Biomedical subjects
Publications and source records attributed to C J Decker.
The 3' untranslated region (3' UTR) can control gene expression by affecting the localization, stability and translation of mRNAs. The recent finding that 3' UTRs can control the decapping rate of mRNAs, in combination with their ability to influence the initiation of translation, suggests that 3' UTRs act through a direct or indirect interaction between the 3' and 5' ends of mRNAs.
The first step in the decay of several yeast mRNAs is the shortening of the poly(A) tail, which for the MFA2 transcript triggers decapping and 5'-to-3' degradation. To understand the basis for differences in mRNA decay rates, it is important to determine if deadenylation-dependent decapping is specific to the unstable MFA2 transcript or is a general mechanism of mRNA degradation. To this end, we analyzed the turnover of the stable PGK1 mRNA by monitoring the decay of a pulse of newly synthesized transcripts while using two strategies to trap decay intermediates. First, we used strains deleted for the XRN1 gene, which encodes a major 5'-to-3' exonuclease in Saccharomyces cerevisiae. In xrn1 delta cells, PGK1 transcripts lacking the 5' cap structure and a few nucleotides at the 5' end were detected after deadenylation. Second, we inserted into the PGK1 5' untranslated region strong RNA secondary structures, which can slow exonucleolytic digestion and thereby trap decay intermediates. These secondary structures led to the accumulation of PGK1 mRNA fragments, following deadenylation, trimmed from the 5' end to the site of the secondary structure. The insertion of strong secondary structures into the 5' untranslated region also inhibited translation of the mRNA and greatly stimulated the decay of the PGK1 transcripts, suggesting that translation of the PGK1 mRNA is required for its normally slow rate of decay. These results suggest that one mechanism of degradation of the PGK1 transcript is deadenylation followed by decapping and subsequent 5'-to-3' exonucleolytic degradation. In addition, by blocking the 5'-to-3' degradation process, we observed PGK1 mRNA fragments that are consistent with a 3'-to-5' pathway of mRNA turnover that is slightly slower than the decapping/5'-to-3' decay pathway. These observations indicate that there are multiple mechanisms by which an individual transcript can be degraded following deadenylation.
The posttranscriptional insertion and deletion of U residues in trypanosome mitochondrial transcripts called RNA editing initiates at the 3' end of precisely defined editing domains that can be identified independently of the cognate guide RNA. The regions where editing initiates in Trypanosoma brucei cytochrome b and cytochrome oxidase subunit II preedited mRNAs are specifically cleaved by a trypanosome mitochondrial endonuclease that acts like mung bean nuclease and therefore is single strand specific. The regions where editing initiates in virtually all examined preedited mRNAs are predicted to form loop structures, suggesting that editing domains could generally be recognized as prominent single-stranded loops. In contrast to preedited mRNA, edited mRNA can be either resistant or sensitive to cleavage by trypanosome mitochondrial endonuclease, depending on the reaction conditions. This selectivity appears dependent on the availability of extract RNAs, and in model reactions, edited mRNA becomes resistant to cleavage upon base pairing with its guide RNA. Natural partially edited mRNAs are also specifically cleaved with a sensitivity like preedited and unlike edited mRNAs, consistent with their being intermediates in editing. These results suggest that in vivo, the structure of editing domains could initially be recognized by the mitochondrial endonuclease, which could target its associated RNA ligase and terminal U transferase to begin cycles of enzymatic editing modifications.
RNA editing in trypanosomes has been proposed to occur through transesterification or endonuclease cleavage and RNA ligation reactions. Both models involve a chimeric intermediate in which a guide RNA (gRNA) is joined through its 3' oligo(U) tail to an editing site of the corresponding mRNA. Velocity centrifugation of Trypanosoma brucei mitochondrial extracts had been reported to completely separate the gRNA-mRNA chimera-forming activity from endonuclease activity (V. W. Pollard, M. E. Harris, and S. L. Hajduk, EMBO J. 11:4429-4438, 1992), appearing to rule out the endonuclease-RNA ligase mechanism. However, we show that an editing-domain-specific endonuclease activity does cosediment with the chimera-forming activity, as does the RNA ligase activity, but detection of the specific endonuclease requires reducing assay conditions. This report further demonstrates that the T. brucei chimera-forming activity is mimicked by mung bean nuclease and T4 RNA ligase. Using cytochrome b (CYb) preedited mRNA and a model CYb gRNA, we found that these heterologous enzymes specifically generate CYb gRNA-mRNA chimeras analogous to those formed in the mitochondrial extract. These combined results provide support for the endonuclease-RNA ligase mechanism of chimera formation.
The first step in the decay of some eukaryotic mRNAs is the shortening of the poly(A) tail. To examine how the transcript body was degraded after deadenylation, we followed the decay of a pulse of newly synthesized MFA2 transcripts while utilizing two strategies to trap intermediates in the degradation pathway. First, we inserted strong RNA secondary structures, which can slow exonucleolytic digestion and thereby trap decay intermediates, into the MFA2 5' UTR. Following deadenylation, fragments of the MFA2 mRNA trimmed from the 5' end to the site of secondary structure accumulated as full-length mRNA levels decreased. In addition, in cells deleted for the XRN1 gene, which encodes a major 5' to 3' exonuclease in yeast, the MFA2 transcript is deadenylated normally but persists as a full-length mRNA lacking the 5' cap structure. These results define a mRNA decay pathway in which deadenylation leads to decapping of the mRNA followed by 5'-->3' exonucleolytic degradation of the transcript body. Because the poly(A) tail and the cap structure are found on essentially all mRNAs, this pathway could be a general mechanism for the decay of many eukaryotic transcripts.
Recent experiments have identified distinct mechanisms of eukaryotic RNA turnover. In one mechanism, deadenylation triggers decapping, exposing the messenger RNA to 5' to 3' degradation. This pathway may act at different rates on the majority of messenger RNAs. There are also degradation mechanisms, such as endonucleolytic cleavage, limited to messenger RNAs containing specific sequence elements.
Derivatives of imidazole-2-thiones block reactions catalyzed by thyroid peroxidase (TPX) and the closely related lactoperoxidase (LPX), and this property is used therapeutically to treat hyperthyroidism. The reactions of a series of benzimidazoline-2-thiones with chemical and enzymatic oxidants were investigated to probe systematically the mechanism of inhibition. Oxidation of benzimidazoline-2-thione (I) and 1-methylbenzimidazoline-2-thione (II) with 3-chloroperbenzoic acid (PBA) yielded reaction products and stoichiometry consistent with benzimidazole-2-sulfenic acids as reactive intermediates. The N,N'-disubstituted nature of 1,3-dimethylbenzimidazoline-2-thione (III) precludes sulfenic acid formation by tautomerization, and the oxidation of III with PBA yielded products and stoichiometry that were consistent with a benzimidazole-2-sulfonyl ylide as the reactive intermediate. I and II are suicide inhibitors of LPX and TPX, but III was found to inhibit only peroxidase-catalyzed iodination reactions by an alternate substrate mechanism. These results provide support for the hypothesis that imidazole-2-sulfenic acids are important reactive intermediates in the suicide inactivation of TPX and LPX and relate the chemical reactivity of the inhibitor with both the potency and mechanism of inhibition. These results suggest that 1,3-disubstituted thiourea derivatives represent a new class of potential antihyperthyroid drugs that block TPX-catalyzed tyrosine iodination but do not cause irreversible enzyme inactivation.
To determine pathways of mRNA turnover in yeast, we have followed the poly(A) tail removal and degradation of a pulse of newly synthesized transcripts from four different genes. Before decay of both stable and unstable mRNAs initiated, there was a temporal lag during which the poly(A) tail was deadenylated to an oligo(A) length. Altering the deadenylation rate of an mRNA led to a corresponding change in the length of this lag. The rate of deadenylation and the stability of the oligo(A) species varied between mRNAs, explaining the differences in mRNA half-lives. To examine how the transcript body was degraded following deadenylation, we used the strategy of inserting strong RNA secondary structures, which can slow exonucleolytic digestion and thereby trap decay intermediates, into the 3' UTR of mRNAs. Fragments lacking the 5' portion of two different mRNAs accumulated after deadenylation as full-length mRNA levels decreased. Therefore, these results define an mRNA decay pathway in which deadenylation leads to either internal cleavage or decapping followed by 5'-->3' exonucleolytic degradation of the mRNA.
OBJECTIVE: To review the general surgical management of mentally handicapped persons with pica. DESIGN: A study of hospital records and a review of the current literature. SETTING: A community hospital. PATIENTS: Thirty-five patients from a large institution for the mentally handicapped were treated for well-documented pica on 56 occasions at the Orillia Soldiers Memorial Hospital between 1976 and 1991. INTERVENTIONS: Endoscopy and laparotomy for the removal of foreign bodies or to close perforations. RESULTS: Fourteen (25%) cases of pica were managed by observation only, but 42 (75%) cases required surgical intervention. There were 34 laparotomies. The complication rate was 30% and the death rate 11%. CONCLUSIONS: Pica is a serious health risk for mentally handicapped patients. Diagnosis and postoperative care can be difficult. Pica should be suspected in mentally handicapped patients with gastrointestinal symptoms.
The covalent binding of a series of 14C- or 35S-labeled benzimidazole-2-thione (MBI) derivatives to rat liver microsomal proteins was studied to determine the mechanisms of hepatic monooxygenase oxidation of model anti-hyperthyroid compounds. All thiocarbamides tested (including methimazole) produced an NADPH-dependent loss of cytochrome P450 (P450) chromophore which could be prevented by the addition of glutathione (GSH). The covalent binding of MBI to liver microsomal proteins from dexamethasone (DEX)-pretreated rats was enhanced 10-fold with NADPH, unaffected by P450 inactivation with 1-aminobenzotriazole (ABT) and attenuated by GSH addition. Heat treatment of microsomes to inactivate the flavin-containing monooxygenase (FMO) decreased the observed binding. Equivalent amounts of [35S]- and [14C]MBI were covalently bound to hepatic microsomal proteins, suggesting retention of both the carbon and sulfur portions of the molecule in the MBI/protein adduct. Thiophilic reagents effected release of covalently bound [14C]- and [35S]MBI in equal amounts suggesting the presence of disulfide bonds between an MBI-derived sulfenic acid and microsomal protein thiols. Coincubation with bovine serum albumin (BSA) resulted in NADPH-dependent binding of [14C]-MBI to BSA sulfhydryls which was blocked by prior treatment of BSA with iodoacetamide. 1-Methyl-benzimidazole-2-thione (MMBI) also covalently bound to microsomal proteins and BSA but at levels lower than with MBI. P450, however, appeared to be more important than FMO in the metabolism of MMBI based on the effects of microsome heat pretreatment or ABT addition. In addition, ca. 1.5-fold more 35S- than 14C-label became bound. The covalent binding of [35S]1,3-dimethyl-benzimidazole-2-thione (DMMBI) to microsomal proteins was ca. six times greater than that of [14C]DMMBI. ABT, catalase and superoxide dismutase had a minimal effect on [35S]DMMBI binding, while FMO inactivation decreased binding by ca. 30%. These findings suggest that both monooxygenases contribute significantly to the hepatic metabolism of thiocarbamides. However, FMO activates thiocarbamides primarily to sulfenic acids, whereas P450 appears to produce both sulfenic acid and other reactive sulfur-derived metabolites. Thiol groups of P450 and other proteins are the molecular targets for these reactive species formed during the hepatic metabolism of anti-hyperthyroid drugs.
A method for application of Avitene is presented. If applied properly, it will avoid the problems that can be associated with its use. It should allow a greater percentage of hemostatic agent to be effectively used on the hemorrhagic surface.
RNA editing is the specific posttranscriptional insertion/deletion of U residues within trypanosomatid mitochondrial transcripts. We used a novel cDNA cloning scheme and analyzed partially edited COIII and CYb RNAs. Our major unanticipated findings are: First, editing appears strikingly indiscriminate within editing domains, inserting and deleting variable numbers of U residues at all sites, both those requiring editing and those that do not. Second, this incomplete editing occurs in precise domains, not in adjacent regions unedited in mature mRNA. Third, editing appears to commence prior to polyadenylation. We propose that within a region being edited, there are cycles of indiscriminate cleavage, U addition or deletion, and religation; sites that become correctly edited would be protected from further modification by duplexing with short RNAs complementary to the edited sequence.
In a preliminary paper [Decker et al. (1986) Biochem. Biophys. Res. Commun. 136, 1162] we have shown that the antimineralocorticoid spironolactone (SPL) preferentially inactivates dexamethasone (DEX) inducible rat hepatic cytochrome P450p isozymes in a suicidal manner. These findings are now confirmed, and the kinetic characteristics of such a process are detailed. In an effort to elucidate the mechanism of SPL-mediated inactivation of cytochrome P450, we have examined the metabolism of SPL in vitro. Incubation of [14C]SPL and NADPH with liver microsomes prepared from DEX-pretreated rats results in the formation of several polar metabolites separable by HPLC with UV detection. This process is found to be dependent on NADPH, O2, SPL, and enzyme concentration, as well as temperature. Furthermore, metabolite formation was significantly attenuated by P450 inhibitors CO and n-octylamine. Mass spectral analysis (thermospray LC/MS, FAB/MS, and FAB/MS/MS) of the two most prominent polar metabolites indicated that these compounds had molecular weights that corresponded to the sulfinic and sulfonic acid derivatives of deacetyl-SPL (SPL-SH). These findings document the formation of previously unreported polar metabolites of SPL by rat liver microsomes enriched in cytochrome P450p and implicate a role for this isozyme in the oxidation of the thiol moiety of deacetyl-SPL. The detection of such metabolites also implicates a catalytic trajectory that includes the thiyl radical and/or sulfenic acid species as a plausible protagonist in drug-mediated inactivation of cytochrome P450p.
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The contributions of the rat hepatic flavin-containing monooxygenase (FMO) and cytochrome P-450 isozymes (P-450) in the ethylenethiourea (ETU) mediated inactivation of P-450 isozymes and covalent binding of the compound to microsomal proteins were investigated. In vitro, ETU was found to inhibit P-450 marker activities in microsomes obtained from untreated (UT) and phenobarbital (PB), beta-naphthoflavone (BNF), and dexamethasone (DEX) pretreated rats. This inhibition was dependent on the presence of NADPH and was completely abolished by coincubation with glutathione (GSH). Heat treatment of microsomes prior to ETU-mediated P-450 inactivation led to diminished loss of P-450 marker activities in microsomes obtained from UT and PB-pretreated, but not BNF- or DEX-pretreated rats, suggesting FMO involvement in the inactivation of some P-450 isozymes. Covalent binding of [14C]ETU to microsomal proteins was found to be NADPH-dependent and enhanced with BNF or DEX pretreatment of rats. This binding was completely inhibited by coincubation with GSH. Heat treatment of microsomes and P-450 inactivation studies indicated a predominant role of FMO in the observed covalent binding. Addition of the sulfhydryl reagents dithiothreitol (DTT) or GSH after the incubation of microsomes, [14C]ETU, and NADPH resulted in the complete release of bound ETU, suggesting the reduction of disulfide bonds between oxidized ETU and protein sulfhydryls. Microsomal heme content was not decreased following incubation of microsomes with ETU and NADPH, and P-450 appeared to be converted to P-420.(ABSTRACT TRUNCATED AT 250 WORDS)
We have previously reported that the diuretic thiosteroid spironolactone (SPL) inactivates rat liver microsomal cytochromes P450 [P450 (P450 3A and P450 2C11)] in a in a mechanism-based fashion, and we have identified two polar SPL metabolites (SPL-sulfinic acid and -sulfonic acid), formed in a partition ratio of approximately 20:1 in such rat liver microsomal incubations [Decker et al. (1989) Biochemistry 28, 5128-5136]. We proposed at the time that these metabolites were most likely derived from further enzymatic (or nonenzymatic) oxidations of the one-electron oxidation product [SPL-thiyl radical (SPL-S.)] and/or the two-electron-oxidized species [SPL-sulfenic acid (SPL-SOH)]. In those studies, glutathione (GSH) was found to attenuate both SPL-mediated P450 loss as well as polar metabolite formation by approximately 40%. We have now reexamined this in greater detail and report that it is due to GSH trapping of an electrophilic oxidized SPL species to form an adduct that we have isolated and unambiguously characterized by mass spectral analyses as the glutathionyl-SPL adduct (SPL-SSG). Moreover, we have found not only that rat liver microsomal formation of this adduct is enhanced at pH 9.0, the pH optimum for flavin-containing monooxygenase (FMO), but also that such adduct formation was indeed efficiently catalyzed by purified hog liver FMO. Because FMO oxidations of thiols are thought to entail a two-electron process to form the corresponding sulfenic acids, we infer that such a SPL-SSG adduct most likely reflects FMO-catalyzed oxidation of SPL to SPL-SOH, which on leaving the FMO active site is then trapped by GSH.(ABSTRACT TRUNCATED AT 250 WORDS)
The metabolism of benzimidazoline-2-thione (I) and the 1-methyl (II) and 1,3-dimethyl (III) derivatives was studied to elucidate the mechanisms of hepatic oxidation for this class of thionosulfur-containing xenobiotics. NADPH-dependent metabolism of I, II, and III to the corresponding benzimidazoles Ia, IIa, and IIIa, respectively, was observed in dexamethasone-pretreated rat hepatic microsomes. III was the only thiocarbamide converted to an amide metabolite (IIIb). The effects of heat and 1-aminobenzotriazole pretreatment suggested that rat hepatic microsomal metabolism of I was catalyzed by the flavin-containing monoxygenase (FMO) only and that of II and III by both FMO and cytochrome P450 isozymes (P450). Addition of 5.0 mM glutathione (GSH) blocked formation of all metabolites from I, II, and III. Highly purified hog liver FMO catalyzed formation of all metabolites observed in rat hepatic microsomal systems. Incubation of III with either rat liver microsomes or with highly purified hog liver FMO in the presence of [18O]water led to ca. 50% incorporation of [18O] into IIIb. When [18O] molecular oxygen was used, ca. 8% incorporation of [18O] into IIIb was observed. Highly purified hog liver FMO also converted I-III to chemically reactive species that covalently bound to protein thiols. In the presence of hog liver FMO, the covalent binding pattern of radiolabeled I-III to bovine serum albumin was essentially identical to that observed for rat hepatic microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)
Sulfonamide antibiotics, typified by sulfamethazine (SMZ), are widely used in veterinary practice. Sulfonamide residues in milk and meat products are of regulatory concern since SMZ is a thyroid carcinogen in rodents and sulfonamide-induced hypersensitivity reactions, including hypothyroidism, have been reported in humans. SMZ and other primary arylamines inhibited iodination reactions catalyzed by thyroid peroxidase (TPO) and the closely related lactoperoxidase (LPO). Inhibition of LPO-catalyzed triiodide ion formation by SMZ and other primary arylamines was complex as both apparent Km and Vmax values were affected, but consistent with a rapid equilibrium binding mechanism. The apparent Ki for SMZ inhibition of TPO- and LPO-catalyzed iodide ion oxidation was approximately 0.42 and 0.11 mM, respectively. The corresponding Ki values for a series of para-substituted anilines correlated with the ease of one-electron N-oxidation as measured by ionization potentials determined from semiempirical molecular orbital calculations. The aniline derivatives containing electron-donating substituents (e.g., p-CH3, p-OEt, p-Cl) were converted by LPO to colored products characteristic of one-electron oxidation. However, sulfonamides were not consumed in such reactions nor were any N-oxygenated derivatives formed in the absence of ascorbate (e.g., hydroxylamino, nitroso, nitro, azoxy). These observations suggest that the primary mechanism for sulfonamide-induced hypothyroidism is reversible inhibition of TPO-mediated thyroid hormone synthesis and not the formation and covalent binding of reactive N-oxygenated metabolites. These results are consistent with a hormonal mechanism for SMZ-induced thyroid carcinogenesis mediated by thyroid-stimulating hormone (TSH).(ABSTRACT TRUNCATED AT 250 WORDS)