Molecular cloning and expression of a human phosphodiesterase 4C.

A cDNA coding for a human phosphodiesterase 4C (PDE4C2) was isolated from the mRNA prepared from the glioblastoma cell line, U87. The cDNA contained an ORF of 1818 bp corresponding to a 605 amino acid polypeptide. The sequence differed at the 5' end from the human PDE4C previously reported (Engels, P. et al, 1995 FEBs Letters 358, 305-310) indicating that it represents a novel splice variant of the human PDE4C gene. Evidence was also obtained for a third 5' splice variant. The PDE4C2 cDNA was transfected into both COS 1 cells and yeast cells, and shown to direct the expression of an 80 kD polypeptide by Western blotting using a PDE4C specific antiserum. The activity of cell lysates was typical of PDE4 being specific for cAMP and inhibitable by the selective inhibitor, rolipram. However, the Km for cAMP of the enzyme produced in COS cells was 0.6 microM compared to 2.6 microM for the yeast 4C activity. In addition the COS cell PDE4 activity was much more sensitive to R rolipram than the yeast PDE4 enzyme (IC50 of 23 nM compared to 1648 nM). This difference in rolipram sensitivity was associated with the detection of a high affinity [3H] R rolipram binding site on the COS cell 4C enzyme but not on the yeast expressed enzyme. The results indicate that the enzyme can adopt more than one active conformation, which are distinguished by their interaction with rolipram.

Does ex vivo vascular resistance reflect viability of non-heart-beating donor livers?

AIMS: The use of non-heart-beating (NHB) donor livers is limited by a higher risk for primary nonfunction and the absence of methods to measure this risk. This study was designed to determine whether ex vivo vascular resistance of livers correlates with the length of warm ischemia (WI), and, thus, with viability of NHB livers. METHODS: Porcine livers were recovered after 0, 45, or 90 minutes WI. Livers were flushed by gravity and cold stored for 3 hours. Thereafter, livers were perfused at 4 degrees C. Portal vein (PV) and hepatic artery (HA) vascular resistance were calculated during liver flush-out and during 24 hours of machine perfusion. RESULTS: During flush-out, PV and HA vascular resistance were higher among livers with longer WI times; however, only in the PV did the results reach statistical significance. During machine perfusion, PV vascular resistance was low from the start and remained fairly constant. In contrast, HA vascular resistance was higher at the start but gradually diminished to reach a more constant value after 4-6 hours. No correlation was observed between HA or PV vascular resistance and WI during machine perfusion. CONCLUSIONS: The vascular resistance during ex vivo machine perfusion of NHB livers does not correlate with the extent of WI damage and, therefore, cannot predict organ viability.

Liver fatty acid-binding protein: an early and sensitive plasma marker of hepatocellular damage and a reliable predictor of graft viability after liver transplantation from non-heart-beating donors.

OBJECTIVE: Liver fatty acid-binding protein (L-FABP) is a small protein (15 kD) involved in the intracellular transport of long-chain fatty acids in the liver. The L-FABP is regarded as a sensitive marker for liver cell damage. In a pig model for liver transplantation (LTx) from non-heart-beating donors (NHBD), we evaluated plasma changes of L-FABP early after reperfusion of grafts exposed to increasing periods of warm ischemia (WI). METHODS: Porcine livers were procured after 0, 15, 30, 45, and 60 minutes' WI. After 4 hours' cold ischemia (CI), LTx was performed. Primary graft nonfunction (PNF) and day 4 survival were recorded. Plasma samples were collected prior to and 15, 60, and 180 minutes after graft reperfusion for determination of L-FABP and aspartate transaminase (AST). RESULTS: Early after reperfusion, levels of L-FABP correlated well with the duration of WI. The PNF developed in 100% of animals after 60 minutes of WI, 50% after 30, and 45 minutes' WI, and was absent after no WI and 15 minutes of WI. Day 4 survival was 100% in 0 minutes' WI, 83% in 15 minutes' WI, 50% in 30 and 45 minutes' WI, and 0% in 60 minutes of WI. CONCLUSIONS: Plasma levels of L-FABP correlated well with WI and concomitant hepatocellular damage in LTx from NHBD. Monitoring of posttransplant L-FABP plasma levels is a valuable new tool to quantify early the extent of parenchymal cell damage of NHBD livers and to predict their viability and function.

The matrix metalloproteinases and their natural inhibitors: prospects for treating degenerative tissue diseases.

Uncontrolled matrix metalloproteinase activity is thought to be a cause of the tissue damage observed in many disease processes. None of the drugs currently in use can prevent tissue destruction, and strategies for the development of synthetic inhibitors have been hampered by a poor understanding of the biochemistry of matrix metalloproteinases. Recent cDNA cloning efforts and characterization of recombinant human matrix metalloproteinases have permitted structure-function analysis of the enzymes and their inhibitors. Progress in this area should help indicate a route to rational strategies for designing lead therapeutic compounds.

An analysis of the conformational changes that accompany the activation and inhibition of gelatinase A.

The latent precursors of the matrix metalloproteinases (MMPs) are converted by (4-aminophenylmercuric)acetate to active forms that lose their propeptide as a result of autolysis. C.D. and an active site mutant of progelatinase A (MMP2) were used to demonstrate that, although propeptide removal is accompanied by a decrease in the enzyme's beta-sheet content, the initial activation is achieved with only minor modifications to the conformation. Mixing activated gelatinase A with the natural inhibitor, TIMP-1, resulted in conformational changes that were absent when a synthetic inhibitor was used. The relevance of these results to MMP activation and inhibition is discussed.

Human progelatinase A can be activated by matrilysin.

The activation of human progelatinase A by other matrix metalloproteinases was studied by following both the loss of its N-terminal propeptide and the accompanying increase in the rate of hydrolysis of a synthetic substrate. Activated stromelysin 1 was unable to cause any activation of progelatinase A beyond that slowly occurring by autolysis, but an 8 h incubation with activated matrilysin was able to produce 64% of the activity generated by incubation with (4-aminophenylmercuric)acetate (APMA). Wild-type progelatinase A and a mutant proenzyme that cannot become active were both cleaved by matrilysin to a lower molecular weight species that had lost the propeptide. This shows that matrilysin activates progelatinase A by removing the propeptide in a process that does not require any autolytic cleavages.

The C-terminal (haemopexin-like) domain structure of human gelatinase A (MMP2): structural implications for its function.

In common with most other matrix metalloproteinases, gelatinase A has a non-catalytic C-terminal domain that displays sequence homology to haemopexin. Crystals of this domain were used by molecular replacement to solve its molecular structure at 2.6 A resolution, which was refined to an R value of 17.9%. This structure has a disc-like shape, with the chain folded into a beta-propeller structure that has pseudo four-fold symmetry. Although the topology and the side-chain arrangement are very similar to the equivalent domain of fibroblast collagenase, significant differences in surface charge and contouring are observable on 1 side of the gelatinase A disc. This difference might be a factor in allowing the gelatinase A C-terminal domain to bind to natural inhibitor TIMP-2.

Matrix metalloproteinases and metastatic cancer.

The rationale for matrix metalloproteinase (MMP) inhibition as a means to treat disease progression in breast cancer stems from the apparent involvement of MMPs in the hydrolysis of basement membranes during tumour cell invasion and subsequent metastasis. MMP-mediated matrix remodelling also appears to promote the growth of tumour cells, possibly by facilitating the proliferation and migration of endothelial cells and the neovascularization of tumour tissue. We found that transfection of the C127 breast cancer cell line by MMP-2 (gelatinase A), but not by MMP-1 or MMP-3 (collagenase and stromelysin respectively), gave rise to an invasive and metastatic phenotype. We were surprised to find that this phenotype depended not only on the catalytic properties of MMP-2 but also on properties associated with the MMP-2 non-catalytic C-terminal domain. Experiments with a synthetic gelatinase inhibitor revealed that a single dose could prevent the lungs of nude mice being colonized by the MMP-2 transfectants, and that the inhibitor had to be administered during or shortly after injection of the cells, indicating that an early event, such as the extravasation of the cells into the lung, is gelatinase-dependent in this system. In other studies employing long-term treatment with CT1746, an orally active gelatinase inhibitor, we have previously demonstrated a reduction in primary tumour growth rates, localized spread, and spontaneous metastasis, even when the treatment was commenced several days after tumour implantation. Furthermore, additive effects were recorded when gelatinase inhibitor therapy was combined with cytotoxic drug treatment. Since the gelatinase inhibitors can also inhibit bone resorption in vitro, these observations point to their potential for delaying disease recurrence and reducing rates of bone loss following conventional therapeutic strategies, in metastatic breast cancer.

CDP840: a novel inhibitor of PDE-4.

We present the in vitro characterization of a novel phosphodiesterase type 4 inhibitor, CDP840 (R-[+]-4-[2-¿3-cyclopentyloxy-4-methoxyphenyl¿-2-phenylethyl]pyridine), which has shown efficacy in a phase II allergen challenge study in asthmatics without adverse effects. CDP840 potently inhibits PDE-4 isoenzymes (IC50 2-30 nM) without any effect on PDE-1, 2, 3, 5, and 7 (IC50 > 100 microM). It exhibited no significant selectivity in inhibiting human recombinant isoenzymes PDE-4A, B, C or D and was equally active against the isoenzymes lacking UCR1 (PDE-4B2 and PDE-4D2). In contrast to rolipram, CDP840 acted as a simple competitive inhibitor of all PDE-4 isoenzymes. Studies with rolipram indicated a heterogeneity within all the preparations of PDE-4 isoenzymes, indicative of rolipram inhibiting the catalytic activity of PDE-4 with both a low or high affinity. These observations were confirmed by the use of a PDE-4A variant, PDE-4A330-886, which rolipram inhibited with low affinity (IC50 = 1022 nM). CDP840 in contrast inhibited this PDE-4A variant with similar potency (IC50 = 3.9 nM), which was in good agreement with the Kd of 4.8 nM obtained from [3H]-CDP840 binding studies. Both CDP840 and rolipram inhibited the high-affinity binding of [3H]-rolipram binding to PDE-4A, B, C, and D with similar Kd app (7-19 nM and 3-5 nM, respectively). Thus, the activity of CDP840 at the [3H]-rolipram binding site was in agreement with the inhibitor's activity at the catalytic site. However, rolipram was approximately 100-fold more potent than CDP840 at inhibiting the binding of [3H]-rolipram to mouse brain in vivo. These data clearly demonstrate that CDP840 is a potent selective inhibitor of all PDE-4 isoenzymes. In contrast to rolipram, CDP840 was well-tolerated in humans. This difference, however, cannot at present be attributed to either isoenzyme selectivity or lack of activity in vitro at the high-affinity rolipram binding site (Sr).

The optimal size of attributor for use with the University of Manchester Drug Misuse Database.

This paper describes the anonymous, attributable code which is applied to different individuals when they are reported to the regional drug misuse databases using the University of Manchester system. It then reports on the effect of removing selected data items included in the attributor on the system's ability to distinguish between different drug users reported to the database. All of the attributable codes that did not use the full set of data items were found to reduce the system's ability to distinguish between the reporting of new drug users and new episodes of drug use for users previously reported to the database.

Validation of the University of Manchester Drug Misuse Database.

OBJECTIVE: The study was conducted to assess the validity and quality of data held by one of the UK regional drug misuse databases (DMD). DESIGN: The research was multi-centred and used retrospective analysis to assess the validity of data held on the database. SETTING: The Regional Database is managed at the University of Manchester Drug Misuse Research Unit and uses data returned by medical and non-medical services within the UK's former North Western Regional Health Authority. MATERIAL: The research was largely based on analysis of the reporting or non-reporting to DMD of 1526 presentations by drug users to four community drug teams (CDTs) during the course of 1993. Two datasets were used: the DMD dataset, based on returns to the regional database from the agencies in question; and agency client records. Additionally the data included on a random sample of 300 database forms returned by these CDTs were compared with information contained in client records. MAIN OUTCOME MEASURES: The study reports on how well DMD is functioning in relation to the correct reporting of episodes of problem drug use and the quality of data held. RESULTS: A very high level of agreement (0.875 +/- 0.017, 95% CI, kappa coefficient 0.728) was established between reports sent in to the database and those expected by examination of agency records. The database figures underestimated the total number of episodes that should have been reported by a factor of 0.008. It was also established that 0.906 (+/- 0.018, 95% CI) of the reports made to the database were made correctly, that 0.178 (+/- 0.030, 95% CI) of eligible presentations were not reported, and that 0.166 (+/- 0.030, 95% CI) of ineligible presentations were mistakenly reported. Lastly, it was established that data were unnecessarily missing or inaccurately recorded in 0.027 of cases and that data entry errors occurred in 0.015 of cases. CONCLUSIONS: The validation project showed that the DMD system is very reliable, providing accurate measures of the extent and nature of presenting problem drug use in the region under study.

An in vivo model for screening peptidomimetic inhibitors of gelatinase A.

Gelatinase A, a matrix metalloproteinase, is frequently associated with human solid tumors, and its secretion and activation in the tumor milieu is considered important in the process of angiogenesis, invasion, and metastasis. Consequently, metalloproteinase inhibitors may be of value in the therapy of cancer as well as other disease states involving tissue remodeling and release of biologically active peptide/protein by proteolytic cleavage. Here we describe the development of a rapid screening assay for in vivo activity of peptidomimetic inhibitors of gelatinase A that involves assessment of inhibition of an enzyme-substrate reaction in a circumscribed body compartment, the mouse pleural cavity. As examples of the utility of this assay, in vivo activity of the aryl sulfonamide, sulfamyl urea, morpholino and carboxylic acid functionality at the P3' position of a series of hydroxamic acid inhibitors was examined after administration both intraperitoneally (ip) (to approximate systemic administration) and orally. For up to 2 h after ip administration, all inhibitors tested showed marked activity (> 90% inhibition) at 17 mumol/kg (approximately 10 mg/kg). This activity declined in a dose-responsive manner to insignificant levels at 0.67 mumol/kg (approximately 0.4 mg/kg). Aryl sulfonamides showed significant inhibition (> 50%) for up to 7 h after administration. A higher dosage (136 mumol/kg, approximately 80 mg/kg) was required to reveal oral activity, which was observed only with morpholino compounds (> 50% inhibition). Thus, the model described may be of value in the identification of orally active gelatinase A inhibitors.

Structural characterization of the interaction of mTOR with phosphatidic acid and a novel class of inhibitor: compelling evidence for a central role of the FRB domain in small molecule-mediated regulation of mTOR.

The mammalian target of rapamycin (mTOR) is a large, multidomain protein kinase, which plays a central role in the regulation of cell growth and has recently emerged as an essential target of survival signals in many types of human cancer cells. Here, we report the solution structures of complexes formed between the FKBP12-rapamycin binding (FRB) domain of mTOR and phosphatidic acid, an important cellular activator of the kinase, and between the FRB domain and a novel inhibitor (HTS-1). The overall structure of the FRB domain is very similar to that seen in the ternary complex formed with FKBP12 and the immunosuppressive drug rapamycin; however, there are significant changes within the rapamycin-binding site with important consequences for rational drug design. The surface of the FRB domain contains a number of distinctive features that have previously escaped attention, including a potential new regulatory site on the opposite face to that involved in the binding of rapamycin, which displays the features expected for a specific binding site for a small molecule. The interaction sites for phosphatidic acid and HTS-1 were found to closely match the site responsible for rapamycin binding. In addition, the structures determined for the FRB-phosphatidic acid and FRB-HTS-1 complexes revealed a striking similarity between the conformations of buried portions of the ligands and that seen for the rapamycin backbone in contact with the domain. Our findings further highlight the importance of the FRB domain in small molecule-mediated regulation of mTOR, demonstrate the ability to identify novel inhibitors of mTOR that bind tightly to the rapamycin-binding site in the absence of FKBP12, and identify a potential new regulatory site that may be exploited in the design of new anticancer drugs.

Exploring the potential of PI3K inhibitors for inflammation and cancer.

Considerable biological evidence has accumulated in support of nominating the Class I PI3Ks (phosphoinositide 3-kinases) as excellent targets for the development of novel pharmaceuticals to treat cancer and inflammatory disease. Although it remains a goal to deliver compounds with precise PI3K isoform selectivity in order to minimize safety risks, it is not yet certain that this approach will deliver suitable benefit against disease when tested in the clinic. The UCB strategy, therefore, has been to generate a range of compounds covering a broad spectrum of PI3K isoform inhibition. Scaffold diversity has been accomplished by identifying hits using both pharmacophore search and high-throughput screening campaigns, while modulation of potency and isoform selectivity has been achieved through exploratory medicinal chemistry. Simple, high-throughput cell assays relevant to either inflammation or cancer have then been employed to establish a blueprint for defining how isoform selectivity affects biological potency. I will focus on two compounds from our collection: a pan-PI3K inhibitor and UCB1311236, a compound with significant potency against only the PI3Kgamma isoform. These examples will be used to illustrate the extent to which isoform selectivity informs on compound potency against other kinases and to highlight the risks and benefits of developing compounds with limited isoform selectivity.

Beyond the racist/hooligan couplet: race, social theory and football culture.

This paper draws on recent research to explore the changing cultures of racism in English football. Starting from a critical analysis of key themes in the literature on football it seeks to show that existing analytical frameworks need to be reworked if they are going to adequately account for the complex forms through which racism is expressed in contemporary football cultures. In the course of this analysis we question some of the ways in which the issue of racism in football is collapsed into broader accounts of 'hooliganism' and other forms of violence among football fans. From this starting point the paper draws on some elements of our empirical research in order to outline an alternative way of framing the issues of racism and multicultrralism in football.

Human progelatinase A can be activated by autolysis at a rate that is concentration-dependent and enhanced by heparin bound to the C-terminal domain.

Activation of the latent precursor of human gelatinase A (progelatinase A) was investigated using recombinant proenzyme purified from culture medium conditioned by transfected mouse myeloma cells. A 4.0 microM progelatinase A solution was activated to a maximum of 48% of the activity produced by 4-aminophenylmercuric acetate (APMA) simply by its incubation at 37 degrees C for 12 h, though at lower starting concentrations the rate and extent of activation were reduced. Activation was shown to be the result of a single autolytic cleavage at the Asn80-Tyr81 peptide bond that removes the propeptide and converts the M(r) = 72,000 proenzyme into the M(r) = 66,000 active species also produced by APMA activation. It is proposed that this cleavage is a bimolecular event catalysed by previously activated gelatinase A. The addition of heparin increased by approximately eightfold the initial rate of progelatinase A autolytic activation but did not affect the activation of a deletion mutant that lacked the C-terminal domain [des-(418-631)progelatinase A]. The inference that this increase resulted from an interaction between heparin and the C-terminal domain was supported by the finding that, unlike des-(418-631)gelatinase A, both full-length gelatinase A and the isolated C-terminal domain were able to bind to heparin-Sepharose CL-6B and that, at NaCl concentrations sufficient to abolish this binding, heparin had no effect. We conclude that heparin is able to enhance autolytic activation by acting as a template that approximates active-->latent gelatinase A and suggest that a similar mechanism may account for the cell-surface activation of this enzyme.

Reciprocated matrix metalloproteinase activation: a process performed by interstitial collagenase and progelatinase A.

Gelatinase A, a member of the matrix metalloproteinase (MMP) family, is secreted possessing an 80 amino acid N-terminal propeptide that must be removed in order to generate the active enzyme. Purified progelatinase A was activated to 38% of maximum by a 6 h incubation at 37 degrees C with equimolar concentrations of trypsin-activated interstitial collagenase (another MMP). The increase in activity was accompanied by cleavage of the M(r) 72,000 progelatinase A to the M(r) 66,000 active enzyme that has Y81 as its N-terminus. At low concentrations, progelatinase A was processed via an inactive intermediate, suggesting that its activation is a biphasic process. This was confirmed by the action of collagenase on proE375-->A (a mutant of progelatinase A that cannot become active) because, in this instance, only an M(r) 68,000 species with L38 as the N-terminus was produced. The remaining propeptide amino acids to Y81 could be readily removed by added active gelatinase A, indicating that collagenase works by generating an intermediate that is susceptible to autolytic activation. Although relatively slow, the rate of activation could be increased approximately 10-fold by the addition of 100 micrograms/mL heparin. This binds to the C-terminal domain of collagenase and progelatinase A and presumably acts as a template that positions the reactants close to one another. Collagenase activated by trypsin retains 8 or 14 amino acids of its propeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical characterization of matrilysin. Activation conforms to the stepwise mechanisms proposed for other matrix metalloproteinases.

The latent precursor of matrilysin (EC 3.4.24.23; punctuated metalloproteinase (PUMP) was purified from transfected mouse myeloma cell conditioned medium and was found to contain one zinc atom per molecule which was essential for catalytic activity. Promatrilysin could be activated to the same specific activity by (4-aminophenyl)mercuric acetate, trypsin, and incubation at elevated temperatures (heat activation). Active matrilysin hydrolyzed the fluorescent substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond with a maximum value for kcat/Km of 1.3 x 10(4) M-1 s-1 at the pH optimum of 6.5 and pKa values of 4.60 and 8.65. Activity is inhibited by the tissue inhibitor of metalloproteinases-1 in a 1:1 stoichiometric interaction. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with N-terminal sequencing revealed that, as with all other matrix metalloproteinases similarly studied, promatrilysin activation was accompanied by the stepwise proteolytic removal of an M(r) 9000 propeptide from the N-terminus. The intermediates generated were dependent on the mode of activation used but, in all cases studied, activation terminated with an autocatalytic cleavage at E77-Y78 to yield the final M(r) 19,000 active matrilysin. From an analysis of the stability of the various intermediates, we propose that the sequence L13-K33 is particularly important in protecting the E77-Y78 site from autocatalytic cleavage, thereby maintaining the latency of the proenzyme.