RESUMEN
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.
Asunto(s)
Ácidos Fosfatidicos/farmacología , Proteína 1A de Unión a Tacrolimus/metabolismo , Factores de Transcripción/efectos de los fármacos , Sitios de Unión , Diseño de Fármacos , Humanos , Ligandos , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos , Estructura Terciaria de Proteína , Proteínas , Sirolimus/metabolismo , Serina-Treonina Quinasas TOR , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/químicaRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Inflamación/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Animales , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/clasificación , Fosfatidilinositol 3-Quinasas/clasificación , Monoéster Fosfórico Hidrolasas/metabolismoRESUMEN
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.
Asunto(s)
Circulación Hepática , Hígado , Preservación de Órganos/métodos , Resistencia Vascular , Animales , Supervivencia Celular , Isquemia , Hígado/citología , Hígado/fisiología , Modelos Animales , PorcinosRESUMEN
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.
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Proteínas Portadoras/sangre , Supervivencia de Injerto/fisiología , Paro Cardíaco , Trasplante de Hígado/fisiología , Hígado/patología , Animales , Biomarcadores/sangre , Proteínas de Unión a Ácidos Grasos , Isquemia , Trasplante de Hígado/patología , Valor Predictivo de las Pruebas , Reperfusión , Análisis de Supervivencia , PorcinosRESUMEN
BACKGROUND/AIMS: We searched for factors implicated in early hepatic stellate cell (HSC) activation in diseased liver, by means of suppression subtractive hybridization (SSH). METHODS: SSH was performed between messenger RNA (mRNA) from normal rat HSC and mRNA from HSC, isolated from rats with acute D-galactosamine (Gal)-induced hepatitis. RESULTS: One of the potentially upregulated factors which we found was alpha B-crystallin (ABCRYS), a small heat-shock protein and a chaperone known to protect the cell against protein degradation in conditions of cellular stress and known to associate with various types of intermediate filaments. Upregulation of ABCRYS mRNA in HSC, following Gal-intoxication (3.5-fold) as well as by culturing the HSC on plastic (20-30-fold), was confirmed by quantitative real-time reverse transcription polymerase chain reaction. The expression of ABCRYS protein in human and rat HSC was demonstrated by immunohistochemistry, in vitro and in vivo, in normal and diseased liver. Double-staining co-localized ABCRYS immunoreactivity with alpha-smooth muscle actin immunoreactivity in human liver and with desmin immunoreactivity in rat liver. In vivo upregulation of ABCRYS protein following Gal-intoxication was also shown, by comparison with desmin expression. CONCLUSIONS: Human and rat HSC express ABCRYS mRNA and protein. Both are rapidly upregulated following HSC activation.
Asunto(s)
Cristalinas/genética , Cristalinas/metabolismo , Hígado/metabolismo , Actinas/metabolismo , Animales , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Desmina/metabolismo , Galactosamina/toxicidad , Expresión Génica , Humanos , Inmunohistoquímica , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Hibridación de Ácido Nucleico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas WKY , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Studies on the in vitro hepatitis C virus (HCV) infection are hampered by the lack of an appropriate system to culture permissive cells to be continuously infected with HCV. Trypsinization required for cell passage can lead to possible temporary loss of permissiveness for infection, whereas refreshment of the medium can result in loss of infectious particles necessary for perpetuation of the infection; it is therefore very difficult to maintain a continuous HCV infection in cell cultures. A new infection method was designed and evaluated in order to prevent these unfavourable circumstances. A cell line derived from the human hepatoblastoma cell line Hep G2 was grown in the extracapillary space of a haemodialysis cartridge, in the presence of a HCV-positive inoculum, while the culture medium was recirculated through the intracapillary space, supplying the cells with nutrients and oxygen. HCV RNA could continuously be detected in the cells up to 77 days of culture. Sequence analysis of the HCV hypervariable region 1 (HVR1) revealed that 56% and 75%, respectively, of the clones obtained from the cells at day 20 and 40 after start of the infection were different from the clones obtained from the original inoculum and that certain nucleotide positions in this region were more susceptible to mutations, leading to an alteration in amino acid sequence. As none of these sequences were present in the clones from the inoculum, it is suggested that new HCV quasispecies have emerged as a result of viral replication in the hepatocytes in vitro. This system seems a valuable tool for the in vitro evaluation of antiviral drugs.
Asunto(s)
Hepacivirus/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Técnicas de Cultivo de Célula/métodos , Línea Celular , Medios de Cultivo , Hepacivirus/química , Humanos , Datos de Secuencia Molecular , ARN Viral/análisis , Análisis de Secuencia de Proteína , Factores de Tiempo , Proteínas Virales/química , Replicación ViralRESUMEN
Activation of the tyrosine kinase ZAP 70 has been shown to be crucial to the transduction of the T-cell receptor signalling pathway, which leads ultimately to proliferation, cytokine gene expression and T-cell effector functions. A series of 2-phenylaminopyrimidines have been identified as potent and selective inhibitors of ZAP 70.
Asunto(s)
Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Pirimidinas/química , Transducción de Señal , Proteína Tirosina Quinasa ZAP-70RESUMEN
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.
Asunto(s)
Bases de Datos Factuales/normas , Trastornos Relacionados con Sustancias/epidemiología , Confidencialidad , Inglaterra/epidemiología , Reproducibilidad de los Resultados , Características de la Residencia , Estudios Retrospectivos , Centros de Tratamiento de Abuso de SustanciasRESUMEN
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.
Asunto(s)
Población Negra , Cultura , Prejuicio , Fútbol , Violencia/psicología , Inglaterra , Humanos , Teoría Psicológica , Política Pública , Investigación , SociologíaRESUMEN
Studies on the pathobiology of chronic (long-term) hepatitis B virus (HBV) infection and in vitro drug testing have been hampered by the lack of appropriate systems for culturing susceptible cells chronically infected with HBV. Most of the in vitro studies of HBV replication have been performed with HBV genome-transduced cell lines. In this system, viral production is mainly the result of chromosomal replication. In an in vitro infection system, owing to medium refreshment (which leads to the removal of infectious particles necessary for the perpetuation of infection) and to trypsinization for cell passages, it is difficult, if not impossible, to maintain chronic HBV infection, despite the use of susceptible cells. To circumvent these unfavourable factors for chronic HBV infection in vitro, we cultured microcarrier-attached immortalized human hepatocytes, infected with HBV, in molecularporous (MW 12,000-14,000) membrane (dialysis) bags for a duration of 2 months. HBV covalently-closed-circular (ccc) DNA, HBV precore/core and X mRNAs were detected in the cells cultured in this system following infection until the end of the experiment (day 58), while in classical culture conditions (monolayer), markers of HBV replication were also detected. Production of hepatitis B surface antigen (HBsAg) and HBV DNA was detected and their levels in culture medium (collected at the end of experiments from the molecularporous membrane bags) were increased 2.86- and 3.28-fold respectively. Using Southern blot analysis, HBV replicative intermediates could also be demonstrated throughout the experiments. However, integrated HBV DNA was not present. In contrast, HBV ccc DNA, HBV precore/core and X mRNAs, and replicative intermediates were not demonstrable in FTO 2B rat hepatoma cells infected in the same manner in parallel experiments. This in vitro infection system, using susceptible, immortalized human hepatocytes, therefore provides a new tool for studying the long-term effect of HBV infection, mainly involving episomal replication in hepatocytes, and for drug testing.
Asunto(s)
Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/fisiopatología , Hígado/citología , Replicación Viral , Southern Blotting , Cápsulas , Células Cultivadas , Células Inmovilizadas , Medios de Cultivo , ADN Circular/análisis , ADN Viral/análisis , Antígenos del Núcleo de la Hepatitis B/biosíntesis , Antígenos de Superficie de la Hepatitis B/biosíntesis , Humanos , Inmunohistoquímica , Hígado/metabolismo , ARN Mensajero/análisis , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Virología/métodosRESUMEN
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).
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa/farmacología , Piridinas/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Animales , Unión Competitiva , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Catálisis/efectos de los fármacos , AMP Cíclico/biosíntesis , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Cobayas , Humanos , Masculino , Ratones , Inhibidores de Fosfodiesterasa/química , Unión Proteica/efectos de los fármacos , Piridinas/química , Pirrolidinonas/antagonistas & inhibidores , Pirrolidinonas/metabolismo , Rolipram , TritioRESUMEN
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.
Asunto(s)
Metaloendopeptidasas/metabolismo , Metástasis de la Neoplasia , Animales , Gelatinasas/análisis , Gelatinasas/genética , Gelatinasas/metabolismo , Humanos , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/patología , Metaloproteinasa 2 de la Matriz , Metaloendopeptidasas/análisis , Metaloendopeptidasas/genética , Invasividad Neoplásica , Metástasis de la Neoplasia/patología , Transfección , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Precursores Enzimáticos/metabolismo , Gelatinasas/metabolismo , Metaloendopeptidasas/metabolismo , Receptores de Superficie Celular/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Animales , Células CHO , Cricetinae , Activación Enzimática , Precursores Enzimáticos/genética , Gelatinasas/genética , Cinética , Metaloendopeptidasas/genética , MutagénesisRESUMEN
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.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/genética , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Catálisis , Clonación Molecular , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , ADN Complementario , Expresión Génica , Humanos , Datos de Secuencia Molecular , Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Sistemas de Información , Sistemas de Registros Médicos Computarizados , Trastornos Relacionados con Sustancias/epidemiología , Recolección de Datos , Procesamiento Automatizado de Datos , Inglaterra/epidemiología , Femenino , Humanos , Masculino , Reproducibilidad de los ResultadosRESUMEN
The expression of the complete human gastric lipase (HGL) gene in Saceharomyces cerevisiae grown in defined medium resulted in the secretion of active recombinant HGL (rec.HGL) to levels of up to approximately 11 mg/liter. Of the total measurable HGL activity, 90% was detected by assaying intact cells, suggesting that the majority of rec.HGL produced was secreted but stayed attached to the cell wall. The remaining 10% was present in the growth medium and from this source active rec.HGL was purified 300-fold by a combination of hydrophobic interaction and ion-exchange chromatography. Rec.HGL migrated on reduced SDS-PAGE as three bands with estimated molecular masses of 47,45, and 43 kDa. All three forms cross-reacted with an antibody raised to natural HGL and their treatment with Endo H showed them to be N-linked glycosylation variants of a single polypeptide. The 47-kDa species was isolated using lentil lectin Sepharose 4B and shown to possess a specific activity comparable to that of the natural enzyme. Rec.HGL had an acid pH activity optimum using either tributyrin or olive oil as substrate and did not lose activity if incubated in the presence of pepsin at pH 2.0. These results demonstrate that HGL secreted by Saccharomyces cerevisiae retained those properties of the natural enzyme required for its use in the treatment of pancreatic insufficiency.
Asunto(s)
Lipasa/biosíntesis , Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae/enzimología , Cromatografía en Agarosa , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Vectores Genéticos , Glicosilación , Hexosaminidasas/metabolismo , Humanos , Lipasa/química , Lipasa/genética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa , Peso Molecular , Saccharomyces cerevisiae/genética , Transformación Genética , Triglicéridos/químicaRESUMEN
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.
Asunto(s)
Gelatinasas/antagonistas & inhibidores , Gelatinasas/química , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/química , Secuencia de Aminoácidos , Dicroismo Circular , Activación Enzimática/efectos de los fármacos , Gelatinasas/metabolismo , Glicoproteínas/farmacología , Humanos , Metaloproteinasa 2 de la Matriz , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Mutación , Acetato Fenilmercúrico/análogos & derivados , Acetato Fenilmercúrico/farmacología , Inhibidores de Proteasas/farmacología , Conformación Proteica , Precursores de Proteínas/química , Inhibidores Tisulares de MetaloproteinasasRESUMEN
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.
Asunto(s)
Gelatinasas/química , Gelatinasas/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Electroquímica , Hemopexina/química , Humanos , Metaloproteinasa 2 de la Matriz , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Inhibidores de Proteasas/metabolismo , Estructura Secundaria de Proteína , Proteínas/metabolismo , Homología de Secuencia , Relación Estructura-Actividad , Porcinos , Inhibidor Tisular de Metaloproteinasa-2RESUMEN
Membrane-type matrix metalloproteinase (MT-MMP) messenger RNA and protein expression were shown to be elevated in human fibroblasts following treatment with concanavalin A, coincident with the induction of the ability to process progelatinase A. CHO cells transfected with the cDNA for MT-MMP were able to process both wild type progelatinase A and a catalytically inactive mutant, E375A progelatinase A. Both proenzymes were converted to a 68-kDa intermediate (reducing gels) form, but only the wild type enzyme was processed further to a 66-kDa end product. In contrast, both forms of progelatinase were processed via the 68-kDa intermediate to 66 kDa by concanavalin A-stimulated fibroblasts. Further study of the processing of E375A progelatinase A by plasma membrane preparations from concanavalin A-stimulated fibroblasts showed that addition of active gelatinase A enhanced processing to the mature form. It was concluded that cell membrane-mediated activation of progelatinase A could be via a cascade involving both MT-MMP and intermolecular autolytic cleavage.
Asunto(s)
Precursores Enzimáticos/metabolismo , Gelatinasas/metabolismo , Expresión Génica , Metaloendopeptidasas/metabolismo , Procesamiento Proteico-Postraduccional , Concanavalina A/farmacología , Activación Enzimática , Inducción Enzimática , Precursores Enzimáticos/biosíntesis , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Gelatinasas/biosíntesis , Expresión Génica/efectos de los fármacos , Glicoproteínas/farmacología , Humanos , Cinética , Metaloendopeptidasas/biosíntesis , Mutagénesis Sitio-Dirigida , Mutación Puntual , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas/farmacología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Inhibidor Tisular de Metaloproteinasa-2 , Inhibidores Tisulares de MetaloproteinasasRESUMEN
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.