Biomedical subjects

D Serra

Publications and source records attributed to D Serra.

At least 19 recordsLinked to original sources

PubMed2005

Characterization of Bordetella pertussis growing as biofilm by chemical analysis and FT-IR spectroscopy.

Although Bordetella pertussis, the etiologic agent of whooping cough, adheres and grows on the ciliated epithelium of the respiratory tract, it has been extensively studied only in liquid cultures. In this work, the phenotypic expression of B. pertussis in biofilm growth is described as a first approximation of events that may occur in the colonization of the host. The biofilm developed on polypropylene beads was monitored by chemical methods and Fourier transform infrared (FT-IR) spectroscopy. Analysis of cell envelopes revealed minimal differences in outer membrane protein (OMP) pattern and no variation of lipopolysaccharide (LPS) expression in biofilm compared with planktonically grown cells. Sessile cells exhibited a 2.4- to 3.0-fold higher carbohydrate/protein ratio compared with different types of planktonic cells. A 1.8-fold increased polysaccharide content with significantly increased hydrophilic characteristics was observed. FT-IR spectra of the biofilm cells showed higher intensity in the absorption bands assigned to polysaccharides (1,200-900 cm(-1) region) and vibrational modes of carboxylate groups (1,627, 1,405, and 1,373 cm(-1)) compared with the spectra of planktonic cells. In the biofilm matrix, uronic-acid-containing polysaccharides, proteins, and LPS were detected. The production of extracellular carbohydrates during biofilm growth was not associated with changes in the specific growth rate, growth phase, or oxygen limitation. It could represent an additional virulence factor that may help B. pertussis to evade host defenses.

Bacterial Adhesion↗

PubMed2005

FTY720 pharmacokinetics in mild to moderate hepatic impairment.

The influence of mild and moderate hepatic impairment on FTY720 pharmacokinetics was assessed. The authors enrolled 32 subjects consisting of 8 with mild and 8 with moderate hepatic impairment based on Child-Pugh criteria and 16 demographically matched control subjects. A single 1-mg oral dose of FTY720 was administered under fasting conditions. Blood, plasma, and urine samples were obtained over a 14-day period for measurement of FTY720 and metabolite concentrations and protein binding. Total blood lymphocyte counts and heart rate were serially monitored to assess pharmacologic responses to FTY720. Peak FTY720 blood concentrations were similar across groups. Oral clearance (CL/F) was reduced 10% in mild hepatic impairment (P = .493) and 31% in moderate hepatic impairment (P = .034). There were no significant differences in blood exposure to the hexanoic or butanoic acid metabolites among groups. The effect of FTY720 on blood lymphocytes was similar across groups, with a mean decrease of 44% from the predose value. Like-wise, the effect of FTY720 on supine heart rate was similar across groups, with a mean 13% decrease from the predose rate occurring 2 to 4 hours postdose and recovering within 1 to 2 days. Although hepatic impairment elicited changes in the disposition of FTY720, the magnitude of these changes suggests that the FTY720 dose does not need to be adjusted in mild or moderate hepatic-impaired patients.

Case-Control Studies↗

PubMed2004

Determination of phenol and o-cresol by GC/MS in a fatal poisoning case.

A fatality due to the ingestion of solution containing phenol and o-cresol is described. The pathological findings were typical of acute substantial poisoning. Blood, urine and stomach content were obtained during post mortem examinations. Phenol and o-cresol were identified using GC/MS. The extractions from autopsy materials were obtained as follows: by gel permeation with cyclohexane/dichloromethane from stomach content, by solid phase extraction (SPE) from urine and by deproteinization with acetonitrile from blood. The phenol and o-cresol concentrations in the samples were found, respectively, as follows: 115.0 and 5.0 microg/g in the stomach contents, 58.3 and 1.9 microg/ml in the blood, 3.3 and 20.5 microg/ml in the urine. Distributions of phenol in fatal poisonings have been reported, but, usually, colorimetry was used as the analytical method and it cannot exclude the interference of other phenolic compounds.

Adult↗

PubMed2004

Recombinant adenovirus vectors activate the alternative complement pathway, leading to the binding of human complement protein C3 independent of anti-ad antibodies.

Recombinant adenoviruses are one of the most common gene transfer vectors utilized in human clinical trials, but it is also clear that systemic administration of this virus will be met by host innate and adaptive antiviral immune responses. One element of innate immunity is the complement system, a group of proteins that has evolved to rapidly recognize foreign microbes and viruses and to clear them from the circulatory system prior to their gaining entry to vulnerable host cells. Excessive complement activation can initiate or propagate a number of deleterious inflammatory responses, by release of potent cytokines and anaphylatoxins and/or by direct cellular toxicity. These reactions can progress rapidly and are factors important in serious complications, including the systemic inflammatory response syndrome and the adult respiratory distress syndrome.

Adenoviridae↗

PubMed2004

Improved efficacy of gene therapy approaches for Pompe disease using a new, immune-deficient GSD-II mouse model.

Glycogen storage disease type II (GSD-II) is a lysosomal storage disorder in which the lack of human acid-alpha glucosidase (hGAA) activity results in massive accumulations of glycogen in cardiac and skeletal muscle fibers. Affected individuals die of cardiorespiratory failure secondary to the skeletal and/or cardiac muscle involvement. Recombinant hGAA enzyme replacement therapy (ERT) is currently in clinical trials and, although promising, ERT may be limited by large-scale production issues and/or the need for frequent infusions. These limitations could be circumvented or augmented by gene therapy strategies. Previous findings in our lab demonstrated that hepatic targeting of a modified adenovirus vector expressing human GAA was able to correct the glycogen accumulation in multiple affected muscles in the GAA-KO mice, by virtue of high-level, hepatic secretion of hGAA. However, although the vector persisted and expressed hGAA for 6 months in the liver, plasma hGAA was not detectable beyond 10 dpi (days postinjection), and reaccumulation of glycogen was observed. Two possibilities may have contributed to this phenomenon, the shut down of the CMV promoter and/or the onset of high levels of anti-hGAA antibodies. In order to test these and other possibilities, we have now developed an immune-deficient mouse model of GSD-II by interbreeding GAA-KO mice with severe combined immune-deficient (SCID) mice, generating double knockout, GAA-KO/SCID mice. In this new mouse model, we evaluated the efficacy of an [E1-, polymerase-] AdhGAA vector, in the absence of anti-hGAA antibody responses. After intravenous injection, GAA detection in the plasma was prolonged for at least 6 months secondary to the lack of anti-hGAA antibody production in all of the treated mice. GAA-KO/SCID mice treated with high doses of viral vector demonstrated longer durations of glycogen correction in both skeletal and cardiac muscles, relative to mice injected with lower doses of the vector. Notably, within 2 weeks of vector injection, muscle strength and coordination was normalized, and the improved muscle function persisted for at least 6 months. In summary, this new mouse model of GSD-II now makes it possible to assess the full potential for efficacy of any GAA-expressing vector (and/or ERT) contemplated for use in GSD-II gene therapy, without the negative influence that anti-hGAA antibodies entail.

Animals↗

PubMed2003

Liver toxicities typically induced by first-generation adenoviral vectors can be reduced by use of E1, E2b-deleted adenoviral vectors.

Adenoviral vectors from which the E1 region has been deleted ([E1(-)] Ad) are known to induce strong immune responses after systemic delivery. In this study we have evaluated liver toxicities in mice after intravenous injection with high doses of [E1(-)] or modified [E1(-), E2b(-)] Ad vectors (both expressing the bacterial beta-galactosidase [lacZ] marker gene) in C57BL/6, BALB/c, and SCID mice. Our data demonstrate a marked reduction in maximal liver toxicities and pathologies (typically noted at 21 days postinjection) with the use of the [E1(-), E2b(-)] modified vector in all strains of mice tested. Our data also demonstrated that despite the use of the [E1(-), E2b(-)] Ad vector, significant liver toxicities were still observed. To address this issue and the fact that the lacZ gene was perceived as a foreign antigen in the immune-competent C57BL/6 and BALB/c mice, we similarly injected mice tolerant of LacZ (lacZ-TG). In contrast to our studies in C57BL/6 and BALB/c mice, LacZ-TG mice exhibited virtually no evidence of hepatotoxicity after intravenous injection with the [E1(-), E2b(-)] vector, in contrast to use of the [E1(-)] Ad vector. Our results demonstrate that the [E1(-), E2b(-)] Ad vector class can reduce liver toxicities typically ascribed to Ad vector-mediated gene transfer after transfer of a highly immunogenic or foreign gene, whereas transfer of a transgene that is perceived as nonforeign by the host can be delivered with virtually no evidence of toxicity. On the basis of a careful review of the literature, these improvements in vector safety rival those noted with other, more significantly modified Ad vectors described to date.

Adenoviridae↗

PubMed2003

Multiple muscles in the AMD quail can be "cross-corrected" of pathologic glycogen accumulation after intravenous injection of an [E1-, polymerase-] adenovirus vector encoding human acid-alpha-glucosidase.

BACKGROUND: Previously, in murine models of acid maltase deficiency (AMD), we demonstrated that intravenous administration of an improved adenovirus (Ad) vector encoding human acid alpha glucosidase (hGAA) resulted in liver transduction, followed by high-level hepatocyte-mediated secretion of hGAA into the plasma space. The hGAA secreted by the liver was taken up and targeted to muscle cell lysosomes. The levels of hGAA achieved by this approach resulted in clearance of lysosomal glycogen accumulations; in some muscle tissues the effect was prolonged (>6 months). We next wished to demonstrate whether this approach could be generalized across divergent species. To accomplish this goal, we determined whether a similar approach would also result in efficacy, but in a quail model of AMD. METHODS: An [E1-, E2b-]Ad vector encoding hGAA was intravenously injected into AMD quails. At several time points thereafter, plasma, liver, and multiple muscle tissues were assayed for evidence of hGAA gene expression, liver-mediated hGAA secretion, uptake of hGAA by skeletal muscles, and evidence of glycogen correction in AMD skeletal muscles. These results were compared with those obtained from mock-injected AMD or wild-type quails. RESULTS: Intravenous [E1-, E2b-]Ad/hGAA vector injection resulted in high-level liver transduction and hepatic secretion of precursor forms of hGAA. The hepatically secreted hGAA was found to not only be efficiently taken up by cardiac and skeletal muscles, but was also proteolytically cleaved and processed equivalently to the quail-GAA protein detected in wild-type quails. The observations suggest that the signals regulating muscle cell uptake (but not proteolytic cleavage) of lysosomal enzymes are conserved and recognized across divergent species of vertebrates. Importantly, once localized to skeletal muscle lysosomes, the hGAA was able to effectively clear the glycogen accumulations present in AMD quail muscles. CONCLUSIONS: Adenovirus-mediated transduction of the hGAA gene, followed by hepatic secretion, uptake, and cross-correction of the pathologic glycogen accumulation noted in multiple muscles of both the AMD mouse and AMD quail, adds support to the notion that gene transfer strategies (Ad-mediated or other agents) targeting liver tissues with the hGAA gene are likely to be highly efficacious in humans affected by AMD.

Adenoviridae↗

PubMed2001

Structural model of the catalytic core of carnitine palmitoyltransferase I and carnitine octanoyltransferase (COT): mutation of CPT I histidine 473 and alanine 381 and COT alanine 238 impairs the catalytic activity.

Carnitine palmitoyltransferase I (CPT I) and carnitine octanoyltransferase (COT) catalyze the conversion of long- and medium-chain acyl-CoA to acylcarnitines in the presence of carnitine. We propose a common three-dimensional structural model for the catalytic domain of both, based on fold identification for 200 amino acids surrounding the active site through a threading approach. The model is based on the three-dimensional structure of the rat enoyl-CoA hydratase, established by x-ray diffraction analysis. The study shows that the structural model of 200 amino acids of the catalytic site is practically identical in CPT I and COT with identical distribution of 4 beta-sheets and 6 alpha-helices. Functional analysis of the model was done by site-directed mutagenesis. When the critical histidine residue 473 in CPT I (327 in COT), localized in the acyl-CoA pocket in the model, was mutated to alanine, the catalytic activity was abolished. Mutation of the conserved alanine residue to aspartic acid, A381D (in CPT I) and A238D (in COT), which are 92/89 amino acids far from the catalytic histidine, respectively (but very close to the acyl-CoA pocket in the structural model), decreased the activity by 86 and 80%, respectively. The K(m) for acyl-CoA increased 6-8-fold, whereas the K(m) for carnitine hardly changed. The inhibition of the mutant CPT I by malonyl-CoA was not altered. The structural model explains the loss of activity reported for the CPT I mutations R451A, W452A, D454G, W391A, del R395, P479L, and L484P, all of which occur in or near the modeled catalytic domain.

Alanine↗

PubMed2001

Localization of an exonic splicing enhancer responsible for mammalian natural trans-splicing.

Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which may lead to the synthesis of two proteins. Generation of the three COT transcripts in rat does not depend on sex, development, fat feeding, the inclusion of the peroxisome proliferator diethylhexyl phthalate in the diet or hyperinsulinemia. In addition, trans-splicing was not detected in COT of other mammals, such as human, pig, cow and mouse, or in Cos7 cells from monkey. Rat COT exon 2 contains two purine-rich sequences. Mutation of the rat COT exon 2 upstream box does not affect the trans-splicing in vitro between two truncated constructs containing exon 2 and its adjacent intron boundaries. In contrast, mutation of the downstream box from the rat sequence (GAAGAAG) to a random sequence or the sequence observed in the other mammals (AAAAAAA) decreased trans-splicing in vitro. In contrast, mutation of the AAAAAAA box of human COT exon 2 to GAAGAAG increases trans-splicing. Heterologous reactions between COT exon 2 from rat and human do not produce trans-splicing. HeLa cells transfected with minigenes of rat COT sequences produced cis- and trans-spliced bands. Mutation of the GAAGAAG box to AAAAAAA abolished trans-splicing and decreased cis-splicing in vivo. We conclude that GAAGAAG is an exonic splicing enhancer that could induce natural trans-splicing in rat COT.

Alternative Splicing↗

PubMed2001

Modulation in vitro of H-ras oncogene expression by trans-splicing.

In man, activated N-, K- and H-ras oncogenes have been found in around 30% of the solid tumours tested. An exon known as IDX, which has been described previously and is located between exon 3 and exon 4A of the c-H-ras pre-mRNA, allows an alternative splicing process that results in the synthesis of the mRNA of a putative protein named p19. It has been suggested that this alternative pathway is less tumorigenic than that which results in the activation of p21. We have used the mammalian trans-splicing mechanism as a tool with which to modulate this particular pre-mRNA processing to produce mRNA similar to that of mature p19 RNA. The E4A exon of the activated H-ras gene was found to be a good target for external trans-splicing. We reprogrammed the rat carnitine octanoyltransferase exon 2 to specifically invade the terminal region of H-ras. Assays performed with this reprogrammed trans-exon showed that the trans-splicing product was obtained in competition with cis-splicing of the D intron of the H-ras gene, and was associated with concomitant down-modulation of D intron cis-splicing. We also found that the exon 4A of the human c-H-ras gene underwent successive trans-splicing rounds with an external exon.

Animals↗

PubMed2001

Long-term efficacy after [E1-, polymerase-] adenovirus-mediated transfer of human acid-alpha-glucosidase gene into glycogen storage disease type II knockout mice.

Glycogen storage disease type II (GSD-II) is a lethal, autosomal recessive metabolic myopathy caused by a lack of acid-alpha-glucosidase (GAA) activity in the cardiac and skeletal muscles. Absence of adequate intralysosomal GAA activity results in massive amounts of glycogen accumulation in multiple muscle groups, resulting in morbidity and mortality secondary to respiratory embarrassment and/or cardiomyopathy. In a mouse model of GSD-II, we demonstrate that infection of the murine liver with a modified adenovirus (Ad) vector encoding human GAA (hGAA) resulted in long-term persistence of the vector in liver tissues for at least 6 months. Despite both a rapid shutdown of hGAA mRNA expression from the vector, as well as the elicitation of anti-hGAA antibody responses (hGAA is a foreign antigen in this model), the hGAA secreted by the liver was taken up by all muscle groups analyzed and, remarkably, persisted in them for at least 6 months. The persistence of the protein also correlated with long-term correction of pathologic intramuscular glycogen accumulations in all muscle groups tested, but most notably the cardiac tissues, which demonstrated a significantly decreased glycogen content for at least 190 days after a single vector injection. The results suggest that gene therapy strategies may have the potential to significantly improve the clinical course for GSD-II patients.

Adenoviridae↗

PubMed2001

Developmental changes in carnitine octanoyltransferase gene expression in intestine and liver of suckling rats.

Carnitine octanoyltransferase (COT), which facilitates the transport of shortened fatty acyl-CoAs from peroxisomes to mitochondria, is expressed in the intestinal mucosa of suckling rats; its mRNA levels increase rapidly after birth, remain steady until day 15, and decrease until weaning, when basal, adult values are established, which remain unchanged thereafter. The process seems to be controlled at the transcriptional level since the developmental pattern of mRNA coincides with that of pre-mRNA values. Dam's milk may influence the intestinal expression of COT, since mRNA levels at birth are low and increase after the first lactation. Moreover, mRNA levels decrease in rats weaned on day 18 or 21. COT is also expressed in the liver of suckling rats. Hepatic COT mRNA is maximal at day 3, remains constant until day 9, and decreases thereafter; this pattern is also similar to that of pre-mRNA values. The profile of expression of COT in intestine and liver strongly resembles that of mitochondrial 3-hydroxy 3-methylglutaryl-coenzyme A synthase and carnitine palmitoyltransferase I, suggesting that analogous transcription factors modulate ketogenesis and mitochondrial and peroxisomal fatty acid oxidation.

Animals↗

PubMed2001

Post-transcriptional regulation of rat carnitine octanoyltransferase.

Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which can lead to the synthesis of two proteins. The occurrence of the three COT transcripts in rat has been found in all tissues examined and does not depend on sex, fat feeding, peroxisome proliferators or hyperinsulinaemia. Rat COT exon 2 contains a putative exonic splicing enhancer (ESE) sequence. Mutation of this ESE (GAAGAAG) to AAAAAAA decreased trans-splicing in vitro, from which it is deduced that this ESE sequence is partly responsible for the formation of the three transcripts. The protein encoded by cis-spliced mRNA of rat COT is inhibited by malonyl-CoA and etomoxir. cDNA species encoding full-length wild-type COT and one double mutant COT were expressed in Saccharomyces cerevisiae. The recombinant enzymes showed full activity towards both substrates, carnitine and decanoyl-CoA. The activity of the doubly mutated H131A/H340A enzyme was similar to that of the rat peroxisomal enzyme but was completely insensitive to malonyl-CoA and etomoxir. These results indicate that the histidine residues His-131 and His-340 are the sites responsible for the interaction of these two inhibitors, which inhibit COT by interacting with the same sites.

Acyl Coenzyme A↗

PubMed2001

Adenovirus vectors with the 100K gene deleted and their potential for multiple gene therapy applications.

The 100K protein has a number of critical roles vital for successful completion of the late phases of the adenovirus (Ad) life cycle. We hypothesized that the introduction of deletions within the 100K gene would allow for the production of a series of new classes of Ad vector, including one that is replication competent but blocked in the ability to carry out many late-phase Ad functions. Such a vector would have potential for several gene therapy applications, based upon its ability to increase the copy number of the transgene encoded by the vector (via genome replication) while decreasing the side effects associated with Ad late gene expression. To efficiently produce 100K-deleted Ad ([100K-]Ad) vectors, an E1- and 100K-complementing cell line (K-16) was successfully isolated. Transfection of an [E1-,100K-]Ad vector genome into the K-16 cells readily yielded high titers of the vector. After infection of noncomplementing cells, we demonstrated that [100K-]Ad vectors have a significantly decreased ability to express several Ad late genes. Additionally, if the E1 gene was present in the infected noncomplementing cells, [100K-]Ad vectors were capable of replicating their genomes to high copy number, but were significantly blocked in their ability to efficiently encapsidate the replicated genomes. Injection of an [E1-,100K-]Ad vector in vivo also correlated with significantly decreased hepatotoxicity, as well as prolonged vector persistence. In summary, the unique properties of [100K-]Ad vectors suggest that they may have utility in a variety of gene therapy applications.

Adenoviridae↗

PubMed2001

Impaired hepatic fatty acid oxidation in rats with short-term cholestasis: characterization and mechanism.

Rats with long-term cholestasis have reduced ketosis during starvation. Because it is unclear whether this is also the case in short-term cholestasis, we investigated hepatic fatty acid metabolism in rats with bile duct ligation for 5 days (BDL5, n = 11) or 10 days (BDL10, n = 11) and compared the findings with those made with pair-fed control rats (CON5 and CON10, n = 11). The plasma beta-hydroxybutyrate concentration was reduced in BDL rats (0.54 +/- 0.10 vs. 0.83 +/- 0.30 mM at 5 days and 0.59 +/- 0.24 vs. 0.88 +/- 0.09 mM at 10 days in BDL and control rats, respectively). In isolated liver mitochondria, state 3 oxidation rates for various substrates were not different between BDL and control rats. Production of ketone bodies from [(14)C]palmitate was reduced by 40% in mitochondria from BDL rats at both time points, whereas production of (14)CO(2) was maintained. These findings indicated intact function of the respiratory chain, Krebs cycle, and beta-oxidation and suggested impaired ketogenesis (HMG-CoA pathway). Accordingly, the formation of acetoacetate from acetyl-CoA by disrupted mitochondria was reduced in BDL rats at 5 days (2.1 +/- 1.0 vs. 4.8 +/- 1.9 nmol/min per mg protein) and at 10 days (1.7 +/- 1.0 vs. 6.2 +/- 1.9 nmol/min per mg protein). The principal defect could be localized at the rate-limiting enzyme of the HMG-CoA pathway, HMG-CoA synthase, which revealed decreased activity, and reduced hepatic mRNA and protein levels. We conclude that short-term cholestasis in rats leads to impaired hepatic fatty acid metabolism due to impaired ketogenesis. Ketogenesis is impaired because of decreased mRNA levels of HMG-CoA synthase, leading to reduced hepatic protein levels and to decreased activity of this key enzyme of ketogenesis. - Lang, C., M. Schäfer, D. Serra, F. G. Hegardt, L. Krähenbühl, and S. Krähenbühl. Impaired hepatic fatty acid oxidation in rats with short-term cholestasis: characterization and mechanism. J. Lipid Res. 2001. 42: 22;-30.

Animals↗

PubMed2000

Inhibition by etomoxir of rat liver carnitine octanoyltransferase is produced through the co-ordinate interaction with two histidine residues.

Rat peroxisomal carnitine octanoyltransferase (COT), which facilitates the transport of medium-chain fatty acids through the peroxisomal membrane, is irreversibly inhibited by the hypoglycaemia-inducing drug etomoxir. To identify the molecular basis of this inhibition, cDNAs encoding full-length wild-type COT, two different variant point mutants and one variant double mutant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae, an organism devoid of endogenous COT activity. The recombinant mutated enzymes showed activity towards both carnitine and decanoyl-CoA in the same range as the wild type. Whereas the wild-type version expressed in yeast was inhibited by etomoxir in an identical manner to COT from rat liver peroxisomes, the activity of the enzyme containing the double mutation H131A/H340A was completely insensitive to etomoxir. Individual point mutations H131A and H340A also drastically reduced sensitivity to etomoxir. Taken together, these results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by etomoxir and that the full inhibitory properties of the drug will be shown only if both histidines are intact at the same time. Our data demonstrate that both etomoxir and malonyl-CoA inhibit COT by interacting with the same sites.

Acyl Coenzyme A↗

PubMed2000

Identification of the two histidine residues responsible for the inhibition by malonyl-CoA in peroxisomal carnitine octanoyltransferase from rat liver.

Carnitine octanoyltransferase (COT), an enzyme that facilitates the transport of medium chain fatty acids through peroxisomal membranes, is inhibited by malonyl-CoA. cDNAs encoding full-length wild-type COT and one double mutant variant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae. Both expressed forms were expressed similarly in quantitative terms and exhibited full enzyme activity. The wild-type-expressed COT was inhibited by malonyl-CoA like the liver enzyme. The activity of the enzyme encoded by the double mutant H131A/H340A was completely insensitive to malonyl-CoA in the range assayed (2-200 microM). These results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by malonyl-CoA. Another mutant variant, H327A, abolishes the enzyme activity, from which it is concluded that it plays an important role in catalysis.

Amino Acid Sequence↗

PubMed2000

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase promoter contains a CREB binding site that regulates cAMP action in Caco-2 cells.

cAMP increases transcription of the mitochondrial (mit.) gene for 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase, which encodes an enzyme that has been proposed as a control site of ketogenesis. The incubation of Caco-2 cells with cAMP increased mit.HMG-CoA synthase mRNA levels 4-fold within 24 h. We have identified an active cAMP-response element (CRE) located 546 bp upstream of the mit. HMG-CoA synthase promoter that is necessary for the induction of expression by dibutyryl cAMP. Co-transfections of constructs, containing the CRE element of the mit.HMG-CoA synthase promoter fused to the gene for chloramphenicol acetyltransferase, with protein kinase A and a dominant-negative mutant of cAMP-response-element-binding protein (CREB) show that the response to cAMP is mediated by the transcription factor CREB. The CRE element confers responsiveness of protein kinase A to a heterologous promoter in transfection assays in Caco-2 cells. Gel-retardation assays revealed that the mit.HMG-CoA synthase CRE binds to recombinant CREB. The shifted band obtained with the putative mit. HMG-CoA synthase CRE sequence and nuclear proteins from Caco-2 cells competed with CRE sequences of other genes such as somatostatin and phosphoenolpyruvate carboxykinase. We conclude that the regulation of the expression of the gene for mit.HMG-CoA synthase in Caco-2 cells by cAMP is mediated by a CRE sequence in the promoter.

Animals↗