The human medium chain acyl-CoA dehydrogenase gene promoter consists of a complex arrangement of nuclear receptor response elements and Sp1 binding sites.
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Acyl-CoA Dehydrogenase↗
Biomedical subjects
D P Kelly
Publications and source records attributed to D P Kelly.
The human medium chain Acyl-CoA dehydrogenase gene promoter consists of a complex arrangement of nuclear receptor response elements and Sp1 binding sites.
Expression of the gene encoding the mitochondrial fatty acid. beta-oxidation enzyme, medium-chain acyl-CoA dehydrogenase (MCAD), is regulated among tissues during development and in response to alterations in substrate availability. To identify and characterize cis-acting MCAD gene promoter regulatory elements and corresponding transcription factors, DNA-protein binding studies and mammalian cell transfection analyses were performed with hjman MCAD gene promoter fragments. DNA:protein binding studies with nuclear protein extracts prepared from hepatoma G2 cells, 3T3 fibroblasts, or Y-1 adrenal tumor cells identified three sequences (nuclear receptor response element 1 or NRRE-1, NRRE-2, and NRRE-3) that bind orphan members of the steroid/thyroid nuclear receptor superfamily including chicken ovalbumin upstream promoter transcription factor and steroidogenic factor 1. Sp1 binding sites (A-C) were identified in close proximity to each of the NRREs. NRRE-3 conferred cell line-specific transcriptional repression by interacting with chicken ovalbumin upstream promoter transcription factor or activation via steroidogenic factor 1. In contrast, the Sp1 binding site A behaved as a transcriptional activator in all cell lines examined. We propose that multiple nuclear receptor transcription factors interact with MCAD gene promoter elements to differentially regulate transcription among a variety of cell types.
3T3 Cells↗
Two-year inhalation toxicity study in rats with hydrochlorofluorocarbon 123.
The potential chronic toxicity and oncogenicity of hydrochlorofluorocarbon 123 (HCFC-123) was evaluated by exposing male and female rats to 0, 300, 1000, or 5000 ppm HCFC-123 for 6 hr/day, 5 days/week, for 2 years. Clinical pathology was evaluated at 6, 12, 18, and 24 months. An interim termination and measurements of hepatic cell proliferation and beta-oxidation activity were conducted at 12 months. The terminal euthanization occurred at 24 months. Males and females exposed to 5000 ppm and females exposed to 300 or 1000 ppm had lower body weights and body weight gains. Serum triglyceride and glucose concentrations were significantly decreased at all exposure concentrations in both sexes. Serum cholesterol was also lower in 300, 1000, and 5000 ppm females and in 5000 ppm males. Alterations in serum protein concentrations occurred at 300, 1000, and 5000 ppm. Survival was higher in 1000 and 5000 ppm males and females. At 24 months, increased relative liver weight occurred in 5000 ppm males, and decreased absolute kidney weight occurred in 5000 ppm males and in 1000 and 5000 ppm females. Benign hepatocellular adenomas were increased in 5000 ppm males and in all test groups of females. Hepatic cholangiofibromas were also increased in 5000 ppm females. Pancreatic acinar cell adenomas were increased in all test groups of males, and acinar cell hyperplasia was increased in the 1000 and 5000 ppm males and females. Benign testicular interstitial adenomas and focal interstitial cell hyperplasia were also increased in all male test groups compared to controls. Diffuse retinal atrophy was increased in all male and female test groups, but it was considered to be an indirect compound-related effect. Hepatic beta-oxidation activity (peroxisome proliferation) was higher in 300, 1000 and 5000 ppm males and 1000 and 5000 ppm females. Compound-related differences in the rate of hepatic cell proliferation were not observed at any exposure concentration. Decreased incidences of a variety of age-related lesions occurred at 1000 and 5000 ppm.
Administration, Inhalation↗
Sudden cardiac death in hypertrophic cardiomyopathy. Variability in phenotypic expression of beta-myosin heavy chain mutations.
BACKGROUND: Recent identification of mutations in the beta-myosin heavy chain gene (MYH7), a major responsible gene for HCM, has provided the opportunity to characterize genotype-phenotype correlation in HCM families. In this study we analysed the phenotypic expression of two beta-myosin heavy chain (beta MHC) mutations in three unrelated HCM families. METHODS: Living individuals from three unrelated HCM families (Families 1, 2, and 3) were screened by history, physical examination, electrocardiography, and two-dimensional echocardiography. Blood was collected from all individuals for DNA extraction. Polymerase chain reaction (PCR), restriction endonuclease digestion and chemical cleavage were utilized for detection of mutations. All mutations were confirmed by sequence analysis. RESULTS: Identification of mutations: A missense mutation in exon 13 of the beta MHC gene (Arg403 Gln) was detected in HCM patients from Families 1 and 2. PCR amplification of the exon 13 DNA, followed by Ddel digestion of the PCR product and gel electrophoresis, showed two fragments of 84 and 70 bp in normal individuals and four fragments of 84, 70, 52 and 32 bp in HCM patients. Sequence analysis showed substitution of an adenine for guanine at coding position 1208. In Family 3, a missense mutation in exon 16 of the beta MHC gene (Val606 Met) was detected in HCM patients. Chemical cleavage of the PCR products showed an uncleaved product of 337 bp in the normal individuals, while in the affected individuals, in addition to the uncleaved product, a 90 bp cleaved product was also detected, indicating the presence of a mismatch in one allele. Sequence analysis showed substitution of an adenine for guanine in coding position 1817. CLINICAL CHARACTERISTICS: Seven members of Family 1 had HCM, of whom five are alive. One patient died from sudden cardiac death (SCD) and another from recurrent cerebral emboli. In Family 2, 15 individuals had HCM of whom nine have died, seven from SCD. The mean age at the time of SCD was 33 years. The third family is comprised of 11 affected individuals and one obligate carrier, of whom one patient died at age 17 from progressive heart failure. Two additional individuals in this family have also succumbed to SCD to age 60. A variety of clinical and echocardiographic manifestations of HCM were present in each family. Logrank test of Kaplan-Meier survival curves indicates that Arg403 Gln mutation was associated with a poor prognosis in HCM families as compared to Val606 Met (P = 0.034). CONCLUSIONS: beta MHC mutations despite showing variable clinical and echocardiographic manifestations of HCM are predictors of survival in HCM families.
Adolescent↗
Insulin-like growth factor (IGF)-I and IGF-binding protein 3 during the first year in term and preterm infants.
Plasma IGF-I and IGF-binding protein 3 (IGFBP-3) were determined by radioimmunoassay in term infants (n = 51) at 0, 2, 4, 6, and 12 mo and preterm infants (n = 51) at -3 (28.4 wk postconceptional age), -2, -1.5, -1, 0, 2, 4, 6, 9, and 12 mo from expected term. The effects of gestational age at birth (term or preterm) and study age were determined by repeated measures analysis of variance and Fisher's least squares difference. In preterm infants, IGF-I increased between -3 and 2 mo from 0.75 to 10.4 nM, decreased between 2 and 9 mo to 7.3 nM, and increased again between 9 and 12 mo to 10.1 nM (p < 0.0001), whereas IGFBP-3 increased relatively little from -3 to 0 mo (14.2 to 30.2 nM, p < 0.05) and plateaued from 2 to 12 mo (49.8 to 62.3 nM). At 0 mo, IGF-I and IGFBP-3 were the same in term and preterm infants, but preterm infants had higher IGF-I from 2 through 12 mo (p < 0.05), higher IGFBP-3 at 2 and 4 mo (p < 0.05), and lower IGFBP-3 at 12 mo (p < 0.05). IGF-I and IGFBP-3 were correlated at most ages. First year IGF-I and IGFBP-3 are influenced by study age and by gestational age, even after adjusting for early birth.
Carrier Proteins↗
The peroxisome proliferator-activated receptor regulates mitochondrial fatty acid oxidative enzyme gene expression.
Medium-chain acyl-CoA dehydrogenase (MCAD) catalyzes a pivotal reaction in mitochondrial fatty acid (FA) beta-oxidation. To examine the potential role of FAs and their metabolites in the regulation of MCAD gene expression, we measured MCAD mRNA levels in animals fed inhibitors of mitochondrial long-chain FA import. Administration of carnitine palmitoyltransferase I inhibitors to mice or rats resulted in tissue-limited increases in steady-state MCAD mRNA levels. HepG2 cell cotransfection experiments with MCAD promoter reporter plasmids demonstrated that this was a transcriptional effect mediated by the peroxisome proliferator-activated receptor (PPAR). The activity mapped to a nuclear receptor response element that functioned in a heterologous promoter context and specifically bound immunoreactive PPAR in rat hepatic nuclear extracts, confirming an in vivo interaction. PPAR-mediated transactions of this promoter and element were also induced by exogenously added FA and fibric acid derivatives. Induction of PPAR transactivation by perturbation of this discrete metabolic step is unusual and indicates that intracellular FA metabolites that accumulate during such inhibition can regulate MCAD expression and are likely candidates for PPAR ligand(s). These results dictate an expanded role for the PPAR in the regulation of FA metabolism.
Acyl-CoA Dehydrogenase↗
Comparative studies of UV-induced DNA cleavage by structural isomers of an iodinated DNA ligand.
PURPOSE: To evaluate the importance of the position of the halogen atom in iodinated DNA-binding bibenzimidazoles, with respect to sensitization of UV-A-induced DNA breakage. METHODS AND MATERIALS: Three analogues of iodoHoechst 33258, denoted ortho-, meta- and paraiodoHoechst, according to the site of iodine substitution, were synthesized. Plasmid DNA (pBR322) was used to assay UV-A-induced DNA single-strand breaks (ssbs). The location of the sites of strand breakage was determined by DNA sequencing gel analysis, using a 32P-endlabelled oligoDNA with a single binding site for the ligands. RESULTS: A clear trend in decreasing activity of sensitization of UV-induced DNA ssbs was established: ortho- > meta-, para- > iodoHoechst 33258. The sequencing gel studies showed that orthoiodoHoechst was distinct from the other three compounds, with respect to the sites of DNA strand breakage and the chemistry of the cleavage reaction. CONCLUSION: The position of iodine substitution in iodinated bibenzimidazoles determines the location of the carbon-centered radical on the ligand in the minor groove of DNA. DNA strand cleavage is mediated by abstraction of a nearby deoxyribosyl H-atom. Hence, the position of the radical species determines: which deoxyribosyl group is attacked (i.e., site of cleavage relative to the ligand binding site); which H-atom is abstracted, more specifically which of the five deoxyribosyl carbons is involved (i.e., the chemistry of the cleavage reaction), and the stereochemistry of the transition state for the H-atom abstraction (and hence the efficiency or extent of strand breakage). The ortho-compound represents the best example to date of iodinated DNA ligands designed as potential radiation sensitizers, as an extension of the well-established sensitization by halogenated DNA precursors.
Base Sequence↗
Inherited cardiomyopathies.
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Cardiomyopathies↗
Subchronic inhalation toxicity studies with hydrochlorofluorocarbon 123 (HCFC 123).
Hydrochlorofluorocarbon 123 (HCFC 123) is one of the chemicals being considered as a replacement for the chlorofluorocarbons. Four subchronic inhalation toxicity studies from 1 to 3 months in duration have been conducted with HCFC 123. One study utilized rats and dogs, while the others were limited to rats only. The exposure levels have ranged from 300 ppm up to 20,000 ppm. Although the studies were conducted over a 14-year period, the results were consistent. In all studies, increases in liver weights were seen at 1000 ppm and above; additionally, one showed this effect at 500 ppm. Histopathological findings were minimal, consisting primarily of focal necrosis in the liver of the dogs at 10,000 ppm. Induction of peroxisomal activity, lowering of serum cholesterol and triglyceride levels, and an increase in urinary fluoride levels were also seen. The 4-hr LC50 in the rat has been reported as 35,000 ppm. At 20,000 ppm for 6 hr, the total daily dose on a concentration times time basis is almost equal to the LC50, yet, in the 4-week study, with 20 exposures at this level, there was no mortality or even marked signs of toxicity. There appeared to be no evidence for cumulative toxicity from multiple exposures in these studies. Overall, HCFC 123 appears to have a low level of toxicity by the inhalation route.
Administration, Inhalation↗
Biological production and consumption of gaseous organic sulphur compounds.
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Bacteria↗
Comparative studies of UV-induced DNA cleavage by analogues of iodoHoechst 33258.
Following the earlier demonstration that iodo-Hoechst 33258 sensitizes DNA and cells to UVA, presumably mediated by formation of a carbon-centred radical on the ligand upon dehalogenation, three isomeric analogues of iodo-Hoechst 33258 have now been studied. The isomers differ in the location of the iodine atom in the phenyl ring of the ligand, relative to the site of attachment of the bibenzimidazole moiety, and are accordingly denoted ortho-, meta- and para-iodoHoechst. Comparison of the ligands with respect to induction of DNA ssb in pBR322 DNA revealed a wide range of activity; (D37's vary by a factor of 37), decreasing in the order: ortho- > meta- and para- > iodoHoechst 33258. Preliminary dehalogenation studies suggest that the higher activity of the ortho isomer results more from increased cross-section for dehalogenation than from increased efficiency of strand breakage per dehalogenation event. However, the chemistry of strand breakage by the ortho-isomer is distinctive, and tentatively assigned to initial attack at the 1'-deoxyribosyl carbon; the other two isomers, like iodo-Hoechst 33258, attack the 5'-carbon. The results are discussed in terms of the spectrum of DNA strand breakage chemistry associated with ionizing radiation, and the potential of DNA strand breaking agents such as the iodoHoechst compounds to study the chemical and biological consequences of the different subclasses of initial DNA damage.
Base Sequence↗
Identification and sequence analysis of the soxB gene essential for sulfur oxidation of Paracoccus denitrificans GB17.
The coding region for lithotrophic sulfur oxidation (Sox) in Paracoccus denitrificans GB17 was identified by isolation of a transposon Tn5-mob mutant with a Sox- phenotype (strain TP19). The corresponding wild-type region was cloned previously (G. Mittenhuber, K. Sonomoto, M. Egert, and C. G. Friedrich, J. Bacteriol. 173:7340-7344, 1991). Sequence analysis of a 2.5-kb subclone that complemented strain TP19 revealed that Tn5-mob was inserted into a coding region for a 553-amino-acid polypeptide named SoxB. This polypeptide had an M(r) of 60.573, including a possible signal peptide. The function of the SoxB protein of P. denitrificans GB17 appeared to be identical to that of enzyme B of the thiosulfate-oxidizing enzyme system of Thiobacillus versutus. The amino acid compositions of the two proteins were identical, and the amino acid sequences of three internal peptides of enzyme B as determined by Edman degradation were identical to corresponding sequences of the deduced SoxB protein of P. denitrificans GB17.
Amino Acid Sequence↗
A pleiotropic element in the medium-chain acyl coenzyme A dehydrogenase gene promoter mediates transcriptional regulation by multiple nuclear receptor transcription factors and defines novel receptor-DNA binding motifs.
We previously identified a complex regulatory element in the medium-chain acyl coenzyme A dehydrogenase gene promoter that confers transcriptional regulation by the retinoid receptors RAR and RXR and the orphan nuclear receptor HNF-4. In this study we demonstrate a trans-repressing regulatory function for the orphan receptor COUP-TF at this same nuclear receptor response element (NRRE-1). The transcriptional regulatory properties and receptor binding sequences of each nuclear receptor response element within NRRE-1 are also characterized. NRRE-1 consists of four potential nuclear hormone receptor hexamer binding sites, arranged as [<--1-(n)s-2-->-3-->(n)4<--4], three of which are used in alternative pairwise binding by COUP-TF and HNF-4 homodimers and by RAR-RXR heterodimers, as demonstrated by mobility shift assays and methylation interference analysis. Binding and transactivation studies with mutant NRRE-1 elements confirmed the existence of distinct retinoid, COUP-TF, and HNF-4 response elements that define novel receptor binding motifs: COUP-TF homodimers bound sites 1 and 3 (two hexamer repeat sequences arranged as an everted imperfect repeat separated by 14 bp or ER14), RAR-RXR heterodimers bound sites 1 and 2 (ER8), and HNF-4 homodimers bound sites 2 and 3 (imperfect DR0). Mixing cotransfection experiments demonstrated that the nuclear receptor dimers compete at NRRE-1 to modulate constitutive and ligand-mediated transcriptional activity. These data suggest a mechanism for the transcriptional modulation of genes encoding enzymes involved in cellular metabolism.
Acyl-CoA Dehydrogenase↗
Current concepts of the molecular basis of heart failure.
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Animals↗
Hepatocyte nuclear factor-4 activates medium chain acyl-CoA dehydrogenase gene transcription by interacting with a complex regulatory element.
We have recently identified a complex transcriptional regulatory element in the medium chain acyl-CoA dehydrogenase (MCAD) gene promoter region that confers response to retinoids through interaction with receptors for all-trans-retinoic acid (RARs) and 9-cis-retinoic acid (RXRs) (Raisher, B. D., Gulick, T., Zhang, Z., Strauss, A. W., Moore, D. D., and Kelly, D. P. (1992) J. Biol. Chem. 267, 20264-20269). We examined the interaction of this element (RAREMCAD) with hepatocyte nuclear factor-4 (HNF-4), an orphan receptor with a tissue expression pattern similar to that of MCAD. Electrophoretic mobility shift assays and cotransfection experiments showed that HNF-4 binds with high affinity to RAREMCAD to activate transcription by an RXR-independent mechanism. Mutational analysis revealed that the MCAD HNF-4 response element consists of an imperfect direct repeat homologous to the consensus sequence for binding to the thyroid receptor/RAR/RXR subgroup of receptors and that distinct sequence requirements dictate HNF-4 binding and transactivation. Mobility shift assays with anti-HNF-4 antiserum demonstrated that the MCAD HNF-4 response element binds endogenous rat liver HNF-4 supporting its role in the regulation of MCAD gene expression in vivo. Thus, HNF-4 activates MCAD gene transcription via a complex regulatory element, the architecture of which carries important implications for the structure of HNF-4 response elements in general.
3T3 Cells↗
Translocation of particle-laden alveolar macrophages and intra-alveolar granuloma formation in rats exposed to Ludox colloidal amorphous silica by inhalation.
Rats were exposed to Ludox colloidal amorphous silica at concentrations of 0, 10, 50 and 150 mg/m3 for 6 h/day, 5 days/week for 4 weeks. The rats were killed after 4 weeks exposure and at 10 days or 3 months post-exposure (PE). Dose-related pulmonary lesions were observed at 50 and 150 mg/m3, but not at 10 mg/m3. Inhaled particles were mostly phagocytized by alveolar macrophages (AMs) in the alveolar duct region and a few free particles were found in Type I pneumocytes in the alveoli. Particle-laden AMs directly penetrated into the bronchiolar interstitium from alveoli and accumulated in bronchus-associated lymphoid tissue (BALT), peribronchiolar, or perivascular interstitium. The particle-laden AMs in the interstitium further migrated into the peribronchial or perivascular lymphatics and accumulated in the tracheo-bronchial lymph nodes (TBLN). Some particle-laden AMs in the BALT transmigrated directly into bronchial lumen through the epithelium. The transmigrated particle laden-AMs in the TBLN were indistinguishable from those seen in the alveoli. They were characterized by slender cytoplasmic processes, phagosomes, myelin figures (alveolar surfactant), cholesterol clefts and lipid droplets. Many migrated particle-laden AMs were necrotic and released particles in the TBLN. The released particles were phagocytized by histiocytes and formed histiocytic granulomas. Silicotic granulomas were initially formed in alveoli with particle-laden AMs and proliferating epithelioid cells. Subsequently, the granulomas were incorporated into the interstitium. However, perivascular silicotic granulomas were developed with accumulation of transmigrated particle-laden AMs and minimal collagenized fibers at 3 months PE. There was no alveolar lipo-proteinosis as seen in crystalline silica exposure.
Administration, Inhalation↗
Detection and assessment by positron emission tomography of a genetically determined defect in myocardial fatty acid utilization (long-chain acyl-CoA dehydrogenase deficiency).
Genetic defects in fatty acid oxidation are important, inherited causes of cardiomyopathy, skeletal myopathies, and childhood sudden death. The clinical manifestations and their severity vary widely among affected subjects and different age groups. Although measurement of serum and urinary fatty acid intermediary metabolites and enzymatic assays establish the diagnosis of a defect in fatty acid oxidation, they do not predict the specific clinical manifestations nor their severity in a given subject. To determine whether impaired myocardial fatty acid utilization, indicative of cardiac phenotypic expression of a specific genetic abnormality in fatty acid oxidation, can be detected, cardiac positron emission tomography with the metabolic tracers carbon-11-labeled palmitate and acetate was performed in 6 patients with long-chain acyl-CoA dehydrogenase (ACD) deficiency and in 9 control subjects. The myocardial extraction of both tracers was similar in patients and controls. The rate of clearance of palmitate from myocardium was significantly prolonged in patients compared with that in control subjects (0.022 +/- 0.012 vs 0.061 +/- 0.033 min-1; p < 0.025), indicative of a decreased rate of oxidation of long-chain fatty acids. Furthermore, the extent of diminution of clearance of palmitate, quantified in terms of the rate of clearance for palmitate divided by that for acetate (to correct for individual differences in overall mitochondrial oxidative metabolic flux), correlated with the clinical severity of the long-chain ACD deficiency. Accordingly, noninvasive evaluation with positron emission tomography may not only facilitate diagnosis, but also enable assessment of the pathogenetic impact and effects of therapeutic interventions in the hearts of subjects with specific, inherited defects in fatty acid oxidative metabolism.
Acetates↗
Attention deficits in children and adolescents with hearing loss. A survey.
OBJECTIVES: To determine the prevalence of reported attention deficits in a sample of children who are deaf or hard of hearing and to examine associated variables. DESIGN: Cross-sectional survey. SETTING: Residential school for the deaf. PARTICIPANTS: All students attending the school (n = 238; median age, 16.0 years; range, 4 to 21 years). Subjects were subgrouped by cause of deafness for further analyses. INTERVENTION: None. MEASUREMENTS/MAIN RESULTS: School personnel completed questionnaires that are commonly used in the assessment of attention problems. Teachers completed the Attention Deficit Disorder With Hyperactivity (ADD-H) Comprehensive Teacher Rating Scale (ACTeRS) and an abbreviated version of the Attention-Activity section of the ANSER (Aggregate Neurobehavioral Student Health and Education Review) System Questionnaires. Dormitory supervisors rated students on the Conners' Parent Rating Scale and the ANSER System Questionnaires. The mean factor scores for the total sample of deaf and hard-of-hearing students on the Attention and Hyperactivity scales of the ACTeRS and Conners' questionnaires did not differ from the normative data. The 64 students with hereditary deafness received significantly better ratings than the 75 with an acquired cause of deafness such as bacterial meningitis or congenital rubella. Raw scores on the ACTeRS were converted to percentile equivalents based on the normative sample. On the Attention Scale, 38.7% of those with acquired deafness were rated below the 20th percentile (indicative of problems); only 14.1% of the students with hereditary deafness were in this range (P = .002). CONCLUSIONS: Ratings of attention for students with hearing loss, attending a residential school, did not differ from the normative data. However, the subgroup of children with acquired sensorineural deafness appear to be at increased risk for attention problems.
Adolescent↗