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1.
Peptides ; 27(4): 905-10, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16274843

ABSTRACT

Two different RNAi methods were used to inhibit the expression of prohormone convertase 1 (PC1) in At-T20 cells. Transient transfection of double stranded RNA and stable expression of a vector expressing hairpin-loop RNA targeting PC1 reduced cholecystokinin (CCK) secretion from At-T20 cells. PC1 mRNA and protein were also decreased in the vector transfected cells. This treatment caused a shift in the forms of cholecystokinin (CCK) secreted, decreasing CCK 22 and increasing CCK 8. Stable expression of RNAi effectively decreased PC1 expression. The observed decrease in CCK seen with these RNAi treatments further supports a role for PC1 in CCK processing in these cells.


Subject(s)
Cholecystokinin/chemistry , Cholecystokinin/metabolism , Gene Expression , Proprotein Convertase 1/deficiency , Amino Acid Sequence , Animals , Cell Line , Gene Expression/drug effects , Mice , Molecular Sequence Data , Proprotein Convertase 1/biosynthesis , Proprotein Convertase 1/genetics , Protein Isoforms/chemistry , Protein Isoforms/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats
2.
J Biol Chem ; 280(46): 38410-5, 2005 Nov 18.
Article in English | MEDLINE | ID: mdl-16174778

ABSTRACT

Prohormone convertase 2 is widely co-localized with cholecystokinin in rodent brain. To examine its role in cholecystokinin processing, cholecystokinin levels were measured in dissected brain regions from prohormone convertase 2 knock-out mice. Cholecystokinin levels were lower in hippocampus, septum, thalamus, mesencephalon, and pons in knock-out mice than wild-type mice. In cerebral cortex, cortex-related structures and olfactory bulb, cholecystokinin levels were higher than wild type. Female mice were more affected by the loss of prohormone convertase 2 than male mice. The decrease in cholecystokinin levels in these brain regions shows that prohormone convertase 2 is important for cholecystokinin processing. Quantitative polymerase chain reaction measurements were performed to examine the relationship between peptide levels and cholecystokinin and enzyme expression. They revealed that cholecystokinin and prohormone convertase 1 mRNA levels in cerebral cortex and olfactory bulb were actually lower in knock-out than wild type, whereas their expression in other brain regions of knock-out mouse brain was the same as wild type. Female mice frequently had higher expression of cholecystokinin and prohormone convertase 1, 2, and 5 mRNA than male mice. The loss of prohormone convertase 2 alters CCK processing in specific brain regions. This loss also appears to trigger compensatory mechanisms in cerebral cortex and olfactory bulb that produce elevated levels of cholecystokinin but do not involve increased expression of cholecystokinin, prohormone convertase 1 or 5 mRNA.


Subject(s)
Brain/metabolism , Cholecystokinin/biosynthesis , Proprotein Convertase 2/genetics , Proprotein Convertase 2/physiology , Amino Acid Sequence , Animals , Brain/pathology , Carboxypeptidases/chemistry , Cerebral Cortex/metabolism , Cholecystokinin/physiology , Chromatography, High Pressure Liquid , Female , Genotype , Hippocampus/metabolism , Male , Mice , Mice, Knockout , Molecular Sequence Data , Olfactory Bulb/metabolism , Phenotype , Polymerase Chain Reaction , Proprotein Convertase 1/metabolism , Proprotein Convertase 5/metabolism , RNA/chemistry , RNA/metabolism , RNA, Messenger/metabolism , Radioimmunoassay , Reverse Transcriptase Polymerase Chain Reaction , Sex Factors , Tissue Distribution , Trypsin/chemistry
3.
Drug Metab Dispos ; 32(8): 800-4, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15258104

ABSTRACT

The role of P-glycoprotein (P-gp) on the distribution of the benzodiazepine triazolam (TRZ) and the azole antifungal agent ketoconazole (KET), and on the TRZ-KET interaction, was studied using mdr1a(-) or mdr1a/b(-/-) mice (P-gp-deficient mice) and matched controls. TRZ and KET also were studied in Caco-2 cells in Transwell culture. After single i.p. injections of TRZ or KET in separate groups of control mice, brain concentrations of TRZ exceeded those in serum [brain/serum area under the concentration curve (AUC) ratio, 5.0], whereas brain/serum AUC ratios for KET were approximately 0.5. On the basis of single time points, brain concentrations of TRZ, or brain/serum ratios, were similar in P-gp-deficient animals compared with controls, whereas P-gp-deficient animals had significantly higher KET brain concentrations and brain/serum ratios. Coadministration of KET with TRZ increased TRZ concentrations in serum, liver, and brain, both in controls and in P-gp-deficient animals, probably attributable to impairment by KET of CYP3A-mediated clearance of TRZ. However, KET did not increase brain/serum ratios of TRZ in either group. In Caco-2 cells, basal-to-apical flux of TRZ was higher than apical-to-basal flux. However, verapamil (100 microM) did not alter flux in either direction. KET inhibited basal-to-apical transport of rho-damine-123, with a 50% inhibitory concentration of 2.7 microM. Thus, TRZ does not appear to undergo measurable blood-brain barrier efflux transport by P-gp in this animal model. KET impairs clearance of TRZ but does not increase tissue uptake. However, KET itself may be a substrate for efflux transport at the blood-brain barrier.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/deficiency , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Ketoconazole/pharmacokinetics , Triazolam/pharmacokinetics , Animals , Caco-2 Cells , Dose-Response Relationship, Drug , Drug Interactions , Humans , Male , Mice , Mice, Knockout
4.
Biochemistry ; 43(29): 9502-11, 2004 Jul 27.
Article in English | MEDLINE | ID: mdl-15260493

ABSTRACT

Cholecystokinin (CCK) is expressed in the central and peripheral nervous systems and functions as a neurotransmitter and neuroendocrine hormone. The in vivo forms of CCK include CCK-83, -58, -39, -33, -22, -12, and -8. Tissues in the periphery produce the larger forms of CCK, such as CCK-58, whereas the brain primarily produces CCK-8. The different biologically active forms of CCK observed in vivo may result from cell-specific differences in endoproteolytic cleavage during post-translational processing. Evidence suggests that cleavages of pro-CCK occur in a specific sequential order. To further delineate the progression of cleavages during pro-CCK maturation, mutagenesis was used to disrupt putative mono- and dibasic cleavage sites. AtT-20 cells transfected with wild-type rat prepro-CCK secret CCK-22 and -8. Mutagenesis of the cleavage sites of pro-CCK had profound effects on the products that were produced. Substitution of basic cleavage sites with nonbasic amino acids inhibits cleavage and leads to the secretion of pathway intermediates such as CCK-83, -33, and -12. These results suggest that CCK-58 is cleaved to both CCK-33 and -22. Furthermore, CCK-8 and -12 are likely derived from cleavage of CCK-33 but not CCK-22. Alanine substitution at the same site completely blocked production of amidated products, whereas serine substitution did not. The cleavages observed at nonbasic residues in this study may represent the activity of enzymes other than PC1 and carboxypeptidase E, such as the enzyme SKI-1. A model for the progression of pro-CCK processing in AtT-20 cells is proposed. The findings in this study further supports the hypothesis that pro-CCK undergoes parallel pathways of proteolytic cleavages.


Subject(s)
Cholecystokinin/metabolism , Protein Precursors/metabolism , Protein Processing, Post-Translational/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cholecystokinin/chemistry , Cholecystokinin/genetics , DNA Primers , Hydrolysis , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Precursors/chemistry , Protein Precursors/genetics , Rats
5.
J Comp Neurol ; 467(3): 307-25, 2003 Dec 15.
Article in English | MEDLINE | ID: mdl-14608596

ABSTRACT

During posttranslational processing to generate CCK 8, pro-cholecystokinin (CCK) undergoes endoproteolytic cleavage at three sites. Several studies using endocrine and neuronal tumor cells in culture and recombinant enzymes and synthetic substrates in vitro have pointed to the subtilisin/kexin-like enzymes prohormone convertase (PC) 1, PC2, and PC5 as potential candidates for these endoproteolytic cleavages. In these experimental models, they all appear to be able to cleave pro-CCK to make the correct products. One rodent model has provided information about the role of PC2. PC2 knockout mouse brains had less CCK 8 than wild-type, although a substantial amount of CCK was still present. The degree to which CCK levels were reduced in these mice was regionally specific. These data indicated that PC2 is important for normal production of CCK but that it is not the only endoprotease that is involved in CCK processing. To evaluate whether PC1 and PC5 are possible candidates for the other enzymes involved in CCK processing, the distribution of PC1, PC2, and PC5 mRNA was studied in rat brain. Their colocalization with CCK mRNA was examined using double-label in situ hybridization. PC2 was the most abundant of these enzymes in terms of the intensity and number of cells labeled. It was widely colocalized with CCK. PC1 and PC5 mRNA-positive cells were less abundant, but they were also widely distributed and strongly colocalized with CCK in the cerebral cortex, hippocampus, amygdala, ventral tegmental area, and substantia nigra zona compacta. The degree of colocalization of the enzymes with CCK was regionally specific. It is clear that PC1 and PC5 are extensively colocalized with CCK and could be participating in CCK processing in the rat brain and may be able to substitute for PC2 in its absence. These three enzymes may represent a redundant system to ensure production of biologically active CCK.


Subject(s)
Brain Chemistry , Cholecystokinin/analysis , Proprotein Convertase 1/analysis , Proprotein Convertase 2/analysis , Proprotein Convertase 5/analysis , Amino Acid Sequence , Animals , Brain/enzymology , Brain Chemistry/physiology , Cholecystokinin/genetics , Cholecystokinin/metabolism , Female , Male , Molecular Sequence Data , Proprotein Convertase 1/genetics , Proprotein Convertase 1/metabolism , Proprotein Convertase 2/genetics , Proprotein Convertase 2/metabolism , Proprotein Convertase 5/genetics , Proprotein Convertase 5/metabolism , Rats , Rats, Sprague-Dawley
6.
J Pharm Pharmacol ; 55(9): 1229-39, 2003 Sep.
Article in English | MEDLINE | ID: mdl-14604466

ABSTRACT

Animals treated with multiple doses of bupropion have had increased bupropion clearance or increased liver weight, suggesting induction of drug-metabolizing activity. The possibility of cytochrome p450 (CYP) induction by bupropion (10 microM) was evaluated in-vitro by comparing catalytic activity, immunoreactive protein and CYP mRNA levels from human hepatocytes in primary culture versus cells treated with vehicle (0.5% methanol) and with rifampicin (rifampin) as a positive control. mRNA levels were analysed using a branched DNA luminescent assay. CYP2B6 activity, protein and mRNA levels were increased by 2.5, 1.5 and 2.1 fold, respectively, by 20 microM rifampicin. However, 10 microM bupropion minimally altered CYP2B6 (1.4, 1.1, 0.8 fold). Although CYP3A4 activity, protein, and mRNA levels were increased by 4.0, 2.3, and 14.0 fold, respectively, by 20 microM rifampicin, 10 microM bupropion minimally altered CYP3A4 (1.4, 1.0, 0.8 fold). Rifampicin (20 microM) increased CYP2E1 protein by 2.1 fold, while 10 microM bupropion minimally altered CYP2E1 protein (1.2 fold). Overall, results of this study suggest that multiple doses of bupropion are not likely to induce CYP2B6, 3A4 or 2E1 in-vivo in man.


Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Bupropion/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Hepatocytes/drug effects , Oxidoreductases, N-Demethylating/metabolism , RNA, Messenger/metabolism , Adult , Antidepressive Agents, Second-Generation/pharmacology , Aryl Hydrocarbon Hydroxylases/genetics , Blotting, Western , Catalysis/drug effects , Cells, Cultured , Cytochrome P-450 CYP2B6 , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , Female , Hepatocytes/cytology , Hepatocytes/metabolism , Humans , Immunohistochemistry , Middle Aged , Oxidoreductases, N-Demethylating/genetics , RNA, Messenger/genetics , Rifampin/pharmacology
7.
Protein Expr Purif ; 31(1): 56-63, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12963341

ABSTRACT

The precursor of cholecystokinin (pro-CCK) was expressed and purified from media of stably transfected D.Mel-2 cell as an V5-His tagged fusion protein. Its identity was confirmed using SDS-PAGE, immunoblotting, gel filtration chromatography, HPLC, and Mass Spectroscopy. Two major forms of pro-CCK were found with a molecular weight of about 14.4 and 11.3 kDa. The smaller form represents the V5-His tagged pro-CCK after cleavage at a single arginine residue at CCK-58. This cleavage is probably being performed by endogenous proteases in these cells. Purification of the desired larger form of pro-CCK is possible using a nickel column with a recovery of about 20%, yielding 500 microg/L media. The purified protein is stable for several months and can be used for further functional studies of pro-CCK.


Subject(s)
Cholecystokinin/biosynthesis , Protein Precursors/biosynthesis , Recombinant Proteins/biosynthesis , Amino Acid Sequence , Animals , Antibodies/immunology , Antibodies/isolation & purification , Blotting, Western , Cell Line , Cholecystokinin/chemistry , Cholecystokinin/isolation & purification , Chromatography, Gel , Chromatography, High Pressure Liquid , Culture Media, Conditioned/chemistry , Culture Media, Serum-Free , Drosophila/cytology , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Gene Expression , Genetic Vectors/genetics , Histidine/genetics , Histidine/isolation & purification , Mass Spectrometry , Molecular Weight , Polymerase Chain Reaction , Protein Precursors/chemistry , Protein Precursors/isolation & purification , Rats , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Transfection
8.
J Neurochem ; 86(3): 556-63, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12859669

ABSTRACT

Alpha-melanocyte-stimulating hormone (alpha-MSH) is a neuropeptide expressed in pituitary and brain that is known to regulate energy balance, appetite control, and neuroimmune functions. The biosynthesis of alpha-MSH requires proteolytic processing of the proopiomelanocortin (POMC) precursor. Therefore, this study investigated the in vivo role of the prohormone convertase 2 (PC2) processing enzyme for production of alpha-MSH in PC2-deficient mice. Specific detection of alpha-MSH utilized radioimmunoassay (RIA) that does not crossreact with the POMC precursor, and which does not crossreact with other adrenocorticotropin hormone (ACTH) and beta-endorphin peptide products derived from POMC. alpha-MSH in PC2-deficient mice was essentially obliterated in pituitary, hypothalamus, cortex, and other brain regions (collectively), compared to wild-type controls. These results demonstrate the critical requirement of PC2 for the production of alpha-MSH. The absence of alpha-MSH was accompanied by accumulation of ACTH, ACTH-containing imtermediates, and POMC precursor. ACTH was increased in pituitary and hypothalamus of PC2-deficient mice, evaluated by RIA and reversed-phase high pressure liquid chromatography (RP-HPLC). Accumulation of ACTH demonstrates its role as a PC2 substrate that can be converted for alpha-MSH production. Further analyses of POMC-derived intermediates in pituitary, conducted by denaturing western blot conditions, showed accumulation of ACTH-containing intermediates in pituitaries of PC2-deficient mice, which implicate participation of such intermediates as PC2 substrates. Moreover, accumulation of POMC was observed in PC2-deficient mice by western blots with anti-ACTH and anti-beta-endorphin. In addition, increased beta-endorphin1-31 was observed in pituitary and hypothalamus of PC2-deficient mice, suggesting beta-endorphin1-31 as a substrate for PC2 in these tissues. Overall, these studies demonstrated that the PC2 processing enzyme is critical for the in vivo production of alpha-MSH in pituitary and brain.


Subject(s)
Brain/metabolism , Pituitary Gland/metabolism , Pro-Opiomelanocortin/metabolism , Subtilisins/deficiency , alpha-MSH/deficiency , Adrenocorticotropic Hormone/analysis , Adrenocorticotropic Hormone/metabolism , Animals , Blotting, Western , Brain Chemistry , Cerebral Cortex/chemistry , Cerebral Cortex/metabolism , Hypothalamus/chemistry , Hypothalamus/metabolism , Mice , Mice, Knockout , Peptide Fragments/analysis , Peptide Fragments/metabolism , Pro-Opiomelanocortin/analysis , Proprotein Convertase 2 , Radioimmunoassay , Subtilisins/genetics , alpha-MSH/analysis , beta-Endorphin/analysis , beta-Endorphin/metabolism
9.
Neuropeptides ; 37(3): 140-8, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12860111

ABSTRACT

The prohormone convertase 2 (PC2) is hypothesized to convert multiple pro-neuropeptides into active peptides that function as neurotransmitters. To examine the in vivo role of PC2 in neuropeptide production, the tissue contents of six different neuropeptides in brain and peripheral nervous tissues were examined in PC2 deficient mice. Specific neuropeptide radioimmunoassays and RP-HPLC (reverse-phase HPLC) provided evaluation of processed, active neuropeptides in brain and neuroendocrine tissues of PC2 deficient mice. Results demonstrated three features with regard to the selective roles of PC2 in determining the production of NPY, somatostatin-28, enkephalin, VIP, galanin, and CRF in neuroendocrine tissues. Firstly, PC2 deficient mice showed changes in several neuropeptides, but not all neuropeptides examined. The absence of active PC2 resulted in altered cellular levels of NPY, somatostatin-28, and (Met)enkephalin; few changes in VIP or galanin occurred in the tissues examined. CRF content was not altered in brains of PC2 deficient mice. Secondly, comparison of a single neuropeptide among different tissues of PC2 deficient mice demonstrated tissue-selective roles for PC2 in production of the neuropeptide. For example, NPY levels were decreased in ileum of PC2 deficient mice, but NPY content was not altered in hypothalamus that is abundant in NPY. In addition, (Met)enkephalin levels in hypothalamus and cortex were decreased in PC2 deficient mice, but no changes were observed in adrenal or intestine. Thirdly, a single tissue region often showed selective alterations among different neuropeptides. For example, the neuropeptide-rich hypothalamus region showed decreased (Met)enkephalin in PC2 deficient mice, but NPY, VIP, galanin, and CRF were not altered. These results demonstrate the selective role of PC2 in neuropeptide production that provides active peptide neurotransmitter or hormones for biological functions in brain and neuroendocrine systems.


Subject(s)
Brain/metabolism , Neuropeptides/metabolism , Neurosecretory Systems/metabolism , Subtilisins/physiology , Animals , Corticotropin-Releasing Hormone/metabolism , Enkephalin, Methionine/metabolism , Galanin/metabolism , Mice , Mice, Knockout , Neuropeptide Y/metabolism , Organ Specificity , Proprotein Convertase 2 , Protein Precursors/metabolism , Radioimmunoassay , Somatostatin/metabolism , Subtilisins/deficiency , Subtilisins/genetics , Vasoactive Intestinal Peptide/metabolism
10.
AIDS ; 17(7): 1092-4, 2003 May 02.
Article in English | MEDLINE | ID: mdl-12700464

ABSTRACT

The effect of lopinavir on P-glycoprotein-mediated rhodamine 123 efflux was studied in Caco-2 monolayer cells. Lopinavir is a potent inhibitor of Rh123 efflux in Caco-2 monolayers (IC50 1.7 microM). Chronic lopinavir exposure (72 h) in LS 180V cells reduced the content of intracellular Rh123 by approximately 50%, indicating increased efflux activity. In LS 180V cells, lopinavir induced P-glycoprotein immunoreactive protein (up to threefold) and messenger RNA levels in a concentration-dependent fashion.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Anti-HIV Agents/pharmacology , HIV Protease Inhibitors/pharmacology , Pyrimidinones/pharmacology , Blotting, Western , Caco-2 Cells , Dose-Response Relationship, Drug , Fluorescent Dyes/metabolism , Humans , Lopinavir , RNA, Messenger/metabolism , RNA, Viral/metabolism , Rhodamine 123/antagonists & inhibitors , Time Factors
11.
Protein Expr Purif ; 24(2): 227-33, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11858717

ABSTRACT

The discovery of the prohormone convertase (PC) family of enzymes has provided several good candidates (PC1, PC2, and PC5) for the enzymes responsible for the endoproteolytic cleavage of procholecystokinin (pro-CCK). Determination of the role of individual pro-hormone convertases in the processing of pro-CCK is complicated because several of these enzymes are found in endocrine tumor cells expressing CCK mRNA and in identified neurons in the brain. Production of active recombinant PC5 permits the determination of its ability to cleave substrates related to pro-CCK. Active PC5, secreted from baculovirus-infected Sf9 cells, was partially purified by ion-exchange chromatography. Western blot analysis confirmed the presence of the active form of the enzyme in infected cell media and its absence from uninfected cell media. The enzyme is most active at acidic pH 6.5 and is maximally activated by 5 mM calcium. PC5 was able to cleave both monobasic and dibasic substrates without a requirement for a basic residue at P-4 and it displayed a K(m) in the micromolar range. The enzyme was inhibited by EDTA, 1,10-phenanthroline, and p-CMS, as well as by two specific PC inhibitors. This is the first reported preparation of active recombinant PC5. Like the other members of its family, it has the correct catalytic characteristics in vitro to play a role in the processing of neuropeptide precursor proteins into their final bioactive forms.


Subject(s)
Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Serine Endopeptidases/biosynthesis , Serine Endopeptidases/isolation & purification , Animals , Baculoviridae/enzymology , Baculoviridae/genetics , Cloning, Molecular , Genetic Vectors , Hydrogen-Ion Concentration , Mice , Proprotein Convertase 5 , Protease Inhibitors/metabolism , Serine Endopeptidases/genetics , Spodoptera/virology
12.
Biochemistry ; 41(2): 570-8, 2002 Jan 15.
Article in English | MEDLINE | ID: mdl-11781096

ABSTRACT

Site-directed mutagenesis in which individual cleavage site P1 amino acids were changed to Ala was performed to delineate their importance in the processing of pro-CCK in mouse pituitary tumor AtT-20 cells. Individual substitution of cleavage sites on pro-CCK, viz., CCK 58 cleavage site R/A to A/A, CCK 33 cleavage site R/K to A/K, CCK 22 cleavage site K/N to A/N, and CCK 8 cleavage site R/D to A/D, did not inhibit pro-CCK expression or the production of some form of amidated CCK. Wild-type CCK cDNA expression in these cells results in production and secretion of CCK 8 and CCK 22. Substitution of the 58R/A cleavage site with A/A produces only CCK 33; 33A/K and 22A/N produce only CCK 8, whereas 8A/D produces CCK 12 and some CCK 22. Where the GRR residues on the C-terminus of CCK 8 were mutated to GAA, no amidated CCK was produced. Significant amounts of the pro-CCK, C-terminal peptide S9S was found in the medium of cells transfected with GAA mutant cDNA, indicating that this pro-CCK was cleaved at the GAA site probably by a nonprohormone convertase enzyme. Further analysis of the cells expressing the GAA mutant demonstrated that it is not extensively cleaved at other sites to produce CCK 8 GAA or larger peptides. In the mutant where the entire pro-CCK, C-terminal S9S was deleted, CCK 8 is processed and secreted normally. Thus, the cleavage at the C-terminal GRR site is essential for subsequent cleavages, and modification of other cleavage sites (58, 33, 22, and 8) has a major impact on pro-CCK processing. These results suggest that there is a temporal order of cleavages, and the structure of pro-CCK has a strong influence on where and whether pro-CCK is processed.


Subject(s)
Cholecystokinin/chemistry , Cholecystokinin/genetics , Protein Precursors/chemistry , Protein Precursors/genetics , Protein Processing, Post-Translational , Amino Acid Sequence , Animals , Binding Sites , Carboxypeptidases/pharmacology , Cathepsin A , Chromatography , Chromatography, Gel , Chromatography, High Pressure Liquid , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Protein Binding , Protein Structure, Tertiary , Radioimmunoassay , Rats , Transfection , Tumor Cells, Cultured
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