154 results on '"Handschumacher RE"'
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2. Bacterial preparation of orotidine-5'-phosphate and uridine-5'-phosphate
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Handschumacher Re
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Multidisciplinary ,Biochemical Phenomena ,Nucleotides ,Uracil ,Nucleosides ,Phosphate ,Uridine ,Phosphates ,chemistry.chemical_compound ,chemistry ,Biochemistry ,Orotidine ,Escherichia coli ,Phosphorylation - Abstract
THE enzymic syntheses of orotidine-5′-phosphate and uridine-5′-phosphate have been reported by Lieberman et al. 1 and others. A chemical phosphorylation of uridine to form the 5′-phosphates has also been described by Hall and Khorana2. This communication describes the preparation of these previously rare compounds from cultures of E. coli B grown in a salts–glucose medium in the presence of the uracil analogue, as-triazine-3,5-dione, referred to as 6-azauracil. more...
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- 1958
Catalog
3. Hepatic homeostasis of plasma L-asparagine
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Woods, JS, primary and Handschumacher, RE, additional
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- 1971
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4. Hepatic regulation of plasma L-asparagine
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Woods, JS, primary and Handschumacher, RE, additional
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- 1973
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5. Identification of two calcineurin B-binding proteins: tubulin and heat shock protein 60.
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Li W and Handschumacher RE
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- Animals, Brain metabolism, Calcineurin genetics, Cattle, Kidney metabolism, Mice, Mitochondria, Liver metabolism, Rats, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Calcineurin metabolism, Chaperonin 60 metabolism, Tubulin metabolism
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Calcineurin (CaN) is a Ca++/calmodulin-dependent protein phosphatase with two subunits: a catalytic subunit (CaNA) and a regulatory subunit (CaNB). With four Ca(++)-binding sites and a sequence homology to calmodulin, CaNB has been defined as the regulatory subunit for CaNA. However, we have shown that mitochondrial expression of CaNB far exceeds that of CaNA. To investigate the role of this excess CaNB, we have generated glutathione-S-transferase-CaNB (GST-CaNB) fusion protein and demonstrated that the fusion protein predominantly bound to alpha-tubulin, a 57 kDa protein in bovine brain extracts, and heat shock protein 60 (Hsp60) in bovine kidney extracts. Their Ca(++)-dependent interactions with CaNB were verified by immunoprecipitation. The binding of CaNB could be demonstrated with purified alpha/beta tubulins and Hsp60, but not GroEL, a bacterial Hsp60 analog. The interaction of CaNB and Hsp60 was not disrupted by the incubation with Hsp10, ATP and Mg++, suggesting that CaNB was not associated with Hsp60 as a misfolded substrate, and may serve as a regulatory protein. Thus, CaNB may play other regulatory roles in Ca(++)-dependent events in addition to its interaction with CaNA, and may be important for Ca(++)-dependent processes in mitochondria. more...
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- 2002
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6. Homeostatic control of uridine and the role of uridine phosphorylase: a biological and clinical update.
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Pizzorno G, Cao D, Leffert JJ, Russell RL, Zhang D, and Handschumacher RE
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- Animals, Antineoplastic Agents administration & dosage, Antineoplastic Agents adverse effects, Biological Transport, Active, Fluorouracil administration & dosage, Fluorouracil adverse effects, Gene Expression Regulation, Enzymologic, Genes, p53, Homeostasis, Humans, Neoplasms drug therapy, Neoplasms genetics, Neoplasms metabolism, Promoter Regions, Genetic, Subcellular Fractions metabolism, Uridine administration & dosage, Uridine Phosphorylase genetics, Vimentin metabolism, Uridine metabolism, Uridine Phosphorylase metabolism
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Uridine, a pyrimidine nucleoside essential for the synthesis of RNA and bio-membranes, is a crucial element in the regulation of normal physiological processes as well as pathological states. The biological effects of uridine have been associated with the regulation of the cardio-circulatory system, at the reproduction level, with both peripheral and central nervous system modulation and with the functionality of the respiratory system. Furthermore, uridine plays a role at the clinical level in modulating the cytotoxic effects of fluoropyrimidines in both normal and neoplastic tissues. The concentration of uridine in plasma and tissues is tightly regulated by cellular transport mechanisms and by the activity of uridine phosphorylase (UPase), responsible for the reversible phosphorolysis of uridine to uracil. We have recently completed several studies designed to define the mechanisms regulating UPase expression and better characterize the multiple biological effects of uridine. Immunohistochemical analysis and co-purification studies have revealed the association of UPase with the cytoskeleton and the cellular membrane. The characterization of the promoter region of UPase has indicated a direct regulation of its expression by the tumor suppressor gene p53. The evaluation of human surgical specimens has shown elevated UPase activity in tumor tissue compared to paired normal tissue. more...
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- 2002
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7. Uridine phosphorylase association with vimentin. Intracellular distribution and localization.
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Russell RL, Cao D, Zhang D, Handschumacher RE, and Pizzorno G
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- 3T3 Cells, Animals, Chromatography, Affinity, Colon, Cytoskeleton ultrastructure, Fluorescent Antibody Technique, Humans, Kinetics, Mice, Peptide Fragments chemistry, Protein Binding, Recombinant Proteins analysis, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Uridine Phosphorylase analysis, Uridine Phosphorylase isolation & purification, Vimentin analysis, Vimentin isolation & purification, Uridine Phosphorylase metabolism, Vimentin metabolism
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Uridine phosphorylase (UPase), a key enzyme in the pyrimidine salvage pathway, is associated with the intermediate filament protein vimentin, in NIH 3T3 fibroblasts and colon 26 cells. Affinity chromatography was utilized to purify UPase from colon 26 and NIH 3T3 cells using the uridine phosphorylase inhibitor 5'-amino benzylacyclouridine linked to an agarose matrix. Vimentin copurification with UPase was confirmed using both Western blot analysis and MALDI-MS methods. Separation of cytosolic proteins using gel filtration chromatography yields a high molecular weight complex containing UPase and vimentin. Purified recombinant UPase and recombinant vimentin were shown to bind in vitro with an affinity of 120 pm and a stoichiometry of 1:2. Immunofluorescence techniques confirm that UPase is associated with vimentin in both NIH 3T3 and colon 26 cells and that depolymerization of the microtubule system using nocodazole results in UPase remaining associated with the collapsed intermediate filament, vimentin. Our data demonstrate that UPase is associated with both the soluble and insoluble pools of vimentin. Approximately 60-70% of the total UPase exists in the cytosol as a soluble protein. Sequential extraction of NIH 3T3 or colon 26 cells liberates an additional 30-40% UPase activity associated with a detergent extractable fraction. All pools of UPase have been shown to possess enzymatic activity. We demonstrate for the first time that UPase is associated with vimentin and the existence of an enzymatically active cytoskeleton-associated UPase. more...
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- 2001
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8. Genomic structure, chromosomal mapping, and promoter region analysis of murine uridine phosphorylase gene.
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Cao D, Nimmakayalu MA, Wang F, Zhang D, Handschumacher RE, Bray-Ward P, and Pizzorno G
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- 3' Untranslated Regions genetics, 5' Untranslated Regions genetics, Animals, Base Sequence, Binding Sites, Cloning, Molecular, DNA-Binding Proteins metabolism, Exons, Introns, Karyotyping, Lymphocytes enzymology, Mice, Molecular Sequence Data, Polymerase Chain Reaction, Restriction Mapping, Spleen enzymology, Chromosome Mapping, Gene Expression Regulation, Enzymologic, Promoter Regions, Genetic, Uridine Phosphorylase genetics
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Uridine phosphorylase (UPase) plays an important role in the activation of 5-fluorouracil and in the regulation of tissue and plasma concentration of uridine, a potential biochemical modulator of 5-fluorouracil therapy. UPase expression is affected by the c-H-ras oncogene and various cytokines through unknown mechanisms. To understand its expression and regulation, we cloned the murine UPase gene, defined its genomic organization, determined its 5'- and 3'-end flanking sequences, and evaluated the promoter activity. The UPase gene contains nine exons and eight introns, spanning a total of approximately 18.0 kb. Its promoter lacks canonical TATA and CCAAT boxes, although a CAATAAAAA TATA-like box is seen from -41 to -49. Furthermore, IFN regulatory factor 1, c/v-Myb, and p53 binding sites are present in the promoter region, indicating that UPase expression may be directly regulated by cytokines and oncogene products. The 1.2-kb flanking fragment showed promoter activity driving the expression of the luciferase gene in various mammalian cells. A TGGGG repeat sequence is seen in the 3'-end flanking region. This element is considered to be a potential recombination consensus hot spot that may contribute to the encoding of different UPase isoforms present in different tissues, both normal and neoplastic. more...
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- 1999
9. beta-alanine and alpha-fluoro-beta-alanine concentrative transport in rat hepatocytes is mediated by GABA transporter GAT-2.
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Liu M, Russell RL, Beigelman L, Handschumacher RE, and Pizzorno G
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- Animals, Biological Transport physiology, Cell Line, Chlorides physiology, Dogs, GABA Plasma Membrane Transport Proteins, Kidney cytology, Kidney metabolism, Liver cytology, Male, Rats, Rats, Sprague-Dawley, Sodium physiology, Uridine metabolism, Carrier Proteins physiology, Liver metabolism, Membrane Transport Proteins, beta-Alanine analogs & derivatives, beta-Alanine metabolism
- Abstract
Studies on the compartmentalization of uridine catabolic metabolism in liver have indicated accumulation of beta-alanine as well as alpha-fluoro-beta-alanine (FbetaAL) for 5-fluorouracil in the hepatocytes. Using preparations of rat hepatocytes we were able to identify a Na+-dependent transport with high affinity for beta-alanine and GABA with Michaelis constant (Km) of 35.3 and 22.5 microM, respectively. A second Na+-dependent kinetic component with Km >1 mM was also identified. The sigmoidal profile of beta-alanine uptake with respect to Na+ shows the involvement of multiple ions of sodium in the transport process. A Hill coefficient of 2.6 +/- 0.4 indicates that at least two sodium ions are cotransported with beta-alanine. The flux of beta-alanine was also shown to be chlorine dependent. The substitution of this anion with gluconate, even in the presence of Na+, reduced the intracellular concentrative accumulation of beta-alanine to passive diffusion level, indicating that both Na+ and Cl- are essential for the activity of this transporter. The transport of beta-alanine was inhibited by GABA, hypotaurine, beta-aminoisobutyric acid, and FbetaAL in a competitive manner. However, concentrations up to 1 mM of L- and D-alanine, taurine, and alpha-aminoisobutyric acid did not affect beta-alanine uptake. Considering the similarities in substrate specificity with the rat GAT-2 transporter, extracts of hepatocytes were probed with the anti-GABA transporter antibody R-22. A 80-kDa band corresponding to GAT-2 was present in the hepatocyte and in the GAT-2 transfected Madin-Darby canine kidney cell extract, confirming the extraneural localization of this transporter. In view of these results, the neurotoxic effects related to the administration of uridine and 5-fluorouracil could be explained with the formation of beta-alanine and FbetaAL and their effect on the cellular reuptake of GABA. more...
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- 1999
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10. Expression, characterization, and detection of human uridine phosphorylase and identification of variant uridine phosphorolytic activity in selected human tumors.
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Liu M, Cao D, Russell R, Handschumacher RE, and Pizzorno G
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- Amino Acid Sequence, Animals, Antibody Specificity, Cloning, Molecular, DNA, Complementary genetics, DNA, Complementary metabolism, Drug Screening Assays, Antitumor, Female, Humans, Kinetics, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Neoplasms drug therapy, Rabbits, Sequence Homology, Amino Acid, Uracil pharmacology, Uridine Phosphorylase biosynthesis, Uridine Phosphorylase isolation & purification, Enzyme Inhibitors pharmacology, Neoplasms enzymology, Uracil analogs & derivatives, Uridine metabolism, Uridine Phosphorylase metabolism
- Abstract
Uridine phosphorylase (UPase) catalyzes the reversible phosphorolysis of uridine to uracil. We purified the enzyme from the murine colon 26 tumor using a two-step procedure through 5-amino-benzylacyclouridine affinity chromatography. Antibodies raised in rabbits against the purified protein revealed single bands in Western blots of normal human tissue and tumor extracts. The polyclonal antibody used to screen a human liver expression library allowed the isolation of a 1.2-kb clone that contained the entire open reading frame of the human UPase. The UPase cDNA has been expressed as a fusion protein in Escherichia coli using the pMal-C2 vector. The kinetic analysis demonstrated that the recombinant UPase preferentially uses uridine, 5-fluorouracil, and uracil as substrates, although lower levels of activity were observed with 2-deoxyuridine and thymidine. Clinical samples of human tumors and adjacent normal tissues were assayed for phosphorolytic activity and sensitivity to 5-benzylacyclouridine (BAU), a potent inhibitor of the enzyme presently in Phase I-II clinical trial. Activity in normal tissues appeared to be low but very sensitive to BAU (approximately 90% inhibition at 10 microM). Tumors had generally 2-3-fold greater activity compared with adjacent normal tissues. In breast cancer specimens and head-neck squamous carcinomas, however, uridine cleavage was only partially inhibited (40-60%) by 10 or 100 microM BAU. The BAU-insensitive activity requires phosphate and pH conditions similar to the normal enzyme, and the new phosphorolytic activity was independent from thymidine phosphorylase. The BAU-insensitive phosphorolytic activity in selected tumors, coupled with the potent inhibitory activity of BAU against the "classical" uridine phosphorylase in normal human tissues, provides the rationale for combining BAU with 5-fluorouracil in the treatment of breast and head-neck tumors. more...
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- 1998
11. Discrete roles of hepatocytes and nonparenchymal cells in uridine catabolism as a component of its homeostasis.
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Liu MP, Beigelman L, Levy E, Handschumacher RE, and Pizzorno G
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- Animals, Biological Transport, Cell Membrane metabolism, Dihydrouracil Dehydrogenase (NAD+), Kinetics, Male, Oxidoreductases metabolism, Rats, Rats, Sprague-Dawley, Sodium pharmacology, Uracil metabolism, Uridine Phosphorylase metabolism, beta-Alanine metabolism, Homeostasis, Liver metabolism, Oxidoreductases Acting on CH-CH Group Donors, Uridine metabolism
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Previous studies indicated that uridine is essentially cleared in a single pass through a rat liver and replaced in a highly regulated manner by uridine formed presumably by de novo synthesis. We report a cellular basis for the catabolic component of this apparent paradox by dissociation of the liver with collagenase into two cell fractions, hepatocytes and a nonparenchymal cell population. Suspensions of the nonparenchymal cells rapidly cleave uridine to uracil, whereas in hepatocytes this activity was <5% of that in nonparenchymal cells. Conversely, hepatocytes cause extensive degradation of uracil to -alanine. These differences correlate with the uridine phosphorylase and dihydrouracil dehydrogenase activity in cell-free extracts of each cell type. We have documented the existence of a Na+-dependent, nitrobenzylthioinosine-insensitive transport system for uridine in the parenchymal cells (Michaelis constant 46 +/- 5 microM) that achieves a three- to fourfold concentration gradient in hepatocytes. A similar system is present in the nonparenchymal cell population. In addition, a highly specific and active Na+-dependent transport system for beta-alanine, the primary catabolic metabolite of uracil, has been demonstrated in hepatocytes. more...
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- 1998
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12. Phase I clinical and pharmacological studies of benzylacyclouridine, a uridine phosphorylase inhibitor.
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Pizzorno G, Yee L, Burtness BA, Marsh JC, Darnowski JW, Chu MY, Chu SH, Chu E, Leffert JJ, Handschumacher RE, and Calabresi P
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- Aged, Aged, 80 and over, Animals, Biological Availability, Dogs, Enzyme Inhibitors adverse effects, Female, Humans, Male, Metabolic Clearance Rate, Middle Aged, Swine, Tissue Distribution, Uracil adverse effects, Uracil pharmacokinetics, Enzyme Inhibitors pharmacokinetics, Uracil analogs & derivatives, Uridine Phosphorylase antagonists & inhibitors
- Abstract
Benzylacyclouridine (BAU, IND 039655) is a potent and specific inhibitor of uridine phosphorylase (UrdPase; EC 2.4.2.3). This enzyme plays a major role in regulating uridine homeostasis and also catalyzes the conversion of fluoropyrimidine nucleosides to their respective bases. Inhibition of UrdPase enzyme activity 18-24 h after 5-fluorouracil (5-FU) administration increased plasma levels of uridine and enhanced the therapeutic index of 5-FU by rescuing normal tissues. Moreover, in vitro preclinical studies have also shown that inhibiting UrdPase enzyme activity by BAU prior to administration of 5-FU increased cytotoxicity in a number of human cancer cell lines. A series of preclinical studies was performed in dogs and pigs to evaluate the pharmacological and pharmacodynamic properties of BAU. These data showed a sustained elevation in plasma uridine concentration in both animal models. The rapid degradation of a tracer dose of uridine into uracil was virtually arrested by BAU administered both p.o. or i.v. The t1/2 of BAU was 1.8-3.6 h in dogs, with bioavailability levels of 85% (30 mg/kg) and 42.5% (120 mg/kg). In pigs, the half-life varied from 1.6 to 2.3 h, with a bioavailability of 40% at 120 mg/kg. The drug was distributed into most tissues with a tissue: plasma ratio of approximately 0.7. On the basis of these preclinical studies, we performed a Phase I clinical trial of BAU in patients with advanced cancer. Patients received 200, 400, 800, and 1600 mg/m2 BAU as a single oral dose. Toxicities included grade 2 anemia, grade 1 fever, grade 1 fatigue, grade 1 constipation, and grade 1 elevation in alkaline phosphatase; none of these toxicities were observed to be dose dependent. The maximum tolerated dose and dose-limiting toxicity were not reached at the doses given. BAU plasma concentrations and area under the curve correlated linearly with the oral dose level. The pharmacokinetics of BAU were consistent with a first-order clearance, with average peak concentrations ranging from 19 microM (200 mg/m2) to 99 microM (1600 mg/m2) and tbeta1/2 ranging from 3.0 to 3.9 h at the four dose levels. Compared with baseline plasma uridine, treatment of patients with 200, 400, 800, and 1600 mg/m2 BAU increased peak uridine concentrations by 120, 150, 250, and 175%, respectively. On the basis of this clinical study, the suggested Phase II starting dose of BAU in combination with 5-FU is 800 mg/m2. Studies combining BAU with 5-FU and incorporating appropriate molecular and biochemical end points to assess the effects of this drug combination on tumor and/or surrogate tumor tissue are under way. more...
- Published
- 1998
13. HIV protease substrate conformation: modulation by cyclophilin A.
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McCornack MA, Kakalis LT, Caserta C, Handschumacher RE, and Armitage IM
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- Catalysis, Cyclosporine pharmacology, Enzyme Inhibitors pharmacology, Isomerism, Kinetics, Magnetic Resonance Spectroscopy, Peptidylprolyl Isomerase, Proline chemistry, Proline metabolism, Protein Conformation, Tyrosine chemistry, Tyrosine metabolism, Amino Acid Isomerases metabolism, Carrier Proteins metabolism, Gene Products, gag metabolism, HIV Protease metabolism, Peptide Fragments metabolism
- Abstract
Cyclophilin A (CyPA), a cytosolic peptidyl-prolyl trans-cis isomerase can accelerate the trans-cis isomerization of Xxx-Pro peptide bonds. One- and two-dimensional 1H-NMR spectroscopy were used to determine that the heptapeptide Ser-Gln-Asn-Tyr-Pro-Ile-Val, a model peptide of an HIV-1 protease cleavage site in the gag polyprotein of HIV-1, is a substrate for CyPA. Experiments revealed a slow exchange about the Tyr-Pro peptide bond with 30 +/- 5% in the cis conformation (pH 1-9). While the interconversion rate is too slow to measure by kinetic NMR methods in the absence of CyPA, these methods, saturation transfer and NOE experiments, established that CyPA enhanced the rate of trans-cis interconversion, a process inhibited by cyclosporin A (CsA). With a substrate:CyPA ratio of 40:1, an interconversion rate of 2.5 s(-1) at 25 degrees C was observed. more...
- Published
- 1997
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14. Pharmacokinetic and phase I trial of intraperitoneal carboplatin and cyclosporine in refractory ovarian cancer patients.
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Chambers SK, Chambers JT, Davis CA, Kohorn EI, Schwartz PE, Lorber MI, Handschumacher RE, and Pizzorno G
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- Antineoplastic Agents blood, Carboplatin blood, Carcinoma drug therapy, Carcinoma metabolism, Cyclosporine blood, Feasibility Studies, Female, Germinoma drug therapy, Germinoma metabolism, Half-Life, Humans, Immunosuppressive Agents blood, Mixed Tumor, Mesodermal drug therapy, Mixed Tumor, Mesodermal metabolism, Pilot Projects, ROC Curve, Retroperitoneal Space, Antineoplastic Agents administration & dosage, Carboplatin administration & dosage, Cyclosporine administration & dosage, Cyclosporine pharmacokinetics, Immunosuppressive Agents administration & dosage, Immunosuppressive Agents pharmacokinetics, Ovarian Neoplasms drug therapy, Ovarian Neoplasms metabolism
- Abstract
Purpose: The feasibility and pharmacokinetics of cyclosporine (CsA) delivered intraperitoneally (IP) have not been previously explored. We performed a pharmacokinetic study of IP CsA followed by a phase I dose-escalation trial of the combination of IP CsA and carboplatin in refractory ovarian cancer patients., Patients and Methods: A pilot study was performed of three patients who received 1, 10, and 20 mg/kg IP CsA alone. Subsequently, a phase I trial of 35 patients was performed between April 1990 and April 1993. Whole-blood and IP fluid CsA concentrations were measured at serial time points. The highest dose delivered IP was 34.6 mg CsA/kg in combination with carboplatin (250 mg/m2 or 300 mg/m2, depending on creatinine clearance), which was not dose-escalated. The area under the concentration-time curve (AUC) for CsA and half-life (T1/2) were calculated. Objective and serologic responses were noted, and toxicity was graded using the National Cancer Institute common toxicity criteria., Results: The feasibility of delivering IP CsA alone was established. We observed a 1,000:1 ratio between IP fluid and blood concentrations at 20 mg CsA/kg. Pharmacokinetic analysis confirmed that at 20 mg CsA/kg, there was an IP fluid-to-blood AUC ratio of 600:1 in favor of peritoneal exposure. At the highest dose delivered, 34.6 mg CsA/kg, the mean IP CsA levels of 1,110 micrograms/ mL were tolerated moderately well and the IP fluid-to-blood ratio of 1,000:1 was maintained. Blood and IP CsA concentrations were analyzed in the presence and absence of IP carboplatin. At 20 mg CsA/kg, there was no difference in either mean blood CsA levels (0.9 microgram/ mL) or mean IP CsA concentrations (1,000 micrograms/mL) obtained in the absence or presence of carboplatin. The most common toxicity in the phase I study was anemia, seen in 66% of patients. Common toxicities at the maximum CsA dose delivered (34.6 mg/kg) were anemia, leukopenia, thrombocytopenia, and hypertension. In this trial, three objective responses (two complete and one partial) were observed for a duration of 3 to 11 months. Control of platinum-resistant ascites was an important feature, noted in five of eight patients., Conclusion: We have established the feasibility of delivering IP CsA up to doses of 34.6 mg/kg in conjunction with carboplatin, and the sustaining of IP fluid to blood ratios of 1,000:1. The IP administration of CsA resulted in a favorable ratio of exposure for the peritoneal cavity compared with systemic exposure, indicating a therapeutic advantage of this approach with a significant decrease in systemic toxicity. We recommend that 34.6 mg/ kg of IP CsA be tested as a phase II dose in combination with carboplatin in refractory ovarian cancer patients. This report provides the groundwork for future studies using IP CsA, both as a chemomodulator of platinum and of multidrug resistance. more...
- Published
- 1997
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15. Regulation of the nuclear factor of activated T cells in stably transfected Jurkat cell clones.
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Li W and Handschumacher RE
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- Alkaline Phosphatase biosynthesis, Bucladesine pharmacology, Clone Cells, Colforsin pharmacology, Cyclosporine pharmacology, DNA-Binding Proteins biosynthesis, Dinoprostone pharmacology, Enzyme Inhibitors pharmacology, Ethers, Cyclic pharmacology, Female, Humans, Immunosuppressive Agents pharmacology, Kinetics, Marine Toxins, NFATC Transcription Factors, Okadaic Acid, Oxazoles pharmacology, Phosphoprotein Phosphatases antagonists & inhibitors, Placenta, Pregnancy, Protein Phosphatase 1, Recombinant Proteins biosynthesis, Recombinant Proteins metabolism, T-Lymphocytes drug effects, T-Lymphocytes immunology, Tacrolimus pharmacology, Transcription Factors biosynthesis, Transfection, Tumor Cells, Cultured, DNA-Binding Proteins physiology, Lymphocyte Activation, Nuclear Proteins, T-Lymphocytes physiology, Tetradecanoylphorbol Acetate pharmacology, Transcription Factors physiology
- Abstract
Two Jurkat cell clones have been stably transfected with a reporter vector for the nuclear factor of activated T cells (NFAT). Upon stimulation, they express high levels of secreted heat stable placental alkaline phosphatase. With these clones, we demonstrated that NFAT activation induced by phorbol 12-myristate 13-acetate and ionomycin was inhibited by both cyclosporin A (CsA) (IC50 = 8 nM) and FK506 (IC50 = 160 pM), presumably by inhibition of calcineurin activity. Selective phosphatase inhibitors for protein phosphatase 1 (PP1) and 2A (PP2A) that do not inhibit calcineurin, such as okadaic acid and calyculin A, also inhibited NFAT activation with IC50s of 87 nM and 4 nM, respectively, suggesting that okadaic acid and related inhibitors may block NFAT activation through the inhibition of PP1, instead of PP2A. NFAT activation was also inhibited by agents that increase cAMP concentrations such as dibutyryl cAMP, forskolin and prostaglandin E2. These stable Jurkat cell clones provide a convenient and sensitive tool to study NFAT regulation. more...
- Published
- 1996
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16. The cyclosporin A-binding immunophilin CyP-40 and the FK506-binding immunophilin hsp56 bind to a common site on hsp90 and exist in independent cytosolic heterocomplexes with the untransformed glucocorticoid receptor.
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Owens-Grillo JK, Hoffmann K, Hutchison KA, Yem AW, Deibel MR Jr, Handschumacher RE, and Pratt WB
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- Animals, Binding Sites, Blotting, Western, CHO Cells, Carrier Proteins isolation & purification, Cattle, Cell-Free System, Cricetinae, Cytosol metabolism, DNA-Binding Proteins isolation & purification, Electrophoresis, Polyacrylamide Gel, HSP90 Heat-Shock Proteins isolation & purification, Heat-Shock Proteins isolation & purification, Mice, Molecular Weight, Rabbits, Receptors, Glucocorticoid isolation & purification, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Reticulocytes metabolism, Tacrolimus Binding Proteins, Transfection, Carrier Proteins metabolism, Cyclosporine metabolism, DNA-Binding Proteins metabolism, HSP90 Heat-Shock Proteins metabolism, Heat-Shock Proteins metabolism, Receptors, Glucocorticoid metabolism
- Abstract
We have recently shown that hsp56, the FK506-binding immunophilin component of both the heat shock protein (hsp90.hsp70.hsp56) heterocomplex and the untransformed glucocorticoid receptor heterocomplex, is bound directly to hsp90 (Czar, M. J., Owens-Grillo, J. K., Dittmar, K. D., Hutchison, K. A., Zacharek, A. M., Leach, K. L., Deibel, M. R., and Pratt, W. B. (1994) J. Biol. Chem. 269, 11155-11161). In this work, we show that both untransformed glucocorticoid receptor and hsp90 heterocomplexes contain CyP-40, a 40-kDa immunophilin of the cyclosporin A-binding class. CyP-40 is present in both native glucocorticoid receptor heterocomplexes and receptor heterocomplexes reconstituted with rabbit reticulocyte lysate, and the presence of CyP-40 in the receptor heterocomplex is stabilized by molybdate. Immunoadsorption of hsp90 from cell lysate yields coimmunoadsorption of both hsp56 and CyP-40, showing that both immunophilins are in native heterocomplex with hsp90. However, immunoadsorption of hsp56 does not yield coimmunoadsorption of CyP-40; thus, the two immunophilins do not exist in the same heterocomplex with hsp90. Both purified CyP-40 and hsp56 bind directly to purified hsp90, and excess CyP-40 blocks the binding of hsp56, consistent with the presence of a common immunophilin binding site on hsp90. Our data also suggest that there are at least two types of untransformed glucocorticoid receptor-hsp90 heterocomplexes, one that contains hsp56 and another that contains CyP-40. The role played by the immunophilins in steroid receptor action is unknown, but it is clear that the peptidylprolyl isomerase activity of immunophilins is not required for glucocorticoid receptor-hsp90 heterocomplex assembly and proper folding of the hormone binding domain by the hsp90-associated protein folding system of reticulocyte lysate. more...
- Published
- 1995
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17. Tamoxifen induces Na+ -dependent uridine transport and dome formation in a human breast tumor cell line.
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Liu MP and Handschumacher RE
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- Biological Transport drug effects, Breast Neoplasms pathology, Cell Growth Processes drug effects, Cell Line, Tumor, Estradiol analogs & derivatives, Estradiol pharmacology, Humans, Polyunsaturated Alkamides, Tamoxifen analogs & derivatives, Breast Neoplasms drug therapy, Breast Neoplasms metabolism, Sodium metabolism, Tamoxifen pharmacology, Uridine metabolism
- Abstract
Purpose: To correlate changes in uridine transport and colony morphology with differentiation of human breast cancer cells by tamoxifen and related agents., Materials and Methods: Cultures of MCF-7 human breast cancer were treated with estradiol or the antiestrogen derivatives tamoxifen, hydroxytamoxifen, and ICI 164, 384. Initial rates of uridine transport and equilibrium concentrations were determined and morphological characteristics of the cultures evaluated., Results: Tamoxifen causes an early induction of a Na+ -dependent transport of uridine characteristic of normal epithelial cells but absent in normal MCF-7 cultures and most human neoplasms examined. The pure antiestrogen ICI 164,384 and the more potent 4-hydroxytamoxifen also induced concentrative uridine transport; estradiol could prevent the expression of this transporter. Associated with induction of transport was a dramatic increase in dome formation in the cultures, a measure of unidirectional inorganic ion transport characteristic of the differentiated state., Conclusions: The induction of a concentrative transport of uridine is a concomitant of cellular differentiation of breast tumor cells. These findings give added weight to evidence that uridine may play a regulatory role in the transition to the neoplastic state. The absence of the transporter and low intracellular uridine concentrations in the undifferentiated state may relate to 5-FU sensitivity of breast tumors. Induction of the transporter by tamoxifen and the consequent major increase in intracellular concentrations of free uridine suggests a potentially negative effect of tamoxifen on regimens containing 5-FU. more...
- Published
- 1995
18. Cyclosporin A potentiates the dexamethasone-induced mouse mammary tumor virus-chloramphenicol acetyltransferase activity in LMCAT cells: a possible role for different heat shock protein-binding immunophilins in glucocorticosteroid receptor-mediated gene expression.
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Renoir JM, Mercier-Bodard C, Hoffmann K, Le Bihan S, Ning YM, Sanchez ER, Handschumacher RE, and Baulieu EE
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- Animals, Cytosol metabolism, Drug Synergism, L Cells, Mice, Recombinant Proteins biosynthesis, Tacrolimus pharmacology, Transcription, Genetic drug effects, Transfection, Triamcinolone Acetonide metabolism, Chloramphenicol O-Acetyltransferase biosynthesis, Cyclosporine pharmacology, Dexamethasone pharmacology, Gene Expression drug effects, Mammary Tumor Virus, Mouse genetics, Receptors, Glucocorticoid metabolism
- Abstract
As previously observed for FK506, we report here that cyclosporin A (CsA) treatment of mouse fibroblast cells stably transfected with the mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter plasmid (LMCAT cells) results in potentiation of dexamethasone (Dex)-induced CAT gene expression. Potentiation by CsA is observed in cells treated with 10-100 nM Dex but not in cells treated with 1 microM Dex, a concentration of hormone which results in maximum CAT activity. At 10 nM Dex, 1-5 microM CsA provokes an approximately 50-fold increase in CAT gene transcription, compared with transcription induced by Dex alone. No induction of CAT gene expression is observed in cells treated with CsA or FK506 in the absence of Dex. The antisteroid RU 486 abolishes effects obtained in the presence of Dex. Using a series of CsA, as well as FK506, analogs, including some devoid of calcineurin phosphatase inhibition activity, we conclude that the potentiation effects of these drugs on Dex-induced CAT gene expression in LMCAT cells do not occur through a calcineurin-mediated pathway. Western-blotting experiments following immunoprecipitation of glucocorticosteroid receptor (GR) complexes resulted in coprecipitation of GR, heat shock protein hsp90 and two immunophilins: the FK506-binding protein FKBP59 and the CsA-binding protein cyclophilin 40 (CYP40). Two separate immunophilin-hsp90 complexes are present in LMCAT cells: one containing CYP40-hsp90, the other FKBP59-hsp90. Thus, both FKBP59 and CYP40 can be classified as hsp-binding immunophilins, and their possible involvement as targets of immunosuppressants potentiating the GR-mediated transcriptional activity is discussed. more...
- Published
- 1995
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19. Cyclophilin-40: evidence for a dimeric complex with hsp90.
- Author
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Hoffmann K and Handschumacher RE
- Subjects
- Amino Acid Isomerases immunology, Amino Acid Sequence, Base Sequence, Binding Sites, Blotting, Western, Carrier Proteins immunology, Peptidyl-Prolyl Isomerase F, Cyclosporine pharmacology, Glutathione Transferase metabolism, HSP70 Heat-Shock Proteins pharmacology, Humans, Molecular Sequence Data, Protein Binding, Recombinant Fusion Proteins metabolism, Amino Acid Isomerases metabolism, Carrier Proteins metabolism, Cyclophilins, HSP90 Heat-Shock Proteins metabolism, Peptidylprolyl Isomerase, Protein Conformation
- Abstract
The expression of human cyclophilin 40 (CyP-40) as a glutathione S-transferase fusion protein has provided a means to identify cellular components that are in association with this ubiquitous protein. When the fusion protein was coupled to a GSH affinity matrix, heat-shock protein 90 (hsp90) was found to be the predominant associated protein in all tissue extracts examined. The relatively high concentration of each of these proteins in various tissues indicates that the dimeric complex exists in concentrations that exceed those of the inactive steroid receptors of which each protein is a component. Association does not occur with heat-shock protein 70 and is not affected by cyclosporin A (CsA). Independent expression of two domains of CyP-40 permitted dissociation of N-terminal isomerase and CsA binding activity from the hsp90 binding site, which is located at the FKBP-59-like C-terminal region. The biological association of CyP-40 with hsp90 in many tissues may reflect a conjoint role in protein folding and trafficking. more...
- Published
- 1995
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- View/download PDF
20. Expression of human cyclophilin-40 and the effect of the His141-->Trp mutation on catalysis and cyclosporin A binding.
- Author
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Hoffmann K, Kakalis LT, Anderson KS, Armitage IM, and Handschumacher RE
- Subjects
- Amino Acid Isomerases genetics, Base Sequence, Binding Sites, Carrier Proteins genetics, Peptidyl-Prolyl Isomerase F, Enzyme Activation, Escherichia coli genetics, Humans, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Binding, Recombinant Proteins metabolism, Amino Acid Isomerases metabolism, Carrier Proteins metabolism, Cyclophilins, Cyclosporine metabolism, Peptidylprolyl Isomerase
- Abstract
The cDNA encoding a human cytosolic 40-kDa cyclophilin (CyP-40) has been inserted into a modified pGEX-3X expression vector and expressed in Escherichia coli to yield recombinant CyP-40 at levels up to 4 mg/l medium. The protein was purified to homogeneity using a cyclosporin affinity matrix and gel filtration. The recombinant CyP-40 showed peptidyl-prolyl cis-trans isomerase activity (kcat/Km = 1.12 x 10(6) M-1.s-1) comparable to that of bovine brain CyP-40. The weak affinity of CyP-40 for cyclosporin A was postulated to arise from a histidine residue that replaces a tryptophan residue critical for cyclosporin A binding and highly conserved in other cyclophilins that have high affinity for cyclosporin A. Site-directed mutagenesis to replace His141 by tryptophan yielded a protein with an approximately 20-fold greater affinity for cyclosporin A (Kdapp 11.5 +/- 2 nM as determined by tryptophan fluorescence measurements). The intrinsic isomerase activity of this mutant protein with succinyl-Ala-Ala-Pro-Phe 4-nitroanilide as substrate was about nine times greater than the value obtained for the nonmutated recombinant CyP-40 and had an activity similar to that of CyP-18. NMR difference spectroscopy and molecular modelling revealed a cyclosporin-A-binding domain that is similar to that of CyP-18. more...
- Published
- 1995
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21. Toxoplasma gondii tachyzoites possess an unusual plasma membrane adenosine transporter.
- Author
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Schwab JC, Afifi Afifi M, Pizzorno G, Handschumacher RE, and Joiner KA
- Subjects
- Adenosine Kinase genetics, Adenosine Kinase metabolism, Animals, Binding, Competitive, Biological Transport, Carrier Proteins drug effects, Dipyridamole pharmacology, Membrane Proteins drug effects, Nucleoside Transport Proteins, Protozoan Proteins drug effects, Purines metabolism, Thioinosine analogs & derivatives, Thioinosine pharmacology, Toxoplasma growth & development, Adenosine metabolism, Carrier Proteins metabolism, Membrane Proteins metabolism, Protozoan Proteins metabolism, Toxoplasma chemistry
- Abstract
Nucleoside transport may play a critical role in successful intracellular parasitism by Toxoplasma gondii. This protozoan is incapable of de novo purine synthesis, and must salvage purines from the host cell. We characterized purine transport by extracellular T. gondii tachyzoites, focusing on adenosine, the preferred salvage substrate. Although wild-type RH tachyzoites concentrated [3H]adenosine 1.8-fold within 30 s, approx. half of the [3H]adenosine was converted to nucleotide, consistent with the known high parasite adenosine kinase activity. Studies using an adenosine kinase deficient mutant confirmed that adenosine transport was non-concentrative. [14C]Inosine, [14C]hypoxanthine and [3H]adenine transport was also rapid and non-concentrative. Adenosine transport was inhibited by dipyridamole (IC50 approx. 0.7 microM), but not nitrobenzylthioinosine (15 microM). Transport of inosine, hypoxanthine and adenine was minimally inhibited by 10 microM dipyridamole, however. Competition experiments using unlabeled nucleosides and bases demonstrated distinct inhibitor profiles for [3H]adenosine and [14C]inosine transport. These results are most consistent with a single, dipyridamole-sensitive, adenosine transporter located in the T. gondii plasma membrane. Additional permeation pathways for inosine, hypoxanthine, adenine and other purines may also be present. more...
- Published
- 1995
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22. Effect of clinically modeled regimens on the growth response and development of resistance in human colon carcinoma cell lines.
- Author
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Pizzorno G and Handschumacher RE
- Subjects
- Cell Division drug effects, Colonic Neoplasms enzymology, Colonic Neoplasms pathology, Drug Resistance, Fluorouracil administration & dosage, Humans, Orotate Phosphoribosyltransferase metabolism, Time Factors, Tumor Cells, Cultured drug effects, Uridine Kinase metabolism, Colonic Neoplasms drug therapy, Fluorouracil pharmacology
- Abstract
Two human colon cell lines, HCT-8 and HT-29, were exposed to 5-fluorouracil (FUra) under conditions similar to the human plasma pharmacokinetic profile achieved by a single bolus dose or a sustained i.v. infusion. The bolus treatment for 5 days caused a substantial cell kill; however, only a moderate inhibition in cell growth was obtained with sustained exposure to the clinically relevant level of 2 microM. To achieve a cell kill equivalent to the bolus method, a sustained concentration of 10 microM was required. This would constitute a 60% increase in the total area under the curve (AUC) compared with the bolus treatment. After three courses of therapy with each of the schedules, emerging cell lines displayed a similar degree of resistance. HT-29 resistant cell lines returned to the original sensitivity within a few weeks, and most of the enzymes involved in the metabolic activation of FUra returned to their pretreatment activities. However, resistance and enzymatic modifications remained in the HCT-8 line for at least 3 months. In the HCT-8 cell line derived from bolus treatment, resistance was associated with a 50-60% reduction in uridine kinase activity. In the line derived from continuous exposure, there was a 35-40% reduction in uridine kinase in addition to a greater reduction in the activity of orotate phosphoribosyltransferase. These changes in both resistant cell lines resulted in a decreased incorporation of [3H]FUra into nucleic acids and a reduced formation of di- and triphosphate nucleotides of FUra. more...
- Published
- 1995
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23. Aberrant cell cycle inhibition pattern in human colon carcinoma cell lines after exposure to 5-fluorouracil.
- Author
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Pizzorno G, Sun Z, and Handschumacher RE
- Subjects
- Colonic Neoplasms drug therapy, DNA biosynthesis, Drug Interactions, Humans, Leucovorin pharmacology, S Phase, Thymidine pharmacology, Thymidylate Synthase metabolism, Tumor Cells, Cultured drug effects, Cell Cycle drug effects, Fluorouracil pharmacology
- Abstract
In this report, we describe the use of two human colon carcinoma cell lines, HCT-8 and HT-29, as potential models to study DNA- and RNA-directed cytotoxicity due to 5-fluorouracil (FUra) exposure by flow microfluorimetric analysis of DNA cell content. The sensitivity of the HT-29 line (EC50 = 0.9 microM) to FUra was somewhat greater than that of the HCT-8 line (EC50 = 4 microM), but each presented a dramatically different DNA histogram after exposure to FUra. In HCT-8, an unexpected and nearly complete disappearance of cells in S-phase occurred, whereas in HT-29 the expected accumulation of cells at the G1-S border was observed. The absence of HCT-8 cells in S-phase also occurred as a result of two RNA polymerase inhibitors: actinomycin D and dichloro-D-ribofuranosylbenzimidazole. However, an accumulation of cells in S-phase was observed in the presence of 5-fluorodeoxyuridine. These results suggest that in the HCT-8 cell line, FUra predominantly causes an RNA-related toxicity. By comparison, the rate of formation of 5-fluorodeoxyuridine monophosphate, the increased dUMP pool size, and low thymidylate synthase activity in the HT-29 line are consistent with its greater susceptibility to DNA-directed toxicity. Further evidence was seen in the prevention of FUra cytotoxicity by thymidine in HT-29, but not in HCT-8 cells. Similarly, Leucovorin synergized the action of FUra in HT-29 but not in HCT-8. Enzymatic correlates supporting these observations are seen in the greater activity of uridine kinase than thymidine kinase (20:1) in HCT-8 cells compared with that in HT-29 cells (4:1). more...
- Published
- 1995
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24. Concentrative transport of adenosine in murine splenocytes: limitation by an ecto-adenosine deaminase.
- Author
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Lee CW and Handschumacher RE
- Subjects
- Animals, Biological Transport physiology, Choline metabolism, Chromatography, High Pressure Liquid, Dipyridamole pharmacology, Female, Inosine metabolism, Mice, Mice, Inbred Strains, Pentostatin pharmacology, Sodium metabolism, Spleen enzymology, Tritium, Uridine metabolism, Adenosine metabolism, Adenosine Deaminase metabolism, Spleen cytology
- Abstract
Coincident with studies of the transport of (3H]adenosine in murine splenocytes, we have evidence for the extracellular degradation of adenosine. A Na(+)-dependent active transport system for nucleosides exists in splenocytes, but no intracellular concentration gradient of adenosine was observed. Inhibition of adenosine transport across the plasma membrane by dipyridamole and a Na(+)-free medium did not prevent the deamination of extracellular adenosine by what has been generally considered to be a cytosolic enzyme. This failure to achieve an adenosine concentration gradient appears to be consequent to the action of a very active ecto-adenosine deaminase. Inhibition of the adenosine deaminase by deoxycoformycin permits a 6-fold increase in intracellular adenosine concentration relative to the medium by the Na(+)-dependent process. Rapid inhibition of adenosine deaminase by deoxycoformycin occurs even in the presence of dipyridamole which prevents the entry of deoxycoformycin as well as adenosine into the cells in a Na(+)-free medium. These results further support the view that this is an ectoenzyme activity. The kinetics of active adenosine transport were Km = 7.8 +/- 1.1 microM with Vmax = 8.2 +/- 2.8 microM/s in a Na+ medium and much less efficiently in a Li+ medium (Km = 250 +/- 50 microM,Vmax = 7.8 +/- 1.3 microM/s). Inhibition of adenosine transport by other nucleosides suggests a single Na(+)-dependent nucleoside transport system in murine splenocytes with narrow substrate specificity. more...
- Published
- 1995
25. Differentiation of HL-60 cells by dimethylsulfoxide activates a Na(+)-dependent nucleoside transport system.
- Author
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Lee CW, Sokoloski JA, Sartorelli AC, and Handschumacher RE
- Subjects
- Biological Transport drug effects, Carrier Proteins drug effects, Cell Differentiation drug effects, Cell Division drug effects, Cell Line, Cell Membrane metabolism, Choline pharmacology, Dipyridamole pharmacology, Humans, Kinetics, Leukemia, Promyelocytic, Acute, Ribonucleosides pharmacology, Sodium pharmacology, Tumor Cells, Cultured, Carrier Proteins metabolism, Cell Differentiation physiology, Dimethyl Sulfoxide pharmacology, Membrane Transport Proteins, Sodium metabolism, Uridine metabolism
- Abstract
Uridine transport in undifferentiated HL-60 cells occurs primarily by facilitated diffusion, but a limited Na(+)-dependent process can be demonstrated (Km = 44 +/- 4.4 microM, Vmax = 0.13 +/- 0.01 microM/s). This latter transport system was inhibited by adenosine and inosine (Ki = 110 and 260 microM, respectively), whereas guanosine and thymidine were less effective (Ki = 1600 and 1200 microM, respectively). Dimethylsulfoxide (DMSO) caused a concentration-dependent decrease in facilitated uridine transport. This change was attributable to a decrease in the number of transporter molecules as determined by the binding of [3H]nitrobenzylthioinosine to cell membranes. Moreover, the Na(+)-dependent transport of uridine was enhanced by DMSO at a concentration of the polar solvent as low as 0.4%. When HL-60 cells were exposed to 1.0% DMSO, a marked increase in Na(+)-dependent uridine transport occurred within 72 hr, a time preceding maximum granulocytic differentiation. This change was attributable to an increase in transport affinity (Km = 1.54 +/- 0.65 microM), with no change in Vmax (Vmax = 0.13 +/- 0.02 microM/s). The consequence of these changes was the generation of a 3- to 4-fold increase in the intracellular concentration of uridine relative to the medium at a physiological concentration of 5 microM uridine. Similar increases in transport affinity were observed for adenosine, inosine, guanosine and thymidine in DMSO-differentiated HL-60 cells (Km values of 2 to 5 microM). These results complement our previous studies with phorbol 12-myristate 13-acetate, in which differentiation to a monocytic phenotype was also associated with enhanced Na(+)-dependent nucleoside transport. more...
- Published
- 1994
26. Isolation, cDNA sequences, and biochemical characterization of the major cyclosporin-binding proteins of Toxoplasma gondii.
- Author
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High KP, Joiner KA, and Handschumacher RE
- Subjects
- Amino Acid Isomerases antagonists & inhibitors, Amino Acid Isomerases chemistry, Amino Acid Isomerases metabolism, Amino Acid Sequence, Animals, Base Sequence, Calcineurin, Calmodulin-Binding Proteins chemistry, Carrier Proteins antagonists & inhibitors, Carrier Proteins chemistry, Carrier Proteins metabolism, Cell Compartmentation, Cyclosporins metabolism, Cyclosporins pharmacology, DNA Primers chemistry, DNA, Complementary, Humans, Molecular Sequence Data, Peptidylprolyl Isomerase, Phosphoprotein Phosphatases chemistry, Sequence Alignment, Sequence Homology, Amino Acid, Toxoplasma genetics, Amino Acid Isomerases genetics, Carrier Proteins genetics, Genes, Protozoan, Protozoan Proteins genetics, Toxoplasma immunology
- Abstract
The activities of the immunosuppressive, antifungal compounds cyclosporin A (CsA), FK-506, and rapamycin are dependent upon high affinity binding proteins collectively termed immunophilins. We report the isolation, biochemical characterization, and amino acid sequences of two major CsA-binding proteins, cyclophilins, from the pathogenic protozoan, Toxoplasma gondii. The 18.5- and 20-kDa molecular mass proteins exhibit peptidylproline cis-trans-isomerase activity, which is inhibitable by 10(-8) M CsA. The amino acid sequences of these two proteins, deduced from cDNA clones, reveal up to 70% amino acid identity to previously isolated cyclophilins. The 18.5-kDa protein appears to be synthesized as a precursor with a 15 amino acid signal peptide. The amino-terminal region of the mature 20-kDa protein has significant homology to the B subunit of the calmodulin-dependent phosphatase, calcineurin. The two T. gondii cyclophilins are products of different genes and appear to have different subcellular distributions. more...
- Published
- 1994
27. Specific interaction of the cyclophilin-cyclosporin complex with the B subunit of calcineurin.
- Author
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Li W and Handschumacher RE
- Subjects
- Animals, Brain enzymology, Calcineurin, Cattle, Cross-Linking Reagents, Cytosol enzymology, Humans, Immunosuppressive Agents pharmacology, Macromolecular Substances, Peptidylprolyl Isomerase, Polyenes pharmacology, Recombinant Proteins metabolism, Sirolimus, Tacrolimus metabolism, Tacrolimus Binding Proteins, Amino Acid Isomerases metabolism, Calmodulin-Binding Proteins metabolism, Carrier Proteins metabolism, Cyclosporine metabolism, Phosphoprotein Phosphatases metabolism
- Abstract
Calcineurin (CaN), a Ca2+/calmodulin-dependent serine/threonine phosphatase, has been shown to be inhibited by the complex of the immunosuppressant cyclosporin A (CsA) and its receptor, cyclophilin (CyP), but not by either alone. In the current study, the topological relationship between cyclophilin and the subunits of CaN has been explored using chemical cross-linking agents. In the presence of cyclosporin, 125I-CyP, shown to bind to CsA, is extensively cross-linked to the B subunit of CaN but not the catalytic A subunit. However, the A subunit is required for binding of the CyP.CsA complex, since cross-linking to recombinant B subunit alone does not occur. The kinetics of association indicate a saturable reaction with a Kd of less than 70 nM. Cross-linking to the B subunit occurs with cross-linkers that span from 0 to 16 A and employ different cross-linking chemistry, indicating direct contact between the B subunit and CyP. Similar cross-linking to the B subunit has been observed with the complex of 125I-labeled FK506 binding protein (FKBP) and FK506 but not with the FKBP-rapamycin complex. CyP.CsA cross-linking to CaN is Ca2+/calmodulin-dependent with intact CaN, but Ca2+/calmodulin-independent after digestion to remove the calmodulin binding and autoinhibitory domain. more...
- Published
- 1993
28. Cyclophilin-40, a protein with homology to the P59 component of the steroid receptor complex. Cloning of the cDNA and further characterization.
- Author
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Kieffer LJ, Seng TW, Li W, Osterman DG, Handschumacher RE, and Bayney RM
- Subjects
- Amino Acid Isomerases genetics, Amino Acid Sequence, Animals, Base Sequence, Brain metabolism, Carrier Proteins genetics, Cattle, Cloning, Molecular, Peptidyl-Prolyl Isomerase F, Cyclosporine metabolism, DNA, Humans, Molecular Sequence Data, Oligodeoxyribonucleotides, Pancreas metabolism, Peptidylprolyl Isomerase, Sequence Homology, Amino Acid, Amino Acid Isomerases chemistry, Carrier Proteins chemistry, Cyclophilins, Receptors, Steroid chemistry
- Abstract
We have reported previously the isolation and preliminary characterization of a 40-kDa cyclosporin A (CsA)-binding protein, cyclophilin-40 (CyP-40). To determine the sequence of this protein, degenerate oligonucleotide primers based on bovine brain CyP-40 tryptic peptides were used to generate a polymerase chain reaction fragment of CyP-40 cDNA. This was used to isolate the complete cDNA from a human pancreatic islet cell library. Northern analysis indicated ubiquitous distribution of CyP-40 mRNA throughout human tissues. The CyP-18 domain of CyP-40 is most similar to maize CyP (64.3% identity), whereas 150 amino acids of the non-CyP-18 domain of CyP-40 share 30.7% identity with P59, a member of the steroid receptor complex. Failure to detect glycosylation and mass spectroscopy with isolated CyP-40 indicate minimal, if any, posttranslational modification. Employing a new assay for calcineurin protein phosphatase activity to compare the effects of CyP-40.CsA and CyP-18.CsA complexes, IC50 values of 320 nM +/- 20 and 195 nM +/- 15, respectively, were obtained. A chemical cross-linking study revealed that CyP-40 competes for 125I-CyP-18 binding to calcineurin in the presence of CsA. The homology of CyP-40 to P59 suggests that CyP-40 might be involved in modulating the activity of biologically important receptors. more...
- Published
- 1993
29. Autoantibodies against cyclophilin in systemic lupus erythematosus and Lyme disease.
- Author
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Kratz A, Harding MW, Craft J, Mackworth-Young CG, and Handschumacher RE
- Subjects
- Adolescent, Adult, Aged, Antibodies, Anti-Idiotypic blood, Antibodies, Monoclonal, Antibody Specificity, Autoantibodies immunology, Child, Enzyme-Linked Immunosorbent Assay, Family Health, Female, Humans, Immunoglobulin G blood, Immunoglobulin Idiotypes blood, Immunoglobulin M blood, Lupus Erythematosus, Systemic genetics, Male, Middle Aged, Peptidylprolyl Isomerase, Protein Denaturation, Amino Acid Isomerases immunology, Autoantibodies blood, Carrier Proteins immunology, Lupus Erythematosus, Systemic immunology, Lyme Disease immunology
- Abstract
Autoantibodies against cyclophilin, a cyclosporin A binding protein, were detected in sera of 29 of 46 (63%) patients with systemic lupus erythematosus and 14 of 40 (35%) Lyme disease patients. The antibodies are directed against the denatured form of both the major and minor isoform of cyclophilin and can be demonstrated in Western blots. Some first-degree relatives of lupus patients also express these antibodies. They are specific for cyclophilin and are not the consequence of hypergammaglobulinaemia. Four monoclonal IgM antibodies from a patient with lepromatous leprosy also bound to cyclophilin. The generation of these antibodies may be of special interest because they are against a protein involved in the control of the immune system not known to be directly associated with DNA or RNA. more...
- Published
- 1992
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30. Immunofluorescent localization and immunochemical determination of cyclophilin-A with specific rabbit antisera.
- Author
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Sarris AH, Harding MW, Jiang TR, Aftab D, and Handschumacher RE
- Subjects
- Animals, Antibody Specificity, Blotting, Western, Cells, Cultured, Cyclosporine pharmacology, Female, Fluorescent Antibody Technique, Immunochemistry, Peptidylprolyl Isomerase, Rabbits, Amino Acid Isomerases analysis, Carrier Proteins analysis
- Abstract
We raised rabbit antisera against homogeneous bovine cyclophilin A (CypA) and we report their use for its immunofluorescent and immunochemical detection without resorting to cyclosporine binding. Indirect immunofluorescence demonstrated that in tissue culture cells CypA is present in the cytoplasm diffusely and also associated with vesicles and the Golgi apparatus. In mitotic cells CypA is increased in amount and redistributed away from cytoplasmic organelles. High levels of CypA were demonstrated in murine splenic erythroblasts and myeloblasts, but they became undetectable during differentiation to mature erythrocytes and granulocytes. Large, often granular, lymphocytes stained very intensely, but small lymphocytes demonstrated variable staining. Dot blot immunoassays demonstrated that murine tissues contain similar amounts of CypA. During CsA treatment murine liver can increase its CypA content much more than spleen. In summary, we demonstrated that cells known to be resistant to the effects of CsA have high levels of CypA. Also tissues that are resistant to CsA can increase their levels more than sensitive tissues upon CsA exposure. Taken together these results suggest that CypA plays a role in cell cycle progression and that sensitivity to CsA may not be simply a reflection of the baseline CypA levels, but may also be affected by the regulation of these levels. Further work is needed in order to delineate the role of CypA in the cell cycle and its relation to the action of CsA. more...
- Published
- 1992
- Full Text
- View/download PDF
31. Leukocyte chemotactic activity of cyclophilin.
- Author
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Xu Q, Leiva MC, Fischkoff SA, Handschumacher RE, and Lyttle CR
- Subjects
- Amino Acid Sequence, Animals, Cattle, Eosinophils cytology, Eosinophils enzymology, Humans, Molecular Sequence Data, Neutrophils cytology, Peptidylprolyl Isomerase, Peroxidases metabolism, Recombinant Proteins pharmacology, Tumor Cells, Cultured, Amino Acid Isomerases physiology, Carrier Proteins physiology, Chemotaxis, Leukocyte physiology
- Abstract
During the purification of eosinophil chemotactic factors synthesized by the uterus in response to estrogen we isolated a protein having an N-terminal (15 amino acids) sequence identical to that of rat cyclophilin. Our data demonstrate that cyclophilin, a cytosolic protein isolated from bovine thymocytes, which specifically binds the immunosuppressive drug cyclosporin A, as well as recombinant human cyclophilin, displays eosinophil chemotactic activity. In addition to its chemotactic activity, cyclophilin stimulated the release of peroxidase activity from eosinophils. Maximal chemotactic activity of cyclophilin was achieved at a concentration of approximately 10 nM. At similar concentrations cyclophilin was also able to stimulate the migration of neutrophils. This chemotactic activity could be prevented by the addition of cyclosporin A, but not by a nonimmunosuppressive analog (1-fur-furyl-cyclosporin A) at similar concentrations. This chemotactic activity may represent an additional mechanism by which immunosuppressive drugs function to prevent tissue rejection. more...
- Published
- 1992
32. Immunity, microbial pathogenesis, and immunophilins: finding the keys, now where are the locks?
- Author
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High KP and Handschumacher RE
- Subjects
- Animals, Anti-Bacterial Agents pharmacology, Cyclosporine pharmacology, Humans, Lymphocyte Activation drug effects, Peptidylprolyl Isomerase, Polyenes pharmacology, Sirolimus, Tacrolimus pharmacology, Tacrolimus Binding Proteins, Virulence, Amino Acid Isomerases physiology, Carrier Proteins physiology, DNA-Binding Proteins physiology, Heat-Shock Proteins physiology, Immunity drug effects, Immunosuppressive Agents pharmacology, Infections etiology
- Abstract
The discovery and clinical use of the immunosuppressants cyclosporin A, FK506, and rapamycin have greatly advanced solid organ and bone marrow transplantation. Though active as antibiotics against a variety of pathogens, their utility has been severely limited by toxicity. Research on the immunophilins, the major binding proteins of these drugs, has given new insights into protein folding and transport as well as mediators of signal transduction in mammalian cells. Microbial immunophilins may also have direct relevance to the intracellular survival of important human pathogens. Defining the mechanisms of enhanced virulence generated by these proteins holds great promise for understanding both the fundamental pathogenesis of these organisms and the immune response generated against them. Such an understanding may provide novel targets for the design of anti-infective agents as well as assist in the development of future immunosuppressives. more...
- Published
- 1992
33. Isolation and partial characterization of membrane-associated cyclophilin and a related 22-kDa glycoprotein.
- Author
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Thalhammer T, Kieffer LJ, Jiang T, and Handschumacher RE
- Subjects
- Amino Acid Isomerases metabolism, Amino Acid Sequence, Animals, Blotting, Western, Brain metabolism, Carrier Proteins metabolism, Cell Membrane metabolism, Cell Nucleus metabolism, Chromatography, Affinity, Cyclosporine metabolism, Cytosol metabolism, Electrophoresis, Polyacrylamide Gel, Humans, Kidney metabolism, Membrane Glycoproteins metabolism, Mice, Microsomes, Liver metabolism, Mitochondria, Liver metabolism, Molecular Sequence Data, Molecular Weight, Peptidylprolyl Isomerase, Rats, Sequence Homology, Nucleic Acid, Spleen metabolism, Subcellular Fractions metabolism, Amino Acid Isomerases isolation & purification, Carrier Proteins isolation & purification, Liver metabolism, Membrane Glycoproteins isolation & purification
- Abstract
The presence of membrane-associated proteins which stereospecifically bind cyclosporin A and react with anti-cyclophilin antibodies has been documented in rat tissues. Extraction of membranes with 6 M urea or 0.5% Chaps releases cyclosporin-binding activity that is 5-12% of that found in cytosol. Cyclosporin-A-binding proteins are present in most subcellular organelles of liver, but microsomes contain the greatest activity. These proteins can be purified by adsorption onto a cyclosporin-A affinity column and elution with cyclosporin A. Two major fractions are resolved on SDS/PAGE: an 18-kDa fraction is comprised of two isoforms that are similar if not identical to the two major cytosolic isoforms of cyclophilin. In addition, in microsomes an approximately equal quantity of a 22-kDa glycoprotein was detected. Based on partial sequencing (five peptides, 89 amino acids) this protein is similar but not identical to human cyclophilin B. This 22-kDa isoform is poorly recognized by affinity-purified anti-cyclophilin antibodies and comprises several predominant isoforms (pI approximately 9.3-9.6). Selective binding of membrane 22-kDa cyclophilin to peanut lectin suggests the oligosaccharides contain a terminal galactosyl-N-galactosamine residue. more...
- Published
- 1992
- Full Text
- View/download PDF
34. Brequinar potentiates 5-fluorouracil antitumor activity in a murine model colon 38 tumor by tissue-specific modulation of uridine nucleotide pools.
- Author
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Pizzorno G, Wiegand RA, Lentz SK, and Handschumacher RE
- Subjects
- Animals, Biphenyl Compounds administration & dosage, Biphenyl Compounds pharmacology, Digestive System drug effects, Digestive System metabolism, Drug Synergism, Female, Fluorouracil administration & dosage, Fluorouracil metabolism, Kidney drug effects, Kidney metabolism, Kinetics, Liver drug effects, Liver metabolism, Mice, Mice, Inbred C57BL, Organ Specificity, Spleen drug effects, Spleen metabolism, Antineoplastic Agents therapeutic use, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Biphenyl Compounds therapeutic use, Colonic Neoplasms drug therapy, Colonic Neoplasms metabolism, Fluorouracil therapeutic use, Thymidylate Synthase metabolism, Uracil Nucleotides metabolism, Uridine metabolism
- Abstract
Modulation of pyrimidine metabolism or the metabolic fate of 5-fluorouracil by a number of different agents has permitted a significant increase in the response rate to this agent, particularly for colorectal cancers. Brequinar, a noncompetitive inhibitor of mitochondrial dihydroorotate dehydrogenase has been shown to achieve a tumor-specific modulation of the therapeutic effect of 5-fluorouracil. A selective decrease of uridine nucleotide pools in Colon tumor 38 compared to normal tissues of C57/BL6 mice was observed after Brequinar administration. This effect was achieved with very low nontherapeutic doses of Brequinar (8 to 27% of the maximum tolerated dose in this model). Pretreatment with Brequinar 4 and 24 h prior to administration of [3H]fluorouracil significantly increased incorporation of the fluoropyrimidine into Colon 38 tumor RNA, while minimal effects were seen in normal tissues of C57/BL6 mice. Brequinar (15, 30, and 50 mg/kg) was administered 4 h prior to fluorouracil (85 mg/kg) on a weekly basis in Colon 38-bearing mice. All combinations potentiated 5-fluorouracil antitumor activity and the lowest dose of Brequinar (15 mg/kg) showed a reduced toxicity (weight loss) compared to the same dose of 5-fluorouracil as a single agent. When Brequinar preceded fluorouracil by 24 h, greater toxicity and less antitumor activity were observed. A comparison of the optimal Brequinar-fluorouracil regimen with a previously optimized N-(phosphonoacetyl)-L-aspartic acid-fluorouracil combination in Colon 38 tumor indicated that Brequinar-fluorouracil was more effective and less toxic. more...
- Published
- 1992
35. Isolation and characterization of a 40-kDa cyclophilin-related protein.
- Author
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Kieffer LJ, Thalhammer T, and Handschumacher RE
- Subjects
- Amino Acid Isomerases metabolism, Amino Acid Sequence, Animals, Blotting, Western, Carrier Proteins metabolism, Cattle, Chickens, Chromatography, Affinity, Chromatography, Gel, Cyclosporins metabolism, Electrophoresis, Polyacrylamide Gel, Immune Sera, Isoelectric Focusing, Molecular Sequence Data, Molecular Weight, Peptidylprolyl Isomerase, Sequence Homology, Nucleic Acid, Thymus Gland metabolism, Amino Acid Isomerases isolation & purification, Brain metabolism, Carrier Proteins isolation & purification
- Abstract
Major and minor isoforms of cyclophilin (CyP-18), a 17.8-kDa protein with peptidyl-prolyl cis-trans isomerase activity, comprise the primary intracellular binding proteins for cyclosporin A. Additional CyP-like proteins with approximate molecular masses of 22 (CyP-22) and 40 kDa (CyP-40) have been recovered from the soluble fraction of calf brain along with CyP-18 by adsorption onto a cyclosporin A affinity column and elution with cyclosporin A. Based on a limited number of peptide sequences from CyP-22, it appears that we have isolated from tissue CyPB, a protein whose sequence was deduced previously from cloned cDNA. The 40-kDa protein was separated from CyP-18 and CyP-22 on a molecular sieving column. Isoelectric focusing of CyP-40 yielded two bands at pI 5.3 and 5.5, in contrast to the basic pI values of CyP-18. Some tryptic peptides from CyP-40 were found to be highly homologous but not identical to bovine CyP-18; others were not significantly homologous to CyP-18 or any other protein in the data base. Unlike the major and minor isoforms of Cyp-18, monospecific polyclonal anti-CyP-18 antibodies did not cross-react with CyP-22 and CyP-40. Likewise, anti-CyP-40 serum minimally cross-reacts with CyP-18 and CyP-22. Cyp-40 possesses peptidyl-prolyl cis-trans isomerase activity which is less sensitive to inhibition by cyclosporin A (IC50 = 300 nM) than is CyP-18 (IC50 = 20 nM). more...
- Published
- 1992
36. Inhibition by pertussis toxin of the activation of Na(+)-dependent uridine transport in dimethyl-sulphoxide-induced HL-60 leukaemia cells.
- Author
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Sokoloski JA, Sartorelli AC, Handschumacher RE, and Lee CW
- Subjects
- Biological Transport drug effects, Cell Differentiation, Dimethyl Sulfoxide pharmacology, Leukemia, Experimental pathology, Tetradecanoylphorbol Acetate pharmacology, Tumor Cells, Cultured, Leukemia, Experimental metabolism, Pertussis Toxin, Sodium metabolism, Uridine metabolism, Virulence Factors, Bordetella pharmacology
- Abstract
The effects of pertussis toxin on the Na(+)-dependent transport of uridine were studied in HL-60 leukaemia cells induced to differentiate along the granulocytic or monocytic pathways by dimethyl sulphoxide (DMSO) or phorbol 12-myristate 13-acetate (PMA) respectively. Pertussis toxin at 50 ng/ml completely inhibited the activation of Na(+)-dependent uridine transport and consequently prevented the formation of intracellular pools of free uridine which occurs in HL-60 cells induced to differentiate by DMSO. The inhibition of Na(+)-dependent uridine transport by pertussis toxin in cells exposed to DMSO was associated with a 14-fold decrease in affinity, with no change in Vmax. Pertussis toxin, however, had no effect on Na(+)-dependent uridine transport in PMA-induced HL-60 cells. Furthermore, 500 ng of cholera toxin/ml had no effect on the Na(+)-dependent uptake of uridine in DMSO-treated HL-60 cells. These results suggest that the activation of the Na(+)-dependent transport of uridine in HL-60 cells induced to differentiate along the granulocytic pathway by DMSO is coupled to a pertussis-toxin-sensitive guanine-nucleotide binding protein (G-protein). more...
- Published
- 1991
- Full Text
- View/download PDF
37. Tissue-specific expansion of uridine pools in mice. Effects of benzylacyclouridine, dipyridamole and exogenous uridine.
- Author
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Darnowski JW, Handschumacher RE, Wiegand RA, Goulette FA, and Calabresi P
- Subjects
- Administration, Oral, Animals, Dipyridamole administration & dosage, Drug Synergism, Female, Injections, Intravenous, Mice, Mice, Inbred Strains, Time Factors, Tissue Distribution, Uracil administration & dosage, Uracil pharmacokinetics, Uracil pharmacology, Uridine administration & dosage, Uridine blood, Uridine pharmacology, Dipyridamole pharmacology, Uracil analogs & derivatives, Uridine metabolism, Uridine Phosphorylase antagonists & inhibitors
- Abstract
The concentration of uridine (Urd) in murine tissues appears to be controlled by Urd catabolism, concentrative Urd transport, and the non-concentrative, facilitated diffusion of Urd. Previous reports document the tissue-specific disruption of these processes, and subsequently intracellular pools of free Urd in mice, by the administration of exogenous Urd (250 mg/kg) or the Urd phosphorylase (EC 2.4.2.3; uracil:ribose-1-phosphate phosphotransferase) inhibitor 5-benzylacyclouridine (BAU) (240 mg/kg). We now report the effect of combinations of BAU (120 mg/kg, p.o.), the nucleoside transport inhibitor dipyridamole (DP) (25 mg/kg, i.p.), and exogenous Urd (250 mg/kg, i.v.) on Urd pools in mice. This dose of BAU increased Urd pools 2- to 6-fold, in a tissue-specific manner, for up to 5 hr. DP increased Urd pools 3-fold in spleen, over a 4-hr period, but did not affect other tissues. Administration of BAU 1 hr prior to exogenous Urd resulted in a 50- to 100-fold expansion of tissue normal after 6 hr. Administration of DP 1 hr prior to exogenous Urd caused a tissue-specific 40- to 100-fold increase in Urd pools which, except in spleen, returned to normal within 2 hr. The marked additive effects of these combinations were in contrast to those obtained following the administration of BAU 1 hr prior to DP. This regimen increased Urd pools from 4- to 9-fold, in a tissue-specific manner. In addition, Urd pools remained elevated for up to 9 hr, except in spleen where the Urd concentration was elevated for up to 15 hr. Analysis of enzyme activities indicated that DP does not enhance the inhibitory effect of BAU against murine liver Urd phosphorylase. However, DP did inhibit plasma clearance of BAU, and this effect may partially explain the apparent synergistic effect of this combination. In spite of the prolonged and dramatic expansion of tissue Urd pools produced by BAU + DP, the total Ura nucleotide content in spleen, gut and colon tumor 38 (CT38) increased by less than 70% over a 12-hr period following administration of this combination. These findings are discussed in light of their biochemical and therapeutic implications. more...
- Published
- 1991
- Full Text
- View/download PDF
38. A phase II trial of cyclosporin A in the treatment of refractory metastatic colorectal cancer.
- Author
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Murren JR, Ganpule S, Sarris A, Durivage H, Davis C, Makuch R, Handschumacher RE, and Marsh JC
- Subjects
- Bone Neoplasms secondary, Colorectal Neoplasms blood, Colorectal Neoplasms mortality, Creatinine blood, Cyclosporins adverse effects, Cyclosporins blood, Drug Evaluation, Female, Humans, Liver Neoplasms secondary, Lung Neoplasms secondary, Male, Neoplasm Recurrence, Local drug therapy, Peritoneal Neoplasms secondary, Survival Rate, Colorectal Neoplasms drug therapy, Cyclosporins therapeutic use
- Abstract
Cyclosporin A (CSA) is an immunosuppressive agent that in experimental models has antiproliferative activity against colon cancer and other human neoplasms. A phase II trial was conducted to evaluate CSA in refractory colorectal malignancies. CSA was administered at a starting dose of 7.0 mg/kg twice daily (total dose, 14 mg/kg/day) and escalated to tolerance. Of 18 patients with measurable disease, 17 were evaluable. All had been treated with one fluorouracil-containing regimen. The European Cooperative Oncology Group (ECOG) performance status was 0 or 1 in 17 of the 18 patients. No objective responses were seen. Four patients maintained stable disease lasting from 10 to 75+ weeks. Significant toxicity occurred in 9 of 17 (53%) patients. Dose reduction was necessary in 10 of 17 (59%). Sustained escalation beyond the initial dose was possible in only two cases. Toxicities included nausea and vomiting (71%), nephrotoxicity (41%), fatigue (35%), flu-like symptoms (29%), and neurotoxicity (18%). In the dose and schedule employed in this trial, CSA is ineffective in refractory colorectal cancer and produces significant toxicity. more...
- Published
- 1991
- Full Text
- View/download PDF
39. Induction of the differentiation of HL-60 cells by phorbol 12-myristate 13-acetate activates a Na(+)-dependent uridine-transport system. Involvement of protein kinase C.
- Author
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Lee CW, Sokoloski JA, Sartorelli AC, and Handschumacher RE
- Subjects
- Alkaloids pharmacology, Biological Transport drug effects, Cell Line, Humans, Kinetics, Leukemia, Promyelocytic, Acute, Protein Kinase C antagonists & inhibitors, Ribonucleosides metabolism, Staurosporine, Cell Differentiation drug effects, Protein Kinase C metabolism, Sodium pharmacology, Tetradecanoylphorbol Acetate pharmacology, Uridine metabolism
- Abstract
The Na(+)-dependent transport and facilitated diffusion of uridine were measured after differentiation of HL-60 leukaemia cells along the monocytic pathway by phorbol 12-myristate 13-acetate (PMA). PMA (200 ng/ml) caused a marked increase in Na(+)-dependent uridine transport within 48 h of exposure that was attributable to an increase in transport affinity (apparent Km values of 1.15 +/- 0.22 and 44 +/- 4.4 microM for PMA-induced and uninduced cells respectively), with no change in Vmax. (0.15 +/- 0.02 and 0.13 +/- 0.01 pmol/s per microliter of cell water for PMA-induced and uninduced cells respectively). A corresponding rapid decrease in both the rate of facilitated diffusion and the formation of uracil nucleotides occurred in PMA-induced cells. As a consequence of these changes, intracellular pools of uridine 3-4-fold greater than those in the medium were generated. A similar increase in Na(+)-dependent transport of adenosine, inosine, guanosine, thymidine and cytidine (Km values of 1-4 microM) was observed. The effects of PMA on the activation of the Na(+)-dependent uridine transporter were inhibited by staurosporine, suggesting the involvement of protein kinase C. The findings indicate that a change in the balance of the cellular mechanisms employed for nucleoside transport occurs during the monocytic differentiation of HL-60 leukaemia cells. more...
- Published
- 1991
- Full Text
- View/download PDF
40. Cyclophilin binding: a more accurate measure of cyclosporine immunosuppressive activity after renal transplantation.
- Author
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Paul K, Harding MW, Marks WH, Handschumacher RE, and Lorber MI
- Subjects
- Chromatography, High Pressure Liquid, Cyclosporins blood, Humans, Immunosuppression Therapy, Peptidylprolyl Isomerase, Amino Acid Isomerases metabolism, Carrier Proteins metabolism, Cyclosporins metabolism, Kidney Transplantation physiology
- Published
- 1991
41. Effects of uridine on the growth and differentiation of HL-60 leukemia cells.
- Author
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Sokoloski JA, Lee CW, Handschumacher RE, Nigam A, and Sartorelli AC
- Subjects
- Cell Line, Dose-Response Relationship, Drug, Fluorescent Antibody Technique, Humans, Leukemia, Promyelocytic, Acute, Nitroblue Tetrazolium, Phenotype, Ribonucleotides analysis, Cell Differentiation drug effects, Cell Division drug effects, Tumor Cells, Cultured drug effects, Uridine pharmacology
- Abstract
HL-60 leukemia cells, induced to differentiate, activate a Na(+)-dependent nucleoside transport system, concomitant with a reduction in the nitrobenzylthioinosine (NBMPR)-sensitive facilitated transport of nucleosides. The consequence of these changes lead to the formation of intracellular pools of uridine. To examine the possible role of accumulated uridine in the commitment of HL-60 leukemia cells to undergo maturation, the effects of uridine on the growth and differentiation of HL-60 cells were monitored. Uridine at millimolar levels caused a concentration-dependent inhibition of cellular growth, resulting in the accumulation of cells in the G2/M phases of the cell cycle, phenomena that preceded the formation of differentiated cells. These effects of uridine were reduced by 10 microM NBMPR, an inhibitor of the facilitated transport of nucleosides. The effects of 24 mM uridine on growth and differentiation of HL-60 cells were also prevented by 5 mM inosine, and partially prevented by either 2 mM hypoxanthine or 20 microM adenosine. Pretreatment of HL-60 cells with 24 mM uridine for 6 days, followed by a 2 h exposure to TPA, resulted in the rapid attachment of cells to the tissue culture dish, and the extension of long processes. Although the concentrations of uridine required for the above effects are greater than those achieved during differentiation, these observations suggest that uridine may play a role in regulating the maturation process. more...
- Published
- 1991
- Full Text
- View/download PDF
42. Bradykinin and its Gly6 analogue are substrates of cyclophilin: a fluorine-19 magnetization transfer study.
- Author
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London RE, Davis DG, Vavrek RJ, Stewart JM, and Handschumacher RE
- Subjects
- Amino Acid Sequence, Animals, Bradykinin chemistry, Cattle, Cyclosporins pharmacology, Fluorine, Glycine chemistry, Kinetics, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Peptidylprolyl Isomerase, Substrate Specificity, Amino Acid Isomerases metabolism, Bradykinin metabolism, Carrier Proteins metabolism, Isomerases metabolism
- Abstract
Fluorine-19 magnetization transfer experiments have been used to determine the rates of cis/trans isomerization about the X-Pro7 peptide bond in [p-fluoro-Phe8]bradykinin (cis/trans ratio approximately 0.1) and its Gly6 analogue (cis/trans ratio approximately 0.4). The measurements were carried out both prior to and after the addition of cyclophilin, which has recently been shown to have peptidyl-proline cis/trans isomerase activity and is the apparent target enzyme of the immunosuppressive agent cyclosporin A. Magnetization transfer measurements over the temperature range 40-75 degrees C in the absence of enzyme give activation energies of 22.8 and 23.0 kcal/mol for [p-fluoro-Phe8]bradykinin and its Gly6 analogue, respectively. The values for the uncatalyzed cis----trans rate constant, kc, are determined by extrapolation to be 4.8 x 10(-2) and 2.1 x 10(-2) s-1 for the two peptides at 25 degrees C. The enzyme-catalyzed enhancement of the cis/trans interconversion rate was proportional to added cyclophilin concentration and was strongly sequence specific, with bradykinin a much better substrate than [Gly6]bradykinin. At a peptide concentration of 2.2 mM, the catalytic activity expressed as kc per micromolar cyclophilin was determined to be 1.2 s-1/microM for [p-fluoro-Phe8]bradykinin and 0.13 s-1/microM for the Gly6 analogue. The increased cis----trans interconversion rates were strongly inhibited by cyclosporin A and the 6-(methylalanine) derivative, which bind to cyclophilin, but not by the 1-(tetrahydrofurfuryl) derivative of cyclosporin that binds weakly. more...
- Published
- 1990
- Full Text
- View/download PDF
43. An overview of recent progress in ligand-receptor research based on nuclear magnetic resonance spectroscopy.
- Author
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Handschumacher RE and Armitage IM
- Subjects
- Humans, Kinetics, Ligands, Macromolecular Substances, Structure-Activity Relationship, Drug Design, Magnetic Resonance Spectroscopy methods, Receptors, Drug
- Published
- 1990
- Full Text
- View/download PDF
44. Structural elements pertinent to the interaction of cyclosporin A with its specific receptor protein, cyclophilin.
- Author
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Hsu VL, Heald SL, Harding MW, Handschumacher RE, and Armitage IM
- Subjects
- Amino Acid Sequence, Animals, Carrier Proteins isolation & purification, Cattle, Humans, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Peptidylprolyl Isomerase, Protein Conformation, Rats, Carrier Proteins metabolism, Cyclosporins metabolism
- Abstract
Cyclophilin (163 amino acids; 17,737 daltons) is a ubiquitous cytosolic protein that specifically binds the potent immunosuppressive drug cyclosporin A (CsA). To characterize the structural details of this interaction, extensive use has been made of two-dimensional (2D) NMR methods. For studies on CsA, these methods are being used to assign the conformational space accessible to CsA by analysis of the spectra from the multiple CsA conformers present in slow exchange in mixed solvent systems. These same 2D NMR methods also have been used for extensive studies of the major bovine thymus cyclophilin (CyP) isoform and its complex with stoichiometric amounts of CsA. In the former case, these studies have revealed 81% of the 156 expected HN-H alpha crosspeaks. The complete spin-coupled spin systems for one-third of these amide resonances have been assigned according to amino acid type. After exhaustive D2O exchange, there remain 44 amide protons which exhibit 2D NMR features indicative of a hydrophobic domain with beta-sheet secondary structure. The CsA-complexed form of CyP exhibits a discrete structure and set of resonances in slow exchange with the drug-free CyP. The amino acids that have been specifically identified to be affected by the interaction are limited in number and include three Phe residues, the unique Trp at position 120, and two Ala residues. more...
- Published
- 1990
- Full Text
- View/download PDF
45. Cyclophilin binding: a receptor-mediated approach to monitoring cyclosporine immunosuppressive activity following organ transplantation.
- Author
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Lorber MI, Paul K, Harding MW, Handschumacher RE, and Marks WH
- Subjects
- Administration, Oral, Bile metabolism, Cadaver, Cyclosporins administration & dosage, Cyclosporins adverse effects, Dose-Response Relationship, Drug, Drug Therapy, Combination, Humans, Immune Tolerance drug effects, Infusions, Intravenous, Kidney drug effects, Kidney Function Tests, Liver drug effects, Liver Function Tests, Methylprednisolone administration & dosage, Peptidylprolyl Isomerase, Prednisone administration & dosage, Structure-Activity Relationship, Carrier Proteins blood, Cyclosporins pharmacokinetics, Graft Rejection drug effects, Kidney Transplantation immunology
- Published
- 1990
46. 1H NMR studies on bovine cyclophilin: preliminary structural characterization of its complex with cyclosporin A.
- Author
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Heald SL, Harding MW, Handschumacher RE, and Armitage IM
- Subjects
- Amino Acid Sequence, Animals, Cattle, Hydrogen, Magnetic Resonance Spectroscopy methods, Molecular Sequence Data, Peptidylprolyl Isomerase, Protein Binding, Thymus Gland, Carrier Proteins metabolism, Cyclosporins metabolism
- Abstract
Cyclophilin (163 residues, Mr 17737), a peptidyl prolyl cis-trans isomerase, is a cytosolic protein that specifically binds the potent immunosuppressant cyclosporin A (CsA). The native form of the major bovine thymus isoform has been analyzed by 2D NMR methods, COSY, HOHAHA, and NOESY, in aqueous media. The 156 main-chain amides in CyP yield 126 observable NH/alpha CH couplings (81%, Gly pairs counted as 1). Following exhaustive D2O exchange, 44 amide resonances remain visible. Further analysis of the NH/NH, NH/alpha CH, and alpha CH/alpha CH regions of the COSY and NOESY data sets indicates that the residual amides in D2O form a coherent hydrophobic domain which yields 2D NMR features suggestive of a beta-sheet. Many (43/126) of the amide resonances have been classified according to amino acid type. In the aromatic region of the spectra, the assignment of the ring spin systems is nearly complete (12/15 Phe, 2/2 Tyr, 1/1 Trp, and 3/4 His). This has successfully lead to the complete assignment of all of their beta CH's, main-chain alpha CH resonances, and many of the backbone amide resonances (8/12 Phe, 2/2 Tyr, 1/1 Trp, and 2/3 His). In other regions of the spectrum, the side-chain and main-chain resonances for 10/23 Gly, 9/9 Ala, 5/11 Thr, 5/9 Val, and 1/6 Leu have been completely assigned. The drug-free cyclophilin and CsA-bound cyclophilin form two discrete protein structures that are in slow exchange on the NMR time scale. Comparison of the fingerprint regions from the COSY spectra obtained from the two forms of the protein reveals a minimum of 16 cross-peaks which are clearly shifted upon complexation. In fact, on the basis of chemical shift changes observed in assigned side-chain and main-chain resonances, only a relatively few of the amino acid residues identified to date are perturbed by complex formation. These include 3 Phe (8, 12, and 14) and the Trp in the aromatic region and 2 Ala (7 and 8) in the Ala/Thr region. In the upfield-shifted methyl region, an assigned Leu and Val spin system and a spin system labeled X10 (an Ile or Leu) are affected by complex formation. In addition, a new aliphatic spin system, labeled X11, which shows a close spatial relationship to the perturbed Phe12, is observed in this region of the spectrum. In summary, the regions of the protein altered by complex formation can be divided into two categories: a hydrophobic and a H2O-accessible domain.(ABSTRACT TRUNCATED AT 400 WORDS) more...
- Published
- 1990
- Full Text
- View/download PDF
47. Effect of herpes simplex virus type 1 infection on nucleoside transport in HeLa S3 cells.
- Author
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Palú G, Handschumacher RE, Parolin C, Stefanelli S, and Palatini P
- Subjects
- Affinity Labels metabolism, Biological Transport drug effects, Dipyridamole pharmacology, HeLa Cells drug effects, HeLa Cells metabolism, Humans, Kinetics, Methionine metabolism, Sucrose metabolism, Thioinosine analogs & derivatives, Thioinosine metabolism, Cell Transformation, Viral, Simplexvirus genetics, Thymidine metabolism
- Abstract
The initial velocity of thymidine uptake was measured in HeLa S3 cells infected with herpes simplex virus type 1 (HSV-1). The rate of nucleoside influx into the cells was shown to increase from as early as 1 h post-infection (p.i.) up to 8 h p.i. This increased uptake was shown to be attributable to a progressively increasing contribution from passive diffusion superimposed upon normal transport. Thus, the specific nucleoside transport system was still operating with unaltered kinetic parameters 8 h after infection. Despite the inhibition of host cell protein synthesis and its replacement by the synthesis of virus-specified proteins, the numbers and affinity of the nucleoside transporters in cells 8 h after infection were virtually unchanged. The increased transport of thymidine in infected cultures was resistant to the nucleoside transport inhibitor dipyridamole, and was correlated with entry of a normally impermeant solute (sucrose) into infected cells. These data suggest that the system for the carrier-mediated facilitated diffusion of nucleosides remains intact in HSV-infected cells, but that progressively increasing passive diffusion takes place. Passive diffusion is the major process operating late after virus infection. more...
- Published
- 1990
- Full Text
- View/download PDF
48. Benzylacyclouridine. Pharmacokinetics, metabolism and biochemical effects in mice.
- Author
-
Darnowski JW and Handschumacher RE
- Subjects
- Administration, Oral, Animals, Chromatography, High Pressure Liquid, Injections, Intravenous, Metabolic Clearance Rate, Mice, Tissue Distribution, Uracil metabolism, Uracil pharmacokinetics, Uracil pharmacology, Uridine blood, Uridine Phosphorylase antagonists & inhibitors, Uracil analogs & derivatives
- Abstract
The pharmacokinetics, tissue distribution and urinary excretion of the uridine (Urd) phosphorylase (EC 2.4.2.3) inhibitor 5-benzylacyclouridine (BAU) were studied in C57BL/6 female mice by reverse-phase HPLC. The plasma clearance of BAU after i.v. administration followed first-order kinetics with a half-life of approximately 36 min. Other pharmacokinetic parameters such as volume of distribution (17 ml), clearance rate (0.3 ml/min) and the elimination rate constant (0.019 hr-1) were relatively constant over a dose range of 5 to 240 mg/kg when based on a first-order clearance model. Following oral administration, BAU was rapidly absorbed from the gut; peak plasma concentrations occurred within 30 min and were approximately 60% of equivalent i.v. doses. The distribution of BAU between plasma and most major organs was rapid and efficient, the exceptions being spleen and brain, which maintained only 40% and 10%, respectively, of the plasma BAU concentration. Approximately 41% of the injected dose of BAU was recovered intact in urine within 24 hr. Another 27% appeared as a more polar metabolite which, at a concentration of 50 microM, did not inhibit murine Urd phosphorylase. A near linear relationship was observed between the injected dose of BAU and its ability to increase the plasma concentration of Urd; i.v. injections of 30, 120 and 240 mg/kg increased plasma Urd 3-, 7- and 15-fold respectively. The utility of these data in the design of combination chemotherapy regimens containing BAU and related compounds is discussed. more...
- Published
- 1988
- Full Text
- View/download PDF
49. Structure of peptide from active site region of Escherichia coli L-asparaginase.
- Author
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Peterson RG, Richards FF, and Handschumacher RE
- Subjects
- Amino Acid Sequence, Asparagine analogs & derivatives, Asparagine metabolism, Binding Sites, Dimethyl Sulfoxide pharmacology, Kinetics, Macromolecular Substances, Peptide Fragments analysis, Protein Binding, Asparaginase metabolism, Escherichia coli enzymology
- Abstract
The L-asparagine analogue 5-diazo-4-oxo-L-[5-14C]norvaline binds irreversibly to the active site of Escherichia coli L-asparaginase. Conditions for optimal labeling in buffers containing 50% dimethylsulfoxide have been developed and kinetic parameters of the inactivation have been determined. After reduction, alkylation and subsequent degradation of the modified enzyme with alpha-chymotrypsin, the principal radioactive decapeptide of sequence Val-Gly-Ala-Met-Arg-Pro-Ser-Thr-Ser-Met was isolated. A second radioactive hexapeptide Arg-Pro-Ser-Thr-Ser-Met resulting from chymotryptic digestion of the decapeptide was also isolated. Evidence is presented for the attachment of the 5-diazo-4-oxo-L-norvaline residue to serine-9 in the decapeptide via an acid-labile linkage. more...
- Published
- 1977
50. The active site of L-asparaginase: dimethylsulfoxide effect of 5-diazo-4-oxo-L-norvaline interactions.
- Author
-
Lachman LB and Handschumacher RE
- Subjects
- Aminolevulinic Acid chemistry, Asparaginase antagonists & inhibitors, Asparagine metabolism, Binding Sites drug effects, Enzyme Activation drug effects, Escherichia coli enzymology, Glutamine metabolism, Hydrolysis drug effects, Kinetics, Aminolevulinic Acid analogs & derivatives, Aminolevulinic Acid metabolism, Asparaginase chemistry, Asparaginase metabolism, Dimethyl Sulfoxide pharmacology
- Abstract
The asparagine analog, 5-diazo-4-oxo-L-norvaline is a substrate and an irreversible inhibitor of L-asparaginase. Covalent attachment occurs at an increased rate at concentrations of dimethylsulfoxide which reduce the catalytic decomposition of diazo-oxo-norvaline. In 55% dimethylsulfoxide asparaginase is inactivated by diazo-oxo-norvaline (0.05 M) with a t 1/2 of twelve seconds. In aqueous buffer the rate of diazo-oxo-norvaline decomposition is increased three-fold in the presence of the nucleophile hydroxylamine; this nucleophile also protects the enzyme against inactivation by diazo-oxo-norvaline in the presence of dimethylsulfoxide. more...
- Published
- 1976
- Full Text
- View/download PDF
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