepharose while the 22,000 M(r) fragment exhibits no such binding, suggesting that the C-terminal domain has an important role in the binding of enzyme to substrate.

Binding Sites↗
PubMed1994

Tissue inhibitor of metalloproteinases (TIMP-1) stimulates the secretion of collagenase from human skin fibroblasts.

This study aimed to investigate possible feedback mechanisms in the control of collagenase (matrix metalloproteinase-1) and tissue inhibitor of metalloproteinases (TIMP-1). Procollagenase, active collagenase, TIMP-1 and collagenase-TIMP complex were applied to human skin and synovial fibroblasts for 48 hours, the cells were washed and the resulting collagenase and TIMP-1 secretion was measured over the following 72 hours using ELISAs. Of the additions, only TIMP-1 showed any measurable effect, stimulating collagenase secretion over a range of 1-8 micrograms/ml. Endotoxin contamination was ruled out as an explanation and the active conformation of the TIMP-1 molecule was shown to be necessary.

Cell Line↗
PubMed1994

Tissue collagenase: serum levels during pregnancy and parturition.

Serum levels of tissue collagenase, matrix metalloproteinase-1, were measured in both longitudinal and cross-sectional studies, in 332 pregnant women and 27 non-pregnant volunteers. The enzyme-linked immunosorbent assay (ELISA) used is the first described to measure collagenase in serum directly, is specific, and is rapid and reproducible. Levels were determined throughout pregnancy, during term and preterm labour, and in the post-partum period. Serum tissue collagenase levels were elevated in pregnancy (P < 0.001). There was no difference between levels of serum collagenase prior to labour at term and those observed during labour. Similarly, there was no significant difference in levels obtained during preterm labour and those at a similar gestation in women who subsequently delivered at term. No significant decrease in levels had occurred by the 4th post-partum day. In view of these findings of unaltered matrix metalloproteinase-1 levels in association with labour, previous reports of raised serum collagenase activity in association with the onset of spontaneous labour, at term and preterm gestation periods, may be due to increased neutrophil collagenase activity.

Collagenases↗
PubMed1994

Using inhibitors of metalloproteinases to treat arthritis. Easier said than done?

Collagenase and stromelysin have a premier role in the irreversible degradation of the extracellular matrix seen in rheumatic disease. It is therefore no surprise that considerable attention has been devoted to developing strategies to reduce their levels in diseased joints. Most efforts have focused on inhibiting the activity of the enzymes, either by increasing the concentration of natural inhibitors such as the TIMPs or by introducing into the joint synthetic compounds that will complex with the enzymes and inactivate them. There have also been studies directed at inhibiting enzyme synthesis. These preclinical studies have been carried out in cell-free and/or cell culture systems and in animal models. Despite promising preclinical data, there have been no stunning successes in the clinical arena. The reasons for this are several. In part, they are rooted in the technical difficulties associated with designing inhibitors of enzyme activity that are of high affinity, and then delivering them to the affected joints while still maintaining specificity and efficacy. The complicated structure of the proteoglycan and collagen that comprise articular cartilage, along with the biochemistry of inflamed synovial tissue, only compound the difficulties. In addition to these technical problems, the lack of fundamental knowledge about the biochemistry and molecular biology of the enzymes has handicapped our efforts. We are just resolving the crystal structure of the metalloproteinases (108) and beginning to understand the mechanisms controlling gene expression (67, 68, 70-72). These advances represent significant achievements in metalloproteinase enzymology and biology and should form the scientific basis for a new generation of effective therapies. For example, knowledge of the active site as derived from the crystal structure of the enzymes may facilitate the development of tightly-binding specific inhibitors which function well in vivo. Similarly, based on our current understanding of mechanisms controlling the regulation of both the TIMP genes and the MMP genes, we are beginning to elucidate how to turn these genes on or off, and hopefully, to modulate disease accordingly. Indeed, although some studies are still at a preclinical level, these possible approaches are becoming a reality (109). Arthritic diseases in general, and rheumatoid arthritis in particular, represent a complicated multifaceted set of clinical disorders. The clinical symptoms and pathologic features result from a cascade of biologic pathways that involve acute and chronic inflammation, the immune response, and metalloproteinase biochemistry.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Tissue inhibitor of metalloproteinases: serum levels during pregnancy and labor, term and preterm.

OBJECTIVE: To determine the levels in serum of tissue inhibitor of metalloproteinases (TIMP) in pregnancy and to examine the possibility of a time course in relation to parturition, both term and preterm. METHODS: Serum tissue inhibitor of metalloproteinases was measured using an enzyme-linked immunosorbent assay. A cross-sectional study was conducted in 333 women during pregnancy, labor, and the postpartum period and in 27 nonpregnant volunteers. Longitudinal data were obtained from 22 women who provided a sample at term, during labor, and in the postpartum period. RESULTS: In uncomplicated pregnancies, serum TIMP levels were low from the onset of pregnancy until 37 weeks' gestation, in comparison to levels in nonpregnant women (P < .001). During the final weeks of pregnancy, levels rose and at 37-42 weeks were similar to nonpregnant levels. The levels did not change with the onset of labor. Serum concentrations of TIMP obtained during preterm labor were elevated compared to a control group of patients at a similar gestation who subsequently delivered at term (P < .01). Serum TIMP levels were significantly higher during the postpartum period than at all other times (P < .001). CONCLUSIONS: Changes in serum TIMP levels during and after pregnancy may parallel the remodeling of the extracellular matrix that takes place throughout this period. Further work is necessary to evaluate the prognostic value of TIMP for preterm labor.

Cross-Sectional Studies↗
PubMed1993

The measurement of collagenase, tissue inhibitor of metalloproteinases (TIMP), and collagenase-TIMP complex in synovial fluids from patients with osteoarthritis and rheumatoid arthritis.

OBJECTIVE: To compare the levels of collagenase, tissue inhibitor of metalloproteinases (TIMP), and collagenase-TIMP complex in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA). This study aims to clarify existing data from previously used enzyme or inhibitor activity assays performed following separation by gel filtration, by using a 1-step enzyme-linked immunosorbent assay (ELISA) for each component. METHODS: Total collagenase, free TIMP, and collagenase-TIMP complex were measured using a newly developed, specific double-antibody sandwich ELISA: RESULTS: Levels of both collagenase and TIMP were significantly higher in RA patients (collagenase 1,560 +/- 150 ng/ml [mean +/- SEM], TIMP 1,610 +/- 130 ng/ml; n = 80) than OA patients (collagenase 420 +/- 90 ng/ml, TIMP 1,050 +/- 60 ng/ml; n = 80), with the difference being especially striking for collagenase. Sixteen RA fluids had detectable levels of collagenase-TIMP complex, compared with only 3 OA fluids. CONCLUSION: The level of total collagenase in SF is greater in RA than OA, while levels of free TIMP show more overlap between the 2 diseases; this may simply reflect the increased inflammation seen in RA, or it may reflect a different disease mechanism.

Arthritis, Rheumatoid↗
PubMed1993

Identification of tissue inhibitor of metalloproteinase-2 (TIMP-2)-progelatinase complex as the third metalloproteinase inhibitor peak in rheumatoid synovial fluid.

The metalloproteinases are a family of enzymes that can degrade all the components of the extracellular matrix. These potent enzymes are often found in proenzyme forms and require activation before the substrate can be digested. To prevent unlimited connective tissue destruction a number of inhibitors exist to limit their activity. In a previous study it was found that metalloproteinases in proenzyme form and metalloproteinase inhibitors were often present in rheumatoid synovial fluids. Two of these inhibitors were identified in rheumatoid synovial fluid as alpha 2 macroglobulin and tissue inhibitor of metalloproteinase (TIMP), the specific metalloproteinase inhibitor. A third inhibitory peak was unidentified. In the study reported here it was shown that this third inhibitor can be purified using gelatin-Sepharose chromatography and consists of TIMP-2 bound to progelatinase (relative molecular weight 72,000) in a similar way to that found in concentrated connective tissue culture medium. The importance of these proteinase inhibitors in synovial fluid is discussed.

Arthritis, Rheumatoid↗
PubMed1992

Large inhibitor of metalloproteinases (LIMP) contains tissue inhibitor of metalloproteinases (TIMP)-2 bound to 72,000-M(r) progelatinase.

Connective-tissue cells in culture produce a family of metalloproteinases which, once activated, can degrade all the components of the extracellular matrix. These potent enzymes are all inhibited by the tissue inhibitor of metalloproteinases (TIMP), and it was thought that this inhibitor was solely responsible for the inhibition of these enzymes within connective tissue. However, other inhibitors have recently been described, including large inhibitor of metalloproteinases (LIMP) present in the culture medium of human foetal lung fibroblasts. Here we show that a large proportion of the inhibitory activity of LIMP consists of 72,000-M(r)-progelatinase bound to TIMP-2, a recently discovered low-M(r) metalloproteinase inhibitor closely related to TIMP. The physiological implications of the secretion of a complex of 72,000-M(r) progelatinase and TIMP-2 are discussed, and the separation of the complex in 6 M-urea is described.

Autoradiography↗
PubMed1992

Polyclonal antibodies against human fibroblast collagenase and the design of an enzyme-linked immunosorbent assay to measure TIMP-collagenase complex.

A polyclonal antibody has been raised against purified human fibroblast collagenase and characterised. This antibody has been used in combination with a monoclonal anti-TIMP antibody in a double antibody sandwich ELISA to measure TIMP-collagenase complex. The assay range was 5-50 ng/ml complex, quantitated in terms of the TIMP component. The assay can measure complex even in the presence of at least a 40-fold excess of free TIMP. The level of TIMP-collagenase complex has been measured in serial samples of synovial fluid from two patients with septic arthritis; high levels of complex are found in some samples, and the level of complex shows an inverse relationship with the level of free TIMP.

Adult↗
PubMed1992

Monoclonal antibodies against human fibroblast collagenase and the design of an enzyme-linked immunosorbent assay to measure total collagenase.

Monoclonal antibodies have been raised against purified human fibroblast collagenase and characterised. One of these antibodies has been used in combination with a polyclonal anticollagenase antibody in a double antibody sandwich ELISA to measure collagenase. The assay range was 2-50 ng/ml collagenase. The assay measures total collagenase, i.e. pro- and active enzyme as well as collagenase in complex with TIMP. The level of collagenase has been measured in sera samples from patients with rheumatoid arthritis and compared with age- and sex-matched controls. The levels measured were: rheumatoid arthritis, 69 +/- 29 ng/ml; normal, 49 +/- 14 ng/ml.

Antibodies, Monoclonal↗