Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mutation of residue W60d of thrombin, located 17 A from the Na+ binding site, suppresses Na+ binding and the functional differences between the slow and fast forms. The molecular basis for the long-range effect of this mutation is provided by a conspicuous network of water molecules which connects the Na+ binding environment to the specificity sites S1 and S2 of the enzyme. The mutation appears to stabilize thrombin in a hybrid conformation that is overall similar to the slow form, but with the fibrinogen recognition site functioning as in the fast form. It also affects the switch in specificity from fibrinogen to protein C linked to the release Na+ and the fast-->slow conversion. Under physiological conditions of pH, temperature and NaCl concentration, the W60dS mutant behaves as an anticoagulant. It has a reduced activity toward fibrinogen by 22-fold, while the reduction of protein C activation in the presence of saturating concentrations of thrombomodulin is less than 2-fold. Even more remarkable is the cleavage of fibrin I monomer leading to release of fibrinopeptide B, which is reduced by more than 130-fold. This property is reminiscent of the snake venom ancrod, which only releases fibrinopeptide A, and adds substantially to the anticoagulant potency of the W60dS mutant. In fact, the clotting time in the presence of this mutant is prolonged more than 40-fold compared to the wild-type.
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PMID:Critical role of W60d in thrombin allostery. 912 41

This article reviews the clinical pharmacokinetics of a water-soluble analogue of camptothecin, irinotecan [CPT-11 or 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxy-camptoth eci n]. Irinotecan, and its more potent metabolite SN-38 (7- ethyl-10-hydroxy-camptothecin), interfere with mammalian DNA topoisomerase I and cancer cell death appears to result from DNA strand breaks caused by the formation of cleavable complexes. The main clinical adverse effects of irinotecan therapy are neutropenia and diarrhoea. Irinotecan has shown activity in leukaemia, lymphoma and the following cancer sites: colorectum, lung, ovary, cervix, pancreas, stomach and breast. Following the intravenous administration of irinotecan at 100 to 350 mg/m2, mean maximum irinotecan plasma concentrations are within the 1 to 10 mg/L range. Plasma concentrations can be described using a 2- or 3-compartment model with a mean terminal half-life ranging from 5 to 27 hours. The volume of distribution at steady-state (Vss) ranges from 136 to 255 L/m2, and the total body clearance is 8 to 21 L/h/m2. Irinotecan is 65% bound to plasma proteins. The areas under the plasma concentration-time curve (AUC) of both irinotecan and SN-38 increase proportionally to the administered dose, although interpatient variability is important. SN-38 levels achieved in humans are about 100-fold lower than corresponding irinotecan concentrations, but these concentrations are potentially important as SN-38 is 100- to 1000-fold more cytotoxic than the parent compound. SN-38 is 95% bound to plasma proteins. Maximum concentrations of SN-38 are reached about 1 hour after the beginning of a short intravenous infusion. SN-38 plasma decay follows closely that of the parent compound with an apparent terminal half-life ranging from 6 to 30 hours. In human plasma at equilibrium, the irinotecan lactone form accounts for 25 to 30% of the total and SN-38 lactone for 50 to 64%. Irinotecan is extensively metabolised in the liver. The bipiperidinocarbonylxy group of irinotecan is first removed by hydrolysis to yield the corresponding carboxylic acid and SN-38 by carboxyesterase. SN-38 can be converted into SN-38 glucuronide by hepatic UDP-glucuronyltransferase. Another recently identified metabolite is 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxy-camptothecin (APC). This metabolite is a weak inhibitor of KB cell growth and a poor inducer of topoisomerase I DNA-cleavable complexes (100-fold less potent than SN-38). Numerous other unidentified metabolites have been detected in bile and urine. The mean 24-hour irinotecan urinary excretion represents 17 to 25% of the administered dose. Recovery of SN-38 and its glucuronide in urine is low and represents 1 to 3% of the irinotecan dose. Cumulative biliary excretion is 25% for irinotecan, 2% for SN-38 glucuronide and about 1% for SN-38. The pharmacokinetics of irinotecan and SN-38 are not influenced by prior exposure to the parent drug. The AUC of irinotecan and SN-38 correlate significantly with leuco-neutropenia and sometimes with the intensity of diarrhoea. Certain hepatic function parameters have been correlated negatively with irinotecan total body clearance. It was noted that most tumour responses were observed at the highest doses administered in phase I trials, which indicates a dose-response relationship with this drug. In the future, these pharmacokinetic-pharmacodynamic relationships will undoubtedly prove useful in minimising the toxicity and maximise the likelihood of tumour response in patients.
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PMID:Clinical pharmacokinetics of irinotecan. 934 1

The structure of an artificial pulmonary surfactant was studied by scanning force- and fluorescence light microscopy (SFM, and FLM, respectively). The surfactant--a mixture of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and recombinant surfactant-associated protein C (SP-C)--was prepared at the air-water interface of a Langmuir film balance and imaged by FLM under various states of compression. In order to visualize their topography by SFM, the films were transferred onto a solid mica support by the Langmuir-Blodgett (LB) technique. We found that a region of high film compressibility of the spread monolayer close to its equilibrium surface pressure (pi = 50 mN/m) was due to the exclusion of layered protrusions with each layer 5.5 to 6.5 nm thick. They remained associated with the monolayer and readily reinserted upon expansion of the film. Comparison with the FLM showed that the protrusions contained the protein in high concentration. The more the film was compressed, the larger was the number of layers on top of each other. The protrusions arose from regions of the monolayer with a distinct microstructure that may have been responsible for their formation. The molecular architecture of the microstructure remains to be elucidated, although some of it can be inferred from spectroscopic data in combination with the SFM topographical images. We illustrate our current understanding of the film structure with a molecular model.
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PMID:A scanning force- and fluorescence light microscopy study of the structure and function of a model pulmonary surfactant. 935 39

Three compounds of the pulmonary surfactant--dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and the surfactant associated protein C (SP-C)--were spread at the air-water interface of a Langmuir trough as a model system to mimic the properties of natural surfactant. Fluorescence microscopical images of the film formed at the interface were obtained during compression using a fluorescence dye bound covalently either to phosphatidylcholine or to SP-C. The images were quantified using statistical methods in respect to relative areas and relative fluorescence intensities of the domains found. In the early stage of compression, film pressure rose slightly and was accompanied by a phase separation which could be recognized in the images by the formation of bright and dark domains. On further compression, after a steep increase of film pressure, a plateau region of constant film pressure started abruptly. During compression in the plateau region, fluorescence intensity of the bright domain formed in the early stage of compression increased. The increasing fluorescence intensity, the non-Gaussian intensity distribution of the bright domain, and the small mean molecular area of the film in the plateau region gave rise to the assumption that multilayer structures were formed in the late stage of compression. The formation of the multilayer structures was fully reversible in repeated compression-expansion cycles including the plateau region of the phase diagram. The ability of lipid/SP-C mixtures to form reversible multilayer structures during compression may be relevant to stability in lungs during expiration and inhalation.
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PMID:The phase behavior of lipid monolayers containing pulmonary surfactant protein C studied by fluorescence light microscopy. 935 40

Irinotecan (CPT-11) is a water-soluble analogue of camptothecin showing activity in colon cancer. Recently, we identified a major metabolite of CPT-11 in patients' plasma, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin (APC), which is produced by the oxidation of the distal piperidine ring (P. Rivory et al, Cancer Res., 56: 3689-3694, 1996). As with all active camptothecin derivatives, CPT-11 is subject to spontaneous interconversion between a lactone and a carboxylate form in aqueous media. The kinetics of biotransformation of the two forms of CPT-11 into APC was studied using pooled human liver microsomes. The formation of APC was characterized by the following parameters: Km = 18.4 +/- 1.4 and 39.7 +/- 11.6 microM; and Vmax = 26.0 +/- 0.6 and 13.4 +/- 1.7 pmol/min/mg protein for the lactone and carboxylate forms of CPT-11, respectively. This reaction was found to be catalyzed principally by cytochrome P-450 (CYP) 3A because of three key results: (a) the CYP 3A-selective inhibitors ketoconazole (1 microM) and troleandomycin (100 microM) inhibited APC formation by 98 and 100%, respectively, mostly in a competitive way; (b) using microsomes from transfected lymphoblastoid cells expressing specific CYPs, we found that only those from CYP 3A4 cDNA-transfected cells transformed CPT-11 into APC; and (c) using 15 individual preparations of human liver microsomes, we observed highly significant correlations between the activity of CPT-11 metabolism into APC and both immunoreactivity with anti-CYP 3A antibodies and testosterone 6beta hydroxylation, an activity specifically mediated by CYP 3A. The effect on this metabolism of 11 drugs used at 100 microM was studied with CPT-11 lactone at 25 microM. Amikacin, Bactrim, ciprofloxacin, rocephine, 5-fluorouracil, metoclopramide, morphine, and paracetamol had no effect, but ondansetron, loperamide, and racecadotril inhibited this pathway by 25, 50, and 50%, respectively. These concentrations exceed those expected in vivo. APC formation in patients may thus be influenced by coadministered ketoconazole therapy and may decline after administration of CPT-11 because of the lactonolysis of the latter.
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PMID:Metabolism of irinotecan (CPT-11) by human hepatic microsomes: participation of cytochrome P-450 3A and drug interactions. 945 91

The inner leaflet of unilamellar lipid vesicles was labeled with fluorescent lysophosphatidylcholines. The probes make a donor-acceptor pair in resonance energy transfer (RET), being labeled with 9-anthrylvinyl (L-APC, donor) and 3-perylenoyl (L-PPC, acceptor) fluorophores. They migrate rapidly between bilayers through the water phase: tau 1/2 of equilibration is approximately 5 min at 37 degrees C. The probe(s) can be removed from the outer leaflet of uniformly labeled medium-size unilamellar vesicles (MUV) by repeated washings with excess unlabeled large unilamellar vesicles (LUV) (separation by centrifugation). The probes flip-flop across bilayers rather slowly. MUV containing the ganglioside GT1b and labeled with the L-APC/L-PPC pair in the inner leaflet were fused with an equal amount of influenza virus; the process was monitored by an increase of the donor fluorescence in RET assay. If inner MUV leaflet was labeled with the anthrylvinyl probe only, the probe fluorescence decreased by half when the probe was removed from the outer leaflets of the fused membranes. This shows that the lipids of the inner and outer leaflets of the MUV randomize in the process of fusion.
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PMID:Selective labeling of the inner liposome leaflet by fluorescent lipid probes, and studies of liposome fusion with influenza virus. 955 39

We have previously reported a mutated protein C, designated protein C Nagoya (PCN), characterized by the deletion of a single guanine residue (8857G). This frameshift mutation results in the replacement of the carboxyl-terminal 39 amino acids of wild-type protein C (G381-P419) by 81 abnormal amino acids. This elongated mutant was not effectively secreted, and was retained in the endoplasmic reticulum. To determine why PCN is not secreted, we constructed a series of mutants from which some or all of the 81 amino acids were deleted. None of these shortened proteins were secreted from producing cells, indicating that the carboxyl-terminal extension is not mainly responsible for the intracellular retention of PCN, and that the 39 carboxyl-terminal amino acids of wild-type protein C are required for secretion. To determine which residues are essential for the secretion of protein C, deletion mutants of the carboxyl-terminal region (D401-P419) were prepared. Metabolic labeling showed that mutants of protein C truncated before W417, Q414, E411, or K410 were efficiently secreted. On the other hand, the mutants truncated before D409 were retained and degraded intracellularly. Immunofluorescence and immunoelectron microscopy showed that truncation before D409 blocks the movement from rough endoplasmic reticulum to the Golgi apparatus. To understand the conformational change in the carboxyl-terminal region, two models of truncated activated protein C were constructed using energy optimization and molecular dynamics with water molecules.
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PMID:The carboxyl-terminal region of protein C is essential for its secretion. 957 15

Humic acid in the drinking water of blackfoot disease endemic areas in Taiwan has been implicated as one of the aetiological factors of the disease. For this report we examined the effects of humic acid on the expression of thrombomodulin (TM) cofactor activity by cultured human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with humic acid (HA) isolated from the drinking water, as a synthetic humic acid polymer (SHA) or with commercial HA, resulted in a dose-dependent reduction of cell surface thrombomodulin activity. Characterization of the mechanism by which humic acid reduced the protein C activation indicated that inhibition was not caused by production or release of a protein C inhibitor. Kinetic analysis showed that binding affinities of TM to thrombin and of TM-thrombin complex to protein C was unchanged upon humic acid treatment. However, the cell surface TM activity was reduced by humic acid, which functions as an irreversible noncompetitive inhibitor of thrombin binding. Down-regulation of TM was inhibited by non-selective protein kinase C inhibitors and a selective inhibitor. These results suggest that protein kinase C is intricately involved in HA-induced TM down-regulation. Down-regulation of TM was also inhibited by free radical scavengers. All these changes occurred in the absence of significant cytotoxic effect. In conclusion, our results suggest that HA induces down-regulation of TM by directly increasing permeability of the cell membrane, thus causing elevation in [Ca2+]i. This species functions as a second messenger to activate protein kinase C, and/or Ca-dependent enzymes eventually inducing down-regulation of TM. Attenuation of vascular endothelial cell TM cofactor activity by humic acid may play a role in the humic acid-induced thrombotic vascular disorders of blackfoot disease.
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PMID:Humic acid reduces protein-C-activating cofactor activity of thrombomodulin of human umbilical vein endothelial cells. 957 76

The use of nonsteroidal anti-inflammatory drugs has been suggested to have a chemopreventive effect against colon carcinoma, through the inhibition of cyclooxygenases 1 and 2, in patients with familial adenomatous polyposis and in animal models. Acarbose, an alpha-glycosidase inhibitor, may also be chemopreventive. In order to examine the effects of these drugs we employed APC gene knockout mice randomized into 3 groups, one for treatment with piroxicam (0.05% concentration in drinking water), one for acarbose (0.04% concentration in food) and another for the control. After 14 weeks of treatment, mice were killed for quantitation of gastric and intestinal adenomas. Tumor multiplicity in the whole gastrointestinal tract decreased from 33.89 +/- 13.07 tumors/mouse in the control group to 17.05 +/- 7 tumors/mouse in the piroxicam-treated group (P < 0.001). The decrease in the acarbose-treated group (29.68 +/- 12.86 tumors/mouse) was not significant (P < 0.05). The number of tumors > or = 3 mm in diameter was also quantified in all gastrointestinal segments. The number of such tumors in the piroxicam group was decreased to 0.56 +/- 1.2 tumors/mouse from the control value of 3.78 +/- 1.17 tumors/mouse (P < 0.001), while in the acarbose-treated group the number decreased to 2.36 +/- 1.7 tumors/mouse (P < 0.01). Thus, piroxicam decreases the size and number of gastrointestinal adenomas in APC 1309 knockout mice, while acarbose decreases only the size.
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PMID:Piroxicam and acarbose as chemopreventive agents for spontaneous intestinal adenomas in APC gene 1309 knockout mice. 961 44

We previously described the accumulation of a 34 kDa thylakoid protein, named CDSP 34 for chloroplastic drought-induced stress protein, in Solanum tuberosum plants subjected to water deficit. A full-length CDSP 34 cDNA has been isolated and we report here that mature CDSP 34 is highly similar to two chromoplastic proteins, fibrillin from Capsicum annuum and CHRC (for chromoplast protein C) from Cucumis sativus, components of carotenoid-accumulating structures. Northern and Western analyses showed that both CDSP 34 transcript and protein accumulated from early stages of water deficit. In water-stressed tomato plants, similar increases in the CDSP 34-related transcript amount were noticed in wild-type and ABA-deficient flacca mutant, but protein accumulation was observed only in wild-type, suggesting a posttranscriptional role of ABA in CDSP 34 synthesis regulation. Substantial increases in CDSP 34 transcript and protein abundances were also observed in potato plants subjected to high illumination. The CDSP 34 protein is proposed to play a structural role in stabilizing stromal lamellae thylakoids upon osmotic or oxidative stress.
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PMID:Molecular characterization of CDSP 34, a chloroplastic protein induced by water deficit in Solanum tuberosum L. plants, and regulation of CDSP 34 expression by ABA and high illumination. 983 68


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