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• W2130069364 abstract "Because S-adenosylmethionine (AdoMet) is required by Pneumocystis carinii in vitro, Pneumocystis infection depletes plasma AdoMet of rats and humans, nicotine reduces AdoMet of guinea pig lungs, and smoking correlates with reduced episodes of Pneumocystis pneumonia (PCP) in AIDS patients, we tested the effect of nicotine treatment on PCP using a rat model. Intraperitoneal infusion of 400 μg of R-(+) nicotine kg-1 h-1 intraperitoneal for 21 days caused a 15-fold reduction in lung AdoMet although neither plasma nor liver were changed. Infusion of 4 and 400 μg kg-1 h-1 into immunosuppressed rats, beginning when rats were inoculated with P. carinii, caused 85 and 99.88% reductions, respectively, in P. carinii cysts at sacrifice 21 days later; P. carinii nuclei were reduced by 91.2 and >99.99%, respectively. This effect was reversed by concomitant administration of AdoMet with nicotine. Treatment with AdoMet alone increased infection intensity. We conclude that AdoMet is a critical and limiting nutrient for Pneumocystis thus can serve as a therapeutic target for PCP. Regarding the mechanism, nicotine treatment caused no change in rat lung activity of AdoMet synthesizing methionine ATP transferase activity nor was there any evidence of increased AdoMet utilization for methylation reactions. Except of a doubling of putrescine, nicotine treatment also did not change lung polyamine content. However, key polyamine anabolic and catabolic enzymes were upregulated, and there were corresponding changes in polyamine metabolic intermediates. We conclude that chronic nicotine treatment increases lung polyamine catabolic/anabolic cycling and/or excretion leading to increased AdoMet-consuming polyamine biosynthesis and depletion of lung AdoMet. Because S-adenosylmethionine (AdoMet) is required by Pneumocystis carinii in vitro, Pneumocystis infection depletes plasma AdoMet of rats and humans, nicotine reduces AdoMet of guinea pig lungs, and smoking correlates with reduced episodes of Pneumocystis pneumonia (PCP) in AIDS patients, we tested the effect of nicotine treatment on PCP using a rat model. Intraperitoneal infusion of 400 μg of R-(+) nicotine kg-1 h-1 intraperitoneal for 21 days caused a 15-fold reduction in lung AdoMet although neither plasma nor liver were changed. Infusion of 4 and 400 μg kg-1 h-1 into immunosuppressed rats, beginning when rats were inoculated with P. carinii, caused 85 and 99.88% reductions, respectively, in P. carinii cysts at sacrifice 21 days later; P. carinii nuclei were reduced by 91.2 and >99.99%, respectively. This effect was reversed by concomitant administration of AdoMet with nicotine. Treatment with AdoMet alone increased infection intensity. We conclude that AdoMet is a critical and limiting nutrient for Pneumocystis thus can serve as a therapeutic target for PCP. Regarding the mechanism, nicotine treatment caused no change in rat lung activity of AdoMet synthesizing methionine ATP transferase activity nor was there any evidence of increased AdoMet utilization for methylation reactions. Except of a doubling of putrescine, nicotine treatment also did not change lung polyamine content. However, key polyamine anabolic and catabolic enzymes were upregulated, and there were corresponding changes in polyamine metabolic intermediates. We conclude that chronic nicotine treatment increases lung polyamine catabolic/anabolic cycling and/or excretion leading to increased AdoMet-consuming polyamine biosynthesis and depletion of lung AdoMet. Pneumocystis is a fungal pathogen that infects the lungs of immunocompromised mammals causing a severe pneumonia known as PCP 1The abbreviations used are: PCP, Pneumocystis pneumonia; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CZE, capillary zone electrophoresis; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; IEF, isoelectric focusing; AdoMet, S-adenosylmethionine; i.p., intraperitoneal; ODC, ornithine decarboxylase; DC, decarboxylase; PMF, protein mass fingerprint. (Pneumocystis pneumonia). Each species of mammal is associated with a particular species of Pneumocystis; for humans it is P. jiroveci and for rats, either P. carinii or P. ratti (1Wakefield A.E. Br. Med. Bull. 2002; 61: 175-188Crossref PubMed Scopus (58) Google Scholar). Most human PCP cases are AIDS-related, and PCP is the most frequent opportunistic infection associated with AIDS (2Jones J.L. Hanson D.L. Dworkin M.S. Kaplan J.E. Ward J.W. J. Infect. Dis. 1998; 178: 114-120Crossref PubMed Scopus (57) Google Scholar, 3Jones J.L. Hanson D.L. Dworkin M.S. Alderton D.L. Fleming P.L. Kaplan J.E. Ward J. Morbidity and Mortality Weekly Report. 48. Centers for Disease Control and Prevention, Atlanta, GA1999: 1-22Google Scholar). However, all immunosuppressed persons are at risk including those treated with corticosteroids for rheumatic disease, cytotoxic agents for cancer, or immunosuppressive drugs for organ transplantation. PCP can also result from severe malnutrition. The rate of HIV infection associated with AIDS has declined in some developed countries because of specific prophylaxis and the wide use of effective anti-HIV drugs, but these medications are not routinely available for the vast majority of world populations where AIDS is rampant. PCP was thought to be infrequent in AIDS patients from developing countries, but recent data show that PCP occurs frequently in Africa (4Zar H.J. Curr. Opin. Pulm. Med. 2004; 10: 176-182Crossref PubMed Scopus (51) Google Scholar, 5Graham S.M. Pediatr. Pulmonol. 2003; 36: 462-468Crossref PubMed Scopus (42) Google Scholar, 6Fisk D.T. Meshnick S. Kazanjian P.H. Clin. Infect. Dis. 2003; 36: 70-78Crossref PubMed Scopus (137) Google Scholar, 7Robberts F.J. Chalkley L.J. Liebowitz L.D. Sadj. 2002; 57: 451-453PubMed Google Scholar). Recently the frequency of PCP cases, often associated with undiagnosed HIV, has increased (8Pulvirenti J. Herrera P. Venkataraman P. Ahmed N. AIDS Patient Care STDS. 2003; 17: 261-265Crossref PubMed Scopus (48) Google Scholar). Even with access to good medical care, the mortality of PCP ranges from 10 to 40%, being higher in patients without AIDS. The two most effective drugs, pentamidine and the combination of trimethoprim and sulfamethoxazole (co-trimoxazole, TMP-SMZ), have significant side effects (9Wilkin A. Feinberg J. Am. Fam. Physician. 1999; 60: 1699-1708PubMed Google Scholar) and there is evidence of developing resistance (10Kessl J.J. Hill P. Lange B.B. Meshnick S.R. Meunier B. Trumpower B.L. J. Biol. Chem. 2004; 279: 2817-2824Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 11Nahimana A. Rabodonirina M. Helweg-Larsen J. Meneau I. Francioli P. Bille J. Hauser P.M. Emerg. Infect. Dis. 2003; 9: 864-867Crossref PubMed Scopus (41) Google Scholar, 12Zingale A. Carrera P. Lazzarin A. Scarpellini P. J. Clin. Microbiol. 2003; 41: 2709-2712Crossref PubMed Scopus (21) Google Scholar, 13Navin T.R. Beard C.B. Huang L. del Rio C. Lee S. Pieniazek N.J. Carter J.L. Le T. Hightower A. Rimland D. Lancet. 2001; 358: 545-549Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 14Kazanjian P.H. Fisk D. Armstrong W. Shulin Q. Liwei H. Ke Z. Meshnick S. J. Infect. Dis. 2004; 189: 1684-1687Crossref PubMed Scopus (48) Google Scholar). Clearly, there is a great need for new therapies that are more effective and less toxic (15Deresinski S.C. Semin. Respir. Infect. 1997; 12: 79-97PubMed Google Scholar). AdoMet is a critical cellular metabolic intermediate. It plays a pivotal role as methyl donor in a myriad of biochemical processes including methyl group transfers for the formation of phosphatidyl choline, regeneration of methionine, methylation of phospholipids, and methylation of other small molecules (16Lu S.C. Int. J. Biochem. Cell Biol. 2000; 32: 391-395Crossref PubMed Scopus (373) Google Scholar). AdoMet-mediated methylation is an important regulatory mechanism for proteins, DNA and RNA (17Ehrlich M. J. Cell. Biochem. 2003; 88: 899-910Crossref PubMed Scopus (181) Google Scholar). AdoMet also interacts with folate metabolism and thus all the reactions involving folate (18Mato J.M. Corrales F.J. Lu S.C. Avila M.A. FASEB J. 2002; 16: 15-26Crossref PubMed Scopus (362) Google Scholar, 19Eloranta T.O. Kajander E.O. Biochem. J. 1984; 224: 137-144Crossref PubMed Scopus (32) Google Scholar). AdoMet is necessary for the synthesis of the essential polyamines spermidine and spermine (20Persson L. Holm I. Stjernborg L. Heby O. Adv. Exp. Med. Biol. 1988; 250: 261-271Crossref PubMed Scopus (13) Google Scholar, 21Heby O. Persson L. Smith S.S. Adv. Exp. Med. Biol. 1988; 250: 291-299Crossref PubMed Scopus (24) Google Scholar). AdoMet is synthesized in a one-step condensation of methionine and ATP catalyzed by AdoMet synthetase (methionine ATP transferase, MAT, EC 2.5.1.6). Pneumocystis is highly unusual in lacking this enzymatic activity (22Merali S. Frevert U. Williams J.H. Chin K. Bryan R. Clarkson A.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2402-2407Crossref PubMed Scopus (89) Google Scholar). With the exception of Pneumocystis, a Rickettsia (23Andersson J.O. Andersson S.G. Mol. Biol. Evol. 1999; 16: 1178-1191Crossref PubMed Scopus (142) Google Scholar, 24Tucker A.M. Winkler H.H. Driskell L.O. Wood D.O. J. Bacteriol. 2003; 185: 3031-3035Crossref PubMed Scopus (54) Google Scholar) and an aberrant protozoan (25Jeon T.J. Jeon K.W. J. Eukaryot. Microbiol. 2003; 50: 61-69Crossref PubMed Scopus (14) Google Scholar, 26Jeon T.J. Jeon K.W. J. Cell Sci. 2004; 117: 535-543Crossref PubMed Scopus (17) Google Scholar), every other cell studied is able to synthesize AdoMet. Pneumocystis does require AdoMet and thus must obtain this key intermediate from its mammalian host. This requirement was first observed in culture and was supported by finding that infection with Pneumocystis causes ≥99% depletion of plasma AdoMet in an animal model of PCP (27Merali S. Vargas D. Franklin M. Clarkson Jr., A.B. J. Biol. Chem. 2000; 275: 14958-14963Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) and in patients with PCP (28Skelly M. Hoffman J. Fabbri M. Holzman R.S. Clarkson A.B. Merali S. Lancet. 2003; 361: 1267-1268Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Reduction of plasma AdoMet is such an unusual occurrence that measurement of AdoMet has potential as a minimally invasive, sensitive and specific diagnostic method for PCP and the rapid recovery of plasma AdoMet after initiation of effective treatment may serve as a measure of response to therapy (28Skelly M. Hoffman J. Fabbri M. Holzman R.S. Clarkson A.B. Merali S. Lancet. 2003; 361: 1267-1268Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Because of an asymmetric carbon atom at position 2 in the pyrrolidine ring, nicotine, 1-methyl-2-(3-pyridyl) pyrrolidine, exists as two optically active isomers. Nicotine produced by the tobacco plant is S(-) isomer, but, when burned in a cigarette, about 5% is pyrolytically converted to the R-(+) isomer (29Godin C.S. Crooks P.A. Toxicol. Lett. 1986; 31: 23-29Crossref PubMed Scopus (10) Google Scholar). Regarding classical nicotine pharmacological effects, the S-(-) isomer is generally more potent (30Yildiz D. Ercal N. Armstrong D.W. Toxicology. 1998; 130: 155-165Crossref PubMed Scopus (122) Google Scholar). On average, about 90% of cigarette smoke is inhaled into the lung and, since the smoke from a cigarette contains about 6–8 mg of nicotine, and a cigar about 8-fold more, smokers can absorb considerable amounts of nicotine (31Larson P. Haag H. Silvette H. Tobacco: Experimental and Clinical Studies. Williams and Wilkins, Baltimore, MD1961Google Scholar). In man, the major metabolite of nicotine is cotinine (70–80%) (32Benowitz N.L. Jacob P. II I Clin. Pharmacol. Therap. 1994; 56: 483-493Crossref PubMed Scopus (396) Google Scholar). Cotinine can be further metabolized by AdoMet-mediated methylation (33Sato M. Crooks P.A. Drug Metab. Disp. 1985; 13: 348-353PubMed Google Scholar, 34Cundy K.C. Godin C.S. Crooks P.A. Biochem. Pharmacol. 1985; 34: 281-284Crossref PubMed Scopus (20) Google Scholar) and N-methyl cotinine is a major urinary metabolite of nicotine (35Pool W.F. Houdi A.A. Damani L.A. Layton W.J. Crooks P.A. Drug Metab. Disp. 1986; 14: 574-579PubMed Google Scholar). Studies with guinea pig tissue showed this methylation to be dependent on aromatic azaheterocycle N-methyltransferase (36Godin C.S. Crooks P.A. J. Pharmacol. Sci. 1986; 75: 949-951Abstract Full Text PDF PubMed Scopus (4) Google Scholar) and to be specific for the R-(+) epimer. The nicotine Km is 14.2 μm (37Cundy K.C. Crooks P.A. Godin C.S. Biochem. Biophys. Res. Commun. 1985; 128: 312-316Crossref PubMed Scopus (20) Google Scholar). Nicotine has many pharmacological effects including modifying spontaneous nerve activity, heart rate, brain excitation, and blood pressure. Although high nicotine dosage can cause convulsions, anti-diuretic effects, and contraception, moderate dosage has been used to treat various illnesses ranging from cardiovascular problems to infections (31Larson P. Haag H. Silvette H. Tobacco: Experimental and Clinical Studies. Williams and Wilkins, Baltimore, MD1961Google Scholar). The most effective nicotine application to date is treatment of veterinary parasitic helminth diseases: 19 mg of nicotine kg-1 per os treats Fasciola, Taenia, and Ascaris infections (38Akubue P.I. J. Pharm. Pharmacol. 1966; 18: 625-626Crossref PubMed Scopus (5) Google Scholar). The IC50 in vitro for various helminths ranges from 30 to 300 μm. Beyond veterinary application, nicotine and nicotine analogues were recently reported to be helpful for patients with Crohn's disease (39Fukada A. Saito H. Inui K. J. Pharmacol. Exp. Ther. 2002; 302: 532-538Crossref PubMed Scopus (38) Google Scholar, 40Thomas G.A. Rhodes J. Green J.T. Richardson C. Postgrad. Med. J. 2000; 76: 273-279Crossref PubMed Scopus (65) Google Scholar), attention deficient/hyperactivity disorder, Alzheimer's disease, and schizophrenia (41Newhouse P.A. Potter A. Kelton M. Corwin J. Biol. Psychiatry. 2001; 49: 268-278Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 42Mihailescu S. Drucker-Colin R. Acta Pharmacol. Sin. 2000; 21: 97-104PubMed Google Scholar, 43Levin E.D. Rezvani A.H. Curr. Drug Target CNS Neurol. Disord. 2002; 1: 423-431Crossref PubMed Scopus (173) Google Scholar). Efforts are being made to identify nicotine analogues with useful activity, less addictive potential, and less toxicity (44Crooks P.A. Ayers J.T. Xu R. Sumithran S.P. Grinevich V.P. Wilkins L.H. Deaciuc A.G. Allen D.D. Dwoskin L.P. Bioorg. Med. Chem. Lett. 2004; 14: 1869-1874Crossref PubMed Scopus (31) Google Scholar, 45Ayers J.T. Dwoskin L.P. Deaciuc A.G. Grinevich V.P. Zhu J. Crooks P.A. Bioorg. Med. Chem. Lett. 2002; 12: 3067-3071Crossref PubMed Scopus (42) Google Scholar). Our interest in nicotine, AdoMet, and PCP began when we associated our findings that Pneumocystis requires AdoMet in culture and depletes plasma AdoMet in the rat model of PCP (22Merali S. Frevert U. Williams J.H. Chin K. Bryan R. Clarkson A.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2402-2407Crossref PubMed Scopus (89) Google Scholar) with the results of a broad clinical study reporting a negative correlation between smoking and recurrent PCP (46Saah A.J. Hoover D.R. Peng Y. Phair J.P. Visscher B. Kingsley L.A. Schrager L.K. J. Am. Med. Assoc. 1995; 273: 1197-1202Crossref PubMed Scopus (82) Google Scholar) and older work showing a dramatic, lung-specific AdoMet reduction in guinea pigs treated with nicotine (29Godin C.S. Crooks P.A. Toxicol. Lett. 1986; 31: 23-29Crossref PubMed Scopus (10) Google Scholar). From these associations, we developed the hypothesis that smoking protects against PCP by the action of nicotine causing a reduction in lung AdoMet. Here we report rat model data supporting this hypothesis and data suggesting the mechanism involved in the effect of nicotine on lung AdoMet. Chemicals and Supplies—Ultra-pure AdoMet, α-cyano-4-hydroxycinnamic acid solution (CHCA), spermidine, spermine, putrescine, and R-(+) nicotine were from Sigma-Aldrich. AccQ.Fluor reagent kits were from Waters Corp. (Milford, MA). Urea, CHAPS, DTT, pharmalyte, glycerol, iodoacetamide, Immobiline Dry Strip (IPG), Plus One silver stain kits, and ExcelGel SDS XL 12–14 gels for two-dimensional separation were from Amersham Biosciences. Lyophilized trypsin powder was from Promega (Madison, WI). Pediatric suspension of trimethoprim and sulfamethoxazole (TMP/SMZ) was from Barre-National (Baltimore, MD). Dexamethasone sodium phosphate, 10 mg ml-1 for injection, was from Elkins-Sinn, Inc. (Cherry Hill, NJ). Cyanoacrylic surgical adhesive was from Henry Schein (Melville, NY) Rat PCP Model—Specific pathogen-free S.D. rats (Taconic Farms, Germantown, NY) were housed in a barrier colony and maintained on multiple antibiotics to avoid other opportunistic infections, as previously described (48Merali S. Chin K. Grady R.W. Weissberger L. Clarkson Jr., A.B. Antimicrob. Agents Chemother. 1995; 39: 1442-1444Crossref PubMed Scopus (24) Google Scholar). The rats were also pretreated with a combination of trimethoprim and sulfamethoxazole (TMP/SMZ) for 21 days to reduce any latent P. carinii infection. This was done by adding 25 ml of a pediatric suspension of TMP/SMZ to each liter of drinking water yielding final concentrations of 0.2 mg of trimethoprim ml-1 and 1.0 mg of sulfamethoxazole ml-1. After TMP/SMZ treatment, infusion pumps were implanted (see below) and delivered saline during the 7 days allowed for recovery before immunosuppression was begun by adding dexamethasone in the drinking water, 1.5 mg liter-1. Inoculation with P. carinii was by intratracheal instillation of a lung homogenate from animals with PCP. Before use for inoculation, lungs were tested for other pathogens by Gram-stained impression smears and by streaking on blood agar plates to detect colony-forming fungi or bacteria (48Merali S. Chin K. Grady R.W. Weissberger L. Clarkson Jr., A.B. Antimicrob. Agents Chemother. 1995; 39: 1442-1444Crossref PubMed Scopus (24) Google Scholar). Inoculation was 4 days after initiation of immunosuppression and was repeated after an additional 2 days. The animals were sacrificed 21 days after the first inoculation. The degree of PCP was determined by counting of cysts and trophozoites in stained smears of lung homogenate as previously described (48Merali S. Chin K. Grady R.W. Weissberger L. Clarkson Jr., A.B. Antimicrob. Agents Chemother. 1995; 39: 1442-1444Crossref PubMed Scopus (24) Google Scholar). Administration of Drugs by Infusion Pumps—Implantation and filling of pumps with 1.0-ml reservoirs and a nominal delivery rate of 0.15 ml day-1 (Veterinary Implant Products Division of Advanced Neuromodulation Systems, Plano, TX) were as previously detailed (48Merali S. Chin K. Grady R.W. Weissberger L. Clarkson Jr., A.B. Antimicrob. Agents Chemother. 1995; 39: 1442-1444Crossref PubMed Scopus (24) Google Scholar) and briefly described here. Steam-sterilized, saline-charged pumps were inserted into subcutaneous pockets in the dorsal thorax area that had been formed by a probe inserted from an incision in the skin of the back. The delivery capillary tubing was tunneled under the skin and inserted into the peritoneum through a small opening in the ventral midline just posterior to the sternum. The capillary was secured in place with cyanoacrylic surgical adhesive (VetBond). Incisions were closed with wound clips. The pump reservoir was accessed by inserting the needle of a 26-gauge infusion set through the skin into the fill port; the flexible tubing of infusion sets helps avoid movement of the needle thereby preventing damage to the fill port. Pump solutions were changed as follows. An infusion set needle was inserted into the fill port and any remaining solution was withdrawn with an attached syringe. The volume remaining in the pump allowed delivery rate calculation and the manufacturer-reported delivery rate of ∼0.15 ml day-1 was confirmed. To add fresh solution, another infusion set was inserted and 3 ml of fresh solution was slowly injected into the pump flushing out any of the remaining old solution through the draining infusion set. The draining infusion set was then removed and 1.0 ml of fresh solution was injected into the pump as a refill. The 1.0-ml volume of the pump reservoir allowed continuous delivery for >6 days without adding solution and, since analysis of the nicotine remaining in the pumps after 6 days indicated less than a 1% loss by degradation, 2S. Merali, unpublished data. nicotine and saline solutions were replaced at 6-day intervals. Because AdoMet degrades rapidly, solutions containing AdoMet were replaced daily. At the time the pumps were implanted, the animals weighed 145–155 g. Immunosuppression by dexamethasone without P. carinii inoculation causes a typical weight drop to 120–130 grams after 4 weeks. P. carinii-inoculated animals typically weigh 90–100 g 21-days post-inoculation unless treated to suppress PCP. Nominal dose rates calculated at the beginning of the experiment are reported in “Results” although the dose rate can increase by as much as 20% by the end of the treatment period because of weight loss. AdoMet Measurement—The AdoMet contents of lung, liver, and plasma samples were measured by HPLC analysis using Waters AccQ. Fluor derivatizing reagent as previously reported (27Merali S. Vargas D. Franklin M. Clarkson Jr., A.B. J. Biol. Chem. 2000; 275: 14958-14963Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). For biological samples, the limit of detection is 0.5 nmol and linearity extends to 5,000 nmol. All samples were analyzed in triplicate, and the coefficient of variation ranged from 5 to 17%, depending on the amount of AdoMet in the sample. Nicotine Measurement—Measurement of R-(+) nicotine in tissues and plasma was done using a modification of a published capillary zone electrophoresis (CZE) method (49Palmer M.E. Smith R.F. Chambers K. Tetler L.W. Rapid Commun. Mass Spectrom. 2001; 15: 224-231Crossref PubMed Scopus (9) Google Scholar). The apparatus was a P/ACE MDQ system equipped with a photodiode array detector allowing electropherograms to be monitored at 257 and 205 nm. We used an amine capillary kit (Beckman, Inc.; 50 mm inner diameter × 60 cm total length, 50 cm to the detector window). Sample preparation involved adding 20 μl of 10% perchloric acid to 80 μl of lung homogenate to precipitate the proteins that were removed by centrifugation at 5000 × g for 10 min. Supernatants were stored for up to 7 days at -20 °C before CZE analysis. Prior to analysis, samples were diluted 1:1 with water. The separation protocol was as follows: 2 min, 20 psi rinse using the kit “amine regenerator solution,” 2 min, 20 psi rinse with Tris buffer (50 mm Tris, pH 8.0), sample injection and separation using 25 kV in reverse polarity mode for 7 min at 25 °C. Specificity was assured by demonstrating that R-(+) nicotine was resolved from all other peaks in cell extracts. Instrument precision was monitored by making triplicate injections from a single pooled standard. Linearity was demonstrated using a series of standard solutions over a range of 0.10–20.00 pmol. Analytical Methods—Lung tissues (500 mg) from treated or normal lung were snap-frozen in liquid nitrogen and were crushed into a fine powder using mortar and pestle. The powder was resuspended in 2 ml of buffer (150 mm KCl, 2 mm dithiothreitol, 25 mm HEPES, 5 mm MgSO4,) and sonicated at 40 watts and 70% duty cycle for about 2 min, then centrifuged at 10,000 × g for 15 min. An aliquot of this lysate was used for Bio-Rad protein assay, and the protein from the rest of the lysate was used for the reaction mixture. Measurement of the AdoMet biosynthesis enzyme MAT was as previously described in (27Merali S. Vargas D. Franklin M. Clarkson Jr., A.B. J. Biol. Chem. 2000; 275: 14958-14963Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Presumptive SSAT activity (pmol of acetylspermidine (mg of protein)-1 h-1), AdoMet DC activity and ODC activity were as described previously (50Merali S. J. Biol. Chem. 1999; 274: 21017-21022Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 51Tabib A. Morgan M.L.D. Polyamine Protocols. Methods in Molecular Biology. 79. Humana Press Inc., 1998: 41-49Google Scholar, 52Merali S. Clarkson Jr., A.B. Antimicrob. Agents Chemother. 1996; 40: 973-978Crossref PubMed Google Scholar). Spermidine, spermine, putrescine, and N-1-acetylated spermidine were analyzed by HPLC using Waters' AccQ.Fluor derivatizing reagent as previously reported (53Merali S. Clarkson Jr., A.B. J. Chromatogr. B: Biomed. Appl. 1996; 675: 321-326Crossref PubMed Scopus (48) Google Scholar). Preparation of Samples for Proteomics Studies—The lungs of two nicotine-treated (400 μgof R-(+) nicotine kg-1 h-1) and two saline-treated animals were washed aseptically three times with sterile saline then soaked for 15 min at 4 °C in saline. Proteins were extracted from 500 mg of aliquots of lung tissue as follows. After freezing with liquid nitrogen, tissue samples were ground to a powder using a mortar and pestle with liquid nitrogen in the mortar. The frozen powder was added directly to 0.8 ml of cold lysis buffer (9.8 m urea, 4% CHAPS, 10% glycerol, 15 mg ml-1 DTT, and 4% ampholytes) and sonicated at 40 watts and 70% duty cycle for about 2 min. The sonicate was centrifuged at 10,000 × g for 15 min to remove non-homogenized cartilage and other debris. A 10-μl aliquot of each lysate was used for a Bradford protein assay (Bio-Rad). Acetone with 10% trichloroacetic acid and 20 mm DTT was added to the balance to precipitate the proteins that were then collected by centrifugation at 10,000 × g for 30 min at 20 °C. The acetone/trichloroacetic acid/DTT solution was poured off and enough lysis buffer added to obtain a protein concentration of 3 mg ml-1. The proteins were re-solubilized by sonication followed by shaking for 3 h at room temperature. After a final centrifugation at 10,000 × g for 15 min, the supernatant was collected for two-dimensional protein electrophoresis. First Dimension Electrophoresis-Isoelectric Focusing (IEF)—IEF was performed using a MultiPhor II system Amersham Biosciences following the manufacturer's instructions. Immobiline dry strip gels (18 cm) were hydrated overnight (∼16 h) in 350 μl of solution containing 2% CHAPS (w/v), 0.4% DTT (w/v), 7 m urea, 2 m thiourea, 2% carrier ampholytes solution (v/v), and 70 μg of sample protein. IEF was performed for 30 min at 150 V, 1 h at 300 V, 1.5 h at 1500 V, and 22.5 h at 3000 V to complete 70,000 VH. IEF focusing ranges were 4–7 and 4.5–5.5. Second Dimension Electrophoresis (SDS-PAGE)—Prior to second dimension separation, Immobiline dry strip gel strips were soaked for 15 min in 10 ml equilibration buffer (6 m urea, 30% glycerol, 2% SDS, 1% DTT, and 0.001% bromphenol blue in 0.05 m Tris-HCl buffer, pH 8.8) (54Wheeler D.L. Ness K.J. Oberley T.D. Verma A.K. Cancer Res. 2003; 63: 3037-3042PubMed Google Scholar) and then soaked for 15 min in 10 ml of equilibration buffer containing 250 mg of iodoacetamide. Following the manufacturer's instructions, the Immobiline dry strip gel strips were placed on an Excel Gel SDS XL 12–14 (Amersham Biosciences) mounted in a Multiphor II electrophoresis unit connected to a Multi Temp III thermostatic circulator (Amersham Biosciences) set to 15 °C. Second dimension electrophoresis was run at 10 mA for 10 min, 20 mA for 20 min then at 40 mA for 1.5 h. The gels were silver-stained following the manufacturer's protocol. Image Analysis of Two-dimensional Gels—Monochrome scans of gels were made at 200 dpi (HP model C7710A scanner) and stored in tiff format for input into the Z3 proteomics software package (Compugen, Jamesburg, NJ). The software automatically detected spots and assigned them numbers. Gel to gel variation was reduced by combining three gels per Immobiline dry strip gel range from each animal to produce Raw Master Gels (RMGs). RMGs of the two animals from the treatment group and the two from the control group were combined to produce Master Reference Gels, which were used to create differential displays. The software identified all differentially expressed protein spots in nicotine-treated animals relative to saline-treated controls including those up-regulated, down-regulated, newly expressed, or no longer expressed. Predigestion Processing—Protein spots of interest were excised from the gel and destained by placing them in 1.5 ml of siliconized microcentrifuge tubes, washing with deionized water (500 μl, 6×), transferring into new 1.5-ml siliconized tubes, breaking into small pieces with a pipette tip (0.5–1 mm2) and incubating in 20 μl of fresh destaining solution (equal parts 30 mm potassium ferricyanide and 100 mm sodium thiosulfate) until the brownish color disappeared (1–2 min). The destaining solution was decanted, and the gel pieces washed with water (20 μl, 5–6×) until all traces of yellow disappeared. They were then incubated for 20 min in 20 μl of 200 mm ammonium bicarbonate, washed with water (20 μl, 1×), and dehydrated with acetonitrile (30 μl, 2×) until opaque white. The gel pieces were dried in a vacuum centrifuge (30 min). In-gel Trypsin Digestion—Purification of tryptic peptides followed a published method (55Zhan X. Desiderio D.M. Clin. Chem. 2003; 49: 1740-1751Crossref PubMed Scopus (38) Google Scholar). A vial of 20 μg (833 pmol) of lyophilized trypsin powder was dissolved in 100 μl of 50 mm acetic acid and diluted to 16 ng μl-1 with 230 μl of 50 mm ammonium bicarbonate. A 20–30-μl volume of trypsin solution (depending on the amount of dried gel in the sample) was added to each set of dried gel pieces. Digestion w" @default.
• W2130069364 created "2016-06-24" @default.
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• W2130069364 date "2005-04-01" @default.
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• W2130069364 title "Effect of Nicotine on Lung S-Adenosylmethionine and Development of Pneumocystis Pneumonia" @default.
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