Inosine and N1-methylinosine within a synthetic oligomer mimicking the anticodon loop of human tRNA(Ala) are major epitopes for anti-PL-12 myositis autoantibodies.
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Sera of some patients afflicted with the inflammatory disease myositis contain antibodies of the anti-PL-12 type. A fraction of these polyclonal autoantibodies specifically precipitates the fully matured human tRNA(Ala) bearing the anticodon IGC (PL-12 antigen). Earlier work (Bunn & Mathews, 1987, Science 238:116-119) had shown that the epitopes are located entirely within the anticodon stem-loop of the tRNA(Ala). Here we demonstrate that human anti-tRNA(Ala) autoantibodies immunoprecipitate a synthetic polyribonucleotide containing inosine (I) and N1-methylinosine (m1I) separated by 2 nt as in the anticodon stem-loop of human tRNA(Ala). The shortest polyribonucleotide that can be immunoprecipitated corresponds to the pentanucleotide IpGpCpm1IpUp, which corresponds to part of the anticodon loop of human tRNA(Ala) and lacks the stem-loop structure. The efficiency of immunoprecipitation was about four times greater with longer polyribonucleotides capable of forming a stem-loop structure, and was abolished by altering the relative positions of I and m1I within the synthetic polynucleotide. Synthetic oligodeoxyribonucleotide analogs of the tRNA(Ala) stem-loop, containing the sequence dIpdGdCdm1Ip, are not antigenic. Our results show that human anti-tRNA(Ala) autoantibodies selectively recognize chemical details of modified nucleotides (the 6-keto group of inosine-34 and the 6-keto group and the N1-methyl groups of N1-methylinosine-37) within an anticodon loop structure of a tRNA molecule. We also describe the chemical synthesis of the phosphoramidite derivatives corresponding to N1-methylinosine and N1-methyl-2'-deoxyinosine for use in the automatic chemical synthesis of oligonucleotides containing N1-methylinosine and N1-methyl-2'-deoxyinosine. (+info)
In vivo analysis of amantadine renal clearance in the uninephrectomized rat: functional significance of in vitro bicarbonate-dependent amantadine renal tubule transport.
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Amantadine transport into renal proximal and distal tubules is bicarbonate dependent. In the present study, we addressed the effects of bicarbonate on renal clearance and urinary excretion of amantadine. Renal clearance of kynurenic acid was also studied to determine whether bicarbonate effects are specific for organic base transport by the kidney. After a moderate diuresis was established, animals received i.v. [(3)H]amantadine or [(3)H]kynurenic acid followed by an acute dose of sodium bicarbonate or physiological saline. Urine and blood samples were analyzed for [(3)H]amantadine or [(3)H]kynurenic acid, blood gases, and pH. Amantadine and kynurenic acid were excreted by the kidneys, and both compounds underwent renal tubular secretion. Amantadine metabolism occurred, and one metabolite was detected in the urine. In the bicarbonate-treated rats, the total amount of amantadine excreted in the urine was decreased, whereas the amount of metabolite recovered was similar in both groups. Bicarbonate treatment caused a sustained increase in blood bicarbonate levels, a mild increase in blood pH, and a decrease in amantadine renal clearance and in the amantadine/creatinine clearance ratio. Only a transient decrease in the renal clearance of kynurenic acid and the kynurenic acid/creatinine clearance ratio was observed. This study demonstrates that short-term changes in bicarbonate concentration may have significant effects on renal organic cation elimination. Coupled with our previous in vitro demonstration of bicarbonate-dependent organic cation transport, the present study suggests that bicarbonate inhibition of renal tubule organic cation secretion may explain the previous observation that bicarbonate dosing decreases amantadine excretion by the kidney. (+info)
Folylpolyglutamyl synthetase gene transfer and glioma antifolate sensitivity in culture and in vivo.
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BACKGROUND: Although antifolates are popular agents for use in chemotherapy, they display minimal toxicity against slow-growing tumors and are toxic to actively replicating cells in normal tissues. These drugs are converted intracellularly into polyglutamate derivatives by the enzyme folylpolyglutamyl synthetase (FPGS). Because tumors with high expression of FPGS often respond to nontoxic antifolate doses, we investigated whether augmenting tumoral FPGS activity by gene delivery would enhance tumoral antifolate sensitivity. METHODS: 9L rat gliosarcoma cells were stably transfected with a human FPGS complementary DNA (cDNA), producing 9L/FPGS cells. The sensitivity of these cells to the antifolates methotrexate and edatrexate was measured in culture and in subcutaneous tumors, as was their ability to increase the chemosensitivity of nearby nontransfected cells, i.e., a bystander effect. The antifolate sensitivity of nonselected cells transduced with a hybrid amplicon vector that expressed FPGS was also ascertained. RESULTS: In comparison with 9L cells, 9L/FPGS cells displayed enhanced sensitivity to 4-hour pulses of antifolate. Subcutaneous 9L/FPGS tumors responded as well to methotrexate given every third day as 9L tumors did to daily treatment. A modest bystander effect was observed with edatrexate treatment in culture and in vivo. The observed bystander effect appeared to result from the release of antifolates by transfected cells after the removal of extracellular drug. In culture, enhanced antifolate sensitivity was also seen in other stably transfected rodent and human glioma cell lines, including one with high pre-existing FPGS activity, and in canine and human glioblastoma cell lines transduced with a vector bearing FPGS cDNA. CONCLUSIONS: FPGS gene delivery enhances the antifolate sensitivity of several glioma cell lines and merits further evaluation as a therapeutic strategy. (+info)
Inhibition of esterolysis of enalapril by paraoxon increases the urinary clearance in isolated perfused rat kidney.
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The effect of competing elimination pathways on the metabolic and excretory clearance estimates was examined with tracer concentrations of [(3)H]enalapril, which was both metabolized and excreted by the rat kidney. Perturbation was achieved with use of the carboxylesterase inhibitor paraoxon, which inhibited [(3)H]enalapril metabolism to [(3)H]enalaprilat in rat renal S9 fraction. At 0.1, 0.5, 1, and 10 microM paraoxon, esterolysis of enalapril was inhibited by 76 +/- 7, 93 +/- 5, 96 +/- 5, and 93 +/- 6%, respectively. The lowest concentration (0.1 microM) of paraoxon was chosen for single-pass isolated perfused kidney (IPK) studies because viability was least compromised, and the sodium and glucose reabsorptive functions of the IPK remained constant. After an equilibration period (15-20 min at constant pressure, 90-100 mm Hg), perfusion of the rat kidney with [(3)H]enalapril was carried out under constant flow (8 ml/min) for 30 min in the absence and presence of paraoxon (0.1 microM). The metabolic (from 1.83 +/- 0.52 to 1.48 +/- 0.47 ml/min/g) and total renal (from 1.87 +/- 0.46 to 1. 57 +/- 0.41 ml/min/g) clearances of [(3)H]enalapril in the IPKs were decreased significantly (p <.05) in the presence of paraoxon when compared with controls. Concomitantly, the urinary clearance (from 0. 04 +/- 0.07 to 0.09 +/- 0.09 ml/min/g) and the fractional excretion (from 0.23 +/- 0.18 to 0.52 +/- 0.25) of [(3)H]enalapril doubled (p <.05). The study illustrates that a reduction in cellular metabolism of the kidney brings forth a rise in the estimate of clearance of its complimentary pathway, estimate of the excretory (urinary) clearance. (+info)
The fluorescence of scorpions and cataractogenesis.
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BACKGROUND: Protein cross-linking and fluorescence are widely recognized markers of oxidative aging in human proteins. Oxidative protein aging is a combinatorial process in which diversity arises from the heterogeneity of the targets and is amplified by the nonselective nature of the reactants. The cross-links themselves defy analysis because they are generally embedded in a covalent matrix. Arthropods rely upon oxidative cross-linking in the hardening of the cuticle - a process known as sclerotization. Among arthropods, scorpions are noteworthy in that the process of sclerotization is accompanied by the buildup of strong visible fluorescence. To date, the nature of the fluorescent species has remained a mystery. RESULTS: We have identified one of the soluble fluorescent components of the scorpions Centuroides vittatus and Pandinus imperator as beta-carboline - a tryptophan derivative that has previously been identified by hydrolysis and oxidation of lens protein. We have also shown that beta-carboline-3-carboxylic acid is released from both scorpion exuvia (the shed cuticle) and human cataracts upon hydrolysis, suggesting that the protein-bound beta-carboline and free beta-carboline have common chemical origins. CONCLUSIONS: Cataractogenesis and cuticular sclerotization are disparate oxidative processes - the former is collateral and the latter is constitutive. The common formation of beta-carbolines shows that similar patterns of reactivity are operative. These fundamental mechanisms provide predictive insight into the consequences of human protein aging. (+info)
Mimicry of the hepatitis delta virus replication cycle mediated by synthetic circular oligodeoxynucleotides.
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BACKGROUND: Hepatitis delta virus (HDV) is a circular single-stranded RNA pathogen whose monomeric form results from self-processing. Although studies have examined minimal HDV ribozyme activities, the mechanism for forming the circular virus remains unclear, and the trans catalytic properties of self-processed forms of HDV ribozymes have not been studied. In addition, HDV ribozymes have not previously been engineered to cleave a non-HDV sequence. RESULTS: Long repeating RNAs have been produced from in vitro rolling-circle transcription of synthetic circular oligodeoxynucleotides encoding catalytically active subsets of the entire antigenomic RNA virus. Like full-length HDV, these multimeric RNAs undergo self-processing to monomer length; importantly, cyclization is found to occur efficiently, but only in the presence of the circular template. Linear and circular monomer ribozymes and engineered variants are shown to be active in cleaving HDV and HIV RNA targets in trans, despite having self-binding domains. CONCLUSIONS: Mimicry of the rolling-circle replication pathway for HDV replication has led to a new proposal for cyclization of HDV RNA. Under these conditions, cyclization is mediated by the complementary circular template. In addition, it has been shown that self-processed HDV ribozymes can be catalytically active in trans despite the presence of antisense sequences built into their structure. (+info)
Occurrence of P-flavin binding protein in Vibrio fischeri and properties of the protein.
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In previous studies involving Photobacterium species we proposed that (i) P-flavin is the product of luciferase, (ii) the physiological function of the lux operon is not to produce light but to produce FP(390) (luxF protein), including its prosthetic group, P-flavin, and (iii) FP(390) reactivates oxidatively inactivated cobalamin-dependent methionine synthase similar to flavodoxin but at relatively high ionic strength. It seems difficult to extend this idea to all luminous bacteria because the luxF gene is not present in the lux operon in Vibrio or Xenorhabdus. But we predicted that a luciferase fragment which binds P-flavin should function like FP(390) in these species. In this study, we isolated P-flavin binding protein from Vibrio fischeri ATCC 7744. The obtained protein was a modified luciferase as expected, in which the beta-subunit was intact but about 25 amino acid residues at the C-terminus of the alpha-subunit were deleted and the prosthetic group was the fully reduced P-flavin. These results strongly support that the physiological function of the lux operon is as described above even in luminous bacteria other than Photobacterium species. We propose that chromophore B reported by Tu and Hastings [Tu, S.-C. and Hastings, J.W. (1975) Biochemistry 14, 1975-1980] is the reduced P-flavin. (+info)
Formation and persistence of nucleotide alterations in rats exposed whole-body to environmental cigarette smoke.
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The assessment of pathological effects produced by environmental tobacco smoke in humans is controversial in epidemiological studies. On the other hand, animal models are poorly sensitive to smoke carcinogenicity. We designed an experimental study assessing the tissueselective formation and persistence of DNA adducts in smoke-exposed rats. Sprague-Dawley rats were exposed for 6 h per day, 5 days per week, to environmental smoke resulting from a mixture of sidestream and mainstream smoke generated from Kentucky 2R1 reference cigarettes. The total particulate matter was in the range of 73-93 mg/m(3). DNA adducts were measured by (32)P-post-labelling in rat organs (lung, heart, liver, bladder and testis), tissues (dissected tracheal epithelium) and cells [isolated bronchoalveolar lavage (BAL) cells]. A time-related increase of (32)P-post-labelled DNA modifications was detectable by autoradiography, in the form of massive diagonal radioactive zones and individual spots. Top levels were reached after 4-5 weeks of exposure. The ratio of smoke-induced DNA adducts to the background levels detected in sham-exposed rats was 11.2 in the tracheal epithelium, 10.4 in BAL cells, 7.3 in the heart, 6.3 in the lung, 5.1 in the bladder, 1.9 in the testis and 1. 1 in the liver. Appearance of DNA adducts in the lung was also revealed by synchronous fluorescence spectrophotometry. Smoke-related oxidative damage was demonstrated by a significant enhancement of 8-hydroxy-2'-deoxyguanosine in lung DNA. In parallel, there was a time-related induction of lung microsomal arylhydrocarbon hydroxylase activity, an elevation in cytosolic glutathione S-transferase activity, and a moderate but progressive and significant depletion of reduced glutathione. After discontinuing exposure to environmental cigarette smoke for 1 week, DNA adduct levels significantly dropped in the lung, tracheal epithelium, heart and bladder. The decrease was evident but not statistically significant in BAL cells, and was negligible in the heart. The selective localization and the differential persistence of these promutagenic nucleotide modifications in rat organs, tissues and cells suggest that exposure to environmental cigarette smoke, at least under the high exposure regimens used in experimental studies, may pose a potential risk of developing mutation-related diseases. (+info)