Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.16.2 (PCP)
 3,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The refolding rate of heat-denatured cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from Pyrococcus furiosus has been reported to be unusually slow under some conditions. To elucidate the structural basis of the unusually slow kinetics of the protein, the denaturation and refolding processes of the PCP-0SH were investigated using a real-time 2D (1)H-(15)N HSQC and CD experiments. At 2 M urea denaturation of the PCP-0SH in the acidic region, all of the native peaks in the 2D HSQC spectrum completely disappeared. The conformation of the PCP-0SH just after removal of 6 M GuHCl could be observed as a stable intermediate (D(1) state) in 2D HSQC and CD experiments, which is similar to a molten globule structure. The D(1) state of the PCP-0SH, which is the initial state of refolding, corresponded to the state at 2 M urea and seemed to be the denatured state in equilibrium with the native state under the physiological conditions. The refolding of PCP-0SH from the D(1) state to the native state could be observed to be highly cooperative without any intermediates between them, even if the refolding rate was quite slow. In the higher concentration of denaturants, PCP-0SH showed HSQC and CD spectra characteristic of completely unfolded proteins called the D(2) state. The unusually slow refolding rate was discussed as originating in the conformations in the transition state and/or the retardation of reorganization in an ensemble of nonrandom denatured structures in the D(1) state.
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PMID:Unusually slow denaturation and refolding processes of pyrrolidone carboxyl peptidase from a hyperthermophile are highly cooperative: real-time NMR studies. 1536 77

It was the aim of this study to develop and evaluate a nasal microparticulate delivery system for human growth hormone (hGH) based on the thiomer polycarbophil-cysteine (PCP-Cys) in combination with the permeation mediator glutathione (GSH). Microparticles were prepared by dissolving PCP-Cys/GSH/hGH (7.5:1:1.5), PCP/hGH (8.5:1.5), and mannitol/hGH (8.5:1.5) in demineralized water, followed by lyophilization and micronization. Particles were evaluated with regard to size distribution and swelling behavior using a laser diffraction particle size analyzer. The release of fluorescence-labelled hGH from microparticles was determined in Franz diffusion chambers. In vivo studies in rats were performed comparing the nasal bioavailability achieved by PCP-Cys/GSH/hGH microparticles with that of unmodified PCP/hGH microparticles and mannitol/hGH powder. PCP-Cys/GSH/hGH and PCP/hGH microparticles showed a comparable size distribution (80% in the range of 4.8-23 microm) and swelled to almost fourfold size in phosphate-buffered saline (PBS). Both formulations exhibited almost identical sustained drug release profiles. The intranasal administration of the PCP-Cys/GSH/hGH microparticulate formulation resulted in a relative bioavailability of 8.11+/-2.15%, which represents a 3-fold and 3.3-fold improvement compared to that of PCP/hGH microparticles and mannitol/hGH powder, respectively. The study suggests that the PCP-Cys/GSH/hGH nasal microparticulate formulation might represent a promising novel tool for the systemic delivery of hGH.
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PMID:Nasal delivery of human growth hormone: in vitro and in vivo evaluation of a thiomer/glutathione microparticulate delivery system. 1549 13

Novel macrolides epothilones, produced by cellulolytic myxobacterium Sorangium cellulosum, have the activity to promote microtubule assembly, and are considered to be a potential successor to the famous antitumor drug taxol. The biosynthetic genes leading to the epothilones are clustered into a large operon. The multi-enzyme complex is a hetero-gene cluster of polyketide synthase (PKS) and non-ribosomal peptide synthetases (NRPS) and contains several functional modules, i.e. a loading module, one NRPS module, eight PKS modules, and a P450 epoxidase. The former ten modules biosynthesize desoxyepothilone (epothilones C and D), which is then epoxidized at C12 and C13 and converted into epothilones (epothilones A and B) by the P450 epoxidase. The NRPS module is responsible for the formation of the thiazole side chain from cysteine. The biosynthesis procedure of epothilones can be divided into 5 stages, i.e. formation of holo-ACP/PCP, chain initiation and thiazole ring formation, chain elongation, termination and epoxidation, and post-modification. The analysis of the gene cluster and the biosynthetic pathway reveals that novel epothilone analogs could not only be produced by chemical synthesis/modification, tranditional microbial technologies, but also can be genetically manipulated through combinatiorial biosynthesis approaches.
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PMID:[The PKS/NRPS hetero-gene cluster of epothilones]. 1596 75

Wnt proteins are cysteine-rich glycosylated proteins named after the Drosophilia Wingless (Wg) and the mouse Int-1 genes that play a role in embryonic cell patterning, proliferation, differentiation, orientation, adhesion, survival, and programmed cell death (PCD). Wnt proteins involve at least two intracellular signaling pathways. One pathway controls target gene transcription through beta-catenin, generally referred to as the canonical pathway and a second pathway pertains to intracellular calcium (Ca(2+)) release which is termed the non-canonical or Wnt/ Ca(2+) pathway. The majority of Wnt proteins activate gene transcription through the canonical signaling pathway regulated by pathways that include the Frizzled transmembrane receptor and the co-receptor LRP-5/6, Dishevelled, glycogen synthase kinase-3beta (GSK-3beta), adenomatous polyposis coli (APC), and beta-catenin. In contrast, the non-canonical Wnt signaling pathway has two intracellular signaling cascades that consist of the Wnt/ Ca(2+) pathway with protein kinase C (PKC) and the Wnt/PCP pathway involving Rho/Rac small GTPase and Jun N-terminal kinase (JNK). Through a series of signaling pathways, Wnt proteins modulate cell development, proliferation, and cell fate. In regards to cell survival and fate through PCD, Wnt may be critical for the prevention of tissue pathology that involves cytokine and growth factor control during disorders such as neuropsychiatric disease, retinal disease, and Alzheimer's disease. Elucidation of the vital elements that shape and control the Wnt-Frizzled signaling pathway may provide significant prospects for the treatment of disorders of the nervous system.
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PMID:Vital elements of the Wnt-Frizzled signaling pathway in the nervous system. 1620 77

The metabolism of phencyclidine (PCP) has been studied previously in cytochrome P450 (P450)-containing microsomal systems. However, the reactive intermediate(s) that covalently binds to the P450 and leads to inactivation or leaves the active site to modify other proteins has not been identified. In this study two electrophilic intermediates of PCP were identified by mass spectrometry and by trapping with reduced glutathione (GSH) or N-acetyl cysteine (NAC). The tentative structures of these electrophilic intermediates were determined using mass spectrometry. P450s 2B1 and 2B4 formed a metabolite that exhibited an m/z of 240 corresponding to the mass of the 2,3-dihydropyridinium species of PCP or its conjugate base, the 1,2-dihydropyridine. Chemical reduction of the incubation mixture using NaBH4 resulted in the disappearance of the signal at m/z 240, consistent with reduction of a 2,3-dihydropyridinium species. Furthermore, the reactive metabolite trapped by GSH resulted in an adduct exhibiting an m/z of 547, consistent with the mass of the 2,3-dihydropyridinium species of PCP (m/z 240), that has reacted with a molecule of GSH (m/z 308). However, P450 2B6 formed a different reactive intermediate of PCP that was isolated as a GSH adduct exhibiting an m/z of 581 and an NAC adduct with an m/z of 437. Liquid chromatography-tandem mass spectrometry analysis of these adducts suggested that a di-oxygenated iminium metabolite of PCP could be the reactive intermediate formed by P450 2B6 but not by the other 2B isoforms. These data suggest that P450 2B6 favors oxidation pathways for PCP metabolism that are different from those of P450s 2B1 and 2B4.
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PMID:Selective pathways for the metabolism of phencyclidine by cytochrome p450 2b enzymes: identification of electrophilic metabolites, glutathione, and N-acetyl cysteine adducts. 1632 15

The aim of this study was to compare different oral delivery systems based on the thiolated polymer polycarbophil-cysteine (PCP-Cys) and to provide evidence for the validity of the hypothesis that unhydrated polymers provide better mucoadhesion in vivo. To achieve dry polymer application, a new, experimental dosage form named Eutex (made of Eudragit L100-55 and latex) capsule has been developed. Magnetic resonance imaging was used to localize the point of release of the thiolated polymer from the application forms via the positive magnetic resonance signal from a gadolinium complex (Gd-DTPA). In vivo mucoadhesion was determined by ascertaining the residence time of the fluorescence-tagged thiomer on intestinal mucosa after 3 h. Results showed that in comparison to conventional application forms the Eutex capsules led to 1.9-fold higher mucoadhesive properties of PCP-Cys when compared to application with a conventional enteric-coated capsule, and to 1.4-fold higher mucoadhesion when compared to administration with an enteric-coated tablet of the thiomer. The findings of this study should contribute to the understanding of mucoadhesion and mucoadhesion influencing parameters in vivo and should therefore be of considerable interest for the development of future mucoadhesive oral drug delivery dosage forms.
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PMID:Comparative in vivo mucoadhesion studies of thiomer formulations using magnetic resonance imaging and fluorescence detection. 1690 69

Cystine-knot microproteins exhibit several properties that make them highly interesting as scaffolds for oral peptide drug delivery. It was therefore the aim of the study to evaluate the novel clinically relevant cystine-knot microprotein McoEeTI regarding its potential for oral delivery. Additionally, based on the gained results, important features of McoEeTI were improved. Enzymatic degradation was caused by chymotrypsin, trypsin and porcine small intestinal juice whereas McoEeTI was stable towards elastase, membrane bound proteases, pepsin and porcine gastric juice. Only minor McoEeTI degradation was observed during a 24h incubation period in rat plasma. In the presence of various physiological ions about 50% of McoEeTI formed di- and/or trimers. P(app) value of McoEeTI was determined to be (7.4+/-0.4)x10(-6)cm/s. Sodium caprate and polycarbophil-cysteine (PCP-Cys) had no beneficial effect on McoEeTI permeation, whereas the utilization of a chitosan-thiobutylamidine (Chito-TBA) system improved McoEeTI permeation 3-fold. Enzymatic stability could be strongly improved by the utilization of Bowman-Birk-Inhibitor (BBI) as well as PCP-Cys. In conclusion, this study indicates that McoEeTI represents a promising candidate as a novel scaffold for oral peptide drug delivery.
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PMID:Evaluation and improvement of the properties of the novel cystine-knot microprotein McoEeTI for oral administration. 1707 Jun 61

The cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile, Pyrococcus furiosus, can be trapped in the denatured state under nondenaturing conditions, corresponding to the denatured structure that exists in equilibrium with the native state under physiological conditions. The denatured state is the initial state (D1 state) in the refolding process but differs from the completely denatured state (D2 state) in the concentrated denaturant. Also, it has been found that the D1 state corresponds to the heat-denatured state. To elucidate the structural basis of the D1 state, H/D exchange experiments with PCP-0SH were performed at pD 3.4 and 4 degrees C. The results indicated that amide protons in the C-terminal alpha6-helix region hardly exchanged in the D1 state with deuterium even after 7 days, suggesting that the alpha6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D1 state. In order to examine the role of the alpha6-helix in folding and stability, H/D exchange experiments with a mutant, A199P, at position 199 in the alpha6-helix region were performed. The alpha6-helix region of A199P in the D1 state was partially unprotected, while some hydrophobic residues were protected against the H/D exchange, although these hydrophobic residues were unprotected in the wild-type protein. These results suggest that the structure of A199P in the D1 state formed a temporary stable denatured structure with a non-native hydrophobic cluster and the unstructured alpha6-helix. Both the stability and the refolding rate decreased by the substitution of Pro for Ala199. We can conclude that the native-like helix (alpha6-helix) of PCP-0SH is already constructed in the D1 state and is necessary for efficient refolding into the native structure and stabilization of PCP-0SH.
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PMID:Characterization of the denatured structure of pyrrolidone carboxyl peptidase from a hyperthermophile under nondenaturing conditions: role of the C-terminal alpha-helix of the protein in folding and stability. 1730 36

The purpose of this study was to investigate the effect of thiolated polycarbophil as an adjuvant to enhance the permeation and improve the stability of a phosphorothioate antisense oligonucleotide (PTO-ODN) on the nasal mucosa. Polycarbophil-cysteine (PCP-Cys) was synthesized by the covalent attachment of L-cysteine to the polymeric backbone. Cytotoxicity tests were examined on human nasal epithelial cells from surgery of nasal polyps confirmed by histological studies. Deoxyribonuclease I activity in respiratory region of the porcine nasal cavity was analyzed by an enzymatic assay. The enzymatic degradation of PTO-ODNs on freshly excised porcine nasal mucosa was analyzed and protection of PCP-cysteine toward DNase I degradation was evaluated. Permeation studies were performed in Ussing-type diffusion chambers. PCP-Cys/GSH did not arise a remarkable mortal effect. Porcine respiratory mucosa was shown to possess nuclease activity corresponding to 0.69 Kunitz units/mL. PTO-ODNs were degraded by incubation with nasal mucosa. In the presence of 0.45% thiolated polycarbophil and 0.5% glutathione (GSH), this degradation process could be lowered. In the presence of thiolated polycarbophil and GSH the uptake of PTO-ODNs from the nasal mucosa was 1.7-fold improved. According to these results thiolated polycarbophil/GSH might be a promising excipient for nasal administration of PTO-ODNs.
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PMID:Thiolated polycarbophil as an adjuvant for permeation enhancement in nasal delivery of antisense oligonucleotides. 1970 62

Aim of the present work was to develop novel thiol-functionalized hydrogel microparticles based on poly(methacrylic acid)-chitosan-poly(ethylene glycol) (PCP) for oral drug delivery applications. PCP microparticles were prepared by a modified ionic gelation process in aqueous medium. Thiol modification of surface carboxylic acid groups of PCP micro particles was carried out by coupling l-cysteine with a water-soluble carbodiimide. Ellman's method was adopted to quantify the sulfhydryl groups, and dynamic light-scattering technique was used to measure the average particle size. Cytotoxicity of the modified particles was evaluated on Caco 2 cells by MTT assay. Effect of thiol modification on permeability of paracellular marker fluorescence dextran (FD4) was evaluated on Caco 2 cell monolayers and freshly excised rat intestinal tissue with an Ussing chamber set-up. Mucoadhesion experiments were carried out by an ex vivo bioadhesion method with excised rat intestinal tissue. The average size of the PCP microparticles was increased after thiol modification. Thiolated microparticles significantly improved the paracellular permeability of FD4 across Caco 2 cell monolayers, with no sign of toxicity. However, the efficacy of thiolated system remained low when permeation experiments were carried out across excised intestinal membrane. This was attributed to the high adhesion of the thiolated particles on the gut mucosa. Nevertheless, it can be concluded that surface thiolation is an interesting strategy to improve paracellular permeability of hydrophilic macromolecules.
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PMID:Surface-functionalized polymethacrylic acid based hydrogel microparticles for oral drug delivery. 1973 14


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