OBJECTIVETo investigate the clinical phenotype and ACAT1 gene mutation in a family affected with beta-ketothiolase deficiency (BKTD).

METHODSClinical features and laboratory test data were collected. The probands were monozygotic twin brothers. Genomic DNA was isolated from peripheral blood leukocytes obtained from the probands and their family members. Molecular genetic testing of the ACAT1 gene was carried out.

RESULTSThe probands have presented with fever, vomiting and severe ketoacidosis. By arterial blood gas testing, pH was determined to be 7.164, bicarbonate was 4.0 mmol/L, and urine ketone was ++++. Urinary organic acid gas chromatography-mass spectrometry analysis showed excessive excretion of 3-hydroxybutyric acid, 2-methyl-3-hydroxybutyric acid and tiglylglycine. Increased 3-hydroxybutyrylcarnitine (C4-OH), tiglylcarnitine(C5:1) and 3-hydroxyisovalerylcarnitine (C5-OH) levels. The clinical phenotype of proband's parents were both normal, but an elder sister turned out to be an affected patient. Genetic analysis has identified two heterozygous mutations [c.622C>T(p.R208X) and c.653C>T (p.S218F)] in the proband, which were respectively detected in the mother and father. The c.653C>T (p.S218F) mutation was not found among the 100 healthy controls and has not been included in the Human Gene Mutation Database(HGMD).

CONCLUSIONThe primary clinical manifestations of BKTD is ketoacidosis. Urine organic acid and blood acylcarnitine analyses play an important role in the diagnosis of the disease. The compound heterozygous of ACAT1 gene mutations probably underlie the BKTD in our patient.

Acetyl-CoA C-Acetyltransferase ; genetics ; Acetyl-CoA C-Acyltransferase ; deficiency ; genetics ; Amino Acid Metabolism, Inborn Errors ; genetics ; Computational Biology ; Female ; Humans ; Infant ; Male ; Mutation ; Phenotype

OBJECTIVE: To explore the genetic etiology of a child suspected for β-ketothiolase deficiency by neonatal screening. METHODS: All coding exons and flanking sequences of the ACAT1 gene were subjected to targeted capture and high-throughput sequencing. Suspected variants were verified by Sanger sequencing and bioinformatic analysis. RESULTS: The child was found to harbor compound heterozygous variants of the ACAT1 gene, namely c.121-3C>G and c.275G>A (p. Gly92Asp). The c.121-3C>G variant was also detected in his father and two sisters, while the c.275G>A (p. Gly92Asp) was a de novo variant. A c.334+ 172C>G (rs12226047) polymorphism was also detected in his mother and two sisters. Sanger sequencing has verified that the c.275G>A (p. Gly92Asp) and c.334+172C>G (rs12226047) variants are located on the same chromosome. Bioinformatics analysis suggested both c.121-3C>G and c.275G>A (p.G92D) variants to be damaging. Based on the American College of Medical Genetics and Genomics standards and guidelines, the c.275G>A variant of the ACAT1 gene was predicted to be pathogenic (PS2+ PM2+ PM3+ PP3+PP4), the c.121-3C>G variant to be likely pathogenic (PM2+ PM3+ PP3+PP4). CONCLUSION The c.121-3C>G and c.275G>A variants of the ACAT1 gene probably underlay the pathogenesis of the child. Above finding has enriched the variant spectrum of the ACAT1 gene.
Acetyl-CoA C-Acetyltransferase/genetics* ; Acetyl-CoA C-Acyltransferase/genetics* ; Amino Acid Metabolism, Inborn Errors/genetics* ; Female ; High-Throughput Nucleotide Sequencing ; Humans ; Infant, Newborn ; Male ; Mutation

Clostridium tyrobutyricum is suitable for simultaneous saccharification and fermentation of lignocellulosic. It can produce butyric acid, acetic acid as its main fermentation products from a wide variety of carbohydrates such as glucose, xylose, cellobiose and arabinose. In order to decrease acetic acid content and increase butyric acid content in C. tyrobutyricum, we replaced genes on the acetic acid fermentation pathway with genes on the butyric acid fermentation pathway. Three genes were selected. They were acetyl-CoA acetylrtansfers gene (thl) which is the key enzyme gene associated with acetic acid fermentation pathway from Clostridium acetobutylicum, erythromycin gene (em) from plasmid pIMP1 and phosphotransacetylase gene (pta) which is the key enzyme gene associated with butyric acid fermentation pathway from C. tyrobutyricum. We fused these genes with pUC19 to construct nonreplicative integrated plasmids pUC19-EPT. Then we transformed pUC19-EPT into C. tyrobutyricum through electroporation. The recombinant transformants grown on plates containing erythromycin were validated by PCR. A mutant whose pta gene was displaced by thl gene on the chromosome was selected. In the fermentation from glucose, the mutant's yield of butyric acid is 0.47, increased by 34% compared with wild type; and the yield of acetic acid is 0.05, decreased by 29% compared with wild type.
Acetic Acid ; analysis ; metabolism ; Acetyl-CoA C-Acetyltransferase ; genetics ; Butyric Acid ; analysis ; metabolism ; Clostridium tyrobutyricum ; genetics ; metabolism ; Fermentation ; Genetic Engineering ; methods ; Glucose ; metabolism ; Industrial Microbiology ; Lignin ; metabolism ; Mutation ; Phosphate Acetyltransferase ; genetics

In this study, based on the transcriptome data, we cloned the full-length cDNAs of TwAACT gene from Tripterygium wilfordii suspension cells, and then analyzed the bioinformation of the sequence, detected the genetic differential expression after being induced by methyl jasmonate (MeJA) by RT-PCR. The full-length cDNA of the TwAACT was 1 704 bp containing a 1 218 bp open reading frame (ORF) encoding a polypeptide of 405 amino acids (GeneBank accession No. KP297934). The deduced isoelectric point (pI) was 6.10, a calculated molecular weight was about 41.20 kDa, and online prediction showed that TwAACT had two catalytic active sites. After the induction of MeJA, the relative expression level of TwAACT increased rapidly. The expression level of TwAACT was highest at 24 h. TwAACT was cloned firstly, that laid the foundation for identifying thegene and illustrating thebiosynthesis mechanism and its synthetic biology.
Acetyl-CoA C-Acetyltransferase ; chemistry ; genetics ; metabolism ; Amino Acid Sequence ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Models, Molecular ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Tripterygium ; chemistry ; classification ; enzymology ; genetics

OBJECTIVEThis study aimed to clone the acetyl-CoA C-acetyl transferase (AACT) gene from Aquilaria sinensis and analyze the bioinformatics and expression of the gene.

METHODOne unique sequence containing partly AACT gene sequence was discovered in our previous transcriptome dataset of A. sinensis. AACT gene was cloned by RT-PCR and RACE strategy with the template of RNA extracted from A. sinensis stem. The bioinformatic analysis of this gene and its corresponding protein was performed. The AsAACT expression in calli was analyzed with GADPH gene as an internal control gene in wounded condition by qRT-PCR technique.

RESULTOne unique sequence of AACT, named as AsAACT, was cloned from A. sinensis. The full length of AsAACT cDNA was containing a 1 236 bp ORF that encoded 411 amino acids. The result of qRT-PCR displayed that the highest expression level was at 4 h. which indicated that it was possibly involved in early-stage response to wound.

CONCLUSIONCloning and analyzing AsAACT gene from A. sinensis provided basic information for study the function and expression regulation of AsAACT in terpenoid biosynthesis.

Acetyl-CoA C-Acetyltransferase ; chemistry ; genetics ; metabolism ; Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Models, Molecular ; Molecular Sequence Data ; Protein Structure, Secondary ; Thymelaeaceae ; enzymology ; genetics

OBJECTIVETo investigate the effects of asymmetric dimethylarginine (ADMA) on ACAT-1 expression and cholesterol content in THP-1-derived macrophages and foam cells.

METHODSTHP-1 cells were induced to differentiate into macrophages and further into foam cells. The macrophages and foam cells were exposed to different concentrations (0, 3.75, 7.5, 15, and 30 µmol/L) of ADMA for varying time lengths (6, 12, and 24 h), and the changes in ACAT-1 mRNA and protein levels in the cells were measured with RT-PCR and Western blotting. The cellular cholesterol content was measured with enzyme-linked colorimetry assay.

RESULTSIn THP-1-derived macrophages and foam cells, the expression levels of ACAT-1 mRNA and protein and cellular cholesterol content increased significantly in response to ADMA treatment in a time- and concentration-dependent manner.

CONCLUSIONADMA may play an important role in inducing foam cell formation from macrophages. ACAT-1 inhibition targeting the macrophages and foam cells may serve as a potential therapeutic target in the treatment of atherosclerosis.

Acetyl-CoA C-Acetyltransferase ; metabolism ; Arginine ; analogs & derivatives ; pharmacology ; Cell Line ; Cholesterol ; analysis ; Foam Cells ; cytology ; metabolism ; Humans ; Macrophages ; cytology ; drug effects ; metabolism ; Monocytes ; cytology ; drug effects ; RNA, Messenger ; genetics ; Up-Regulation

OBJECTIVEThe aim of this study was to explore the genetic features of a family with 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency (MHBDD) which may provide the basis for the diagnosis and genetic counseling.

METHODClinical data of the proband was collected, total RNA and genomic DNA were extracted from the peripheral blood. The whole coding region of the ACAT1 gene was amplified by RT-PCR. 5' noncoding region of the ACAT1 gene and all 6 exons and flanking intron regions of the HADH2 gene were amplified by PCR. All amplification products were directly sequenced and compared with the reference sequence.

RESULT(1) The patient was a one-year-old boy who presented with psychomotor retardation and astasia when he was admitted to the hospital. Biochemical test revealed slight hyperlactatemia (3.19 mmol/L) and magnetic resonance imaging showed delayed myelination. 2-Methylacetoacetyl-CoA thiolase deficiency was suggested by gas chromatography-mass spectrometry. (2) There was no mutation in the ACAT1 gene and a hemizygous missense mutation c.388C > T was found in the 4 exon of the HADH2 gene which resulted in p. R130C. Proband's mother was the heterozygote and the father was normal.

CONCLUSIONThis is the first report on MHBDD patient and HADH2 mutation in China. p.R130C is responsible for the pathogenesis of the disease in the infant.

3-Hydroxyacyl CoA Dehydrogenases ; genetics ; Acetyl-CoA C-Acetyltransferase ; deficiency ; genetics ; Acyl Coenzyme A ; genetics ; metabolism ; Base Sequence ; DNA Mutational Analysis ; Dyskinesias ; Heterozygote ; Humans ; Infant ; Intellectual Disability ; enzymology ; genetics ; pathology ; Lipid Metabolism, Inborn Errors ; genetics ; pathology ; Male ; Mental Retardation, X-Linked ; Mutation, Missense ; Pedigree ; Reverse Transcriptase Polymerase Chain Reaction

Acetyl-CoA C-acetyltransferase (AACT) is the first enzyme in the terpene synthesis pathway, catalyzed two units of acetyl-CoA to acetoacetyl-CoA. In order to study the tanshinone biosynthesis in Salvia miltiorrhiza, a novel AACT gene, SmAACT, was cloned using cDNA microarray and RACE strategy. The full length cDNA of SmAACT is 1 623 bp (accession No. EF635969), which contained a 1 200 bp open reading frame (ORF) encoding a 399 amino acid protein. Nine introns were found in the genomic sequence. SmAACT was upregulated by YE and Ag+ elicitors both with cDNA microarray and quantitative RT-PCR analyses along with the accumulation of tanshinones. Sequence homology comparison and phylogenetic analysis all suggested that SmAACT belonged to the class of acetyl-CoA C-acetyltransferase. The transcription level of SmAACT was relatively higher in root than that in stem and leaf tissues. SNP analysis revealed that SmAACT was highly variable in the region of 6 to 9 introns with 33 SNPs in the 600 bp region, there are 5 SNPs in the cDNA region while they are all synonymous cSNPs. Some special genotypes were found in Salvia miltiorrhiza from different areas. SmAACT will be an useful gene for further analyze the mechanism of gene regulation among the tanshinones biosynthesis.
Acetyl-CoA C-Acetyltransferase ; genetics ; metabolism ; Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Gene Expression Regulation, Plant ; Genotype ; Introns ; Oligonucleotide Array Sequence Analysis ; Open Reading Frames ; Phylogeny ; Plant Leaves ; enzymology ; genetics ; Plant Roots ; enzymology ; genetics ; Plant Stems ; enzymology ; genetics ; Plants, Medicinal ; classification ; enzymology ; genetics ; Polymorphism, Single Nucleotide ; Reverse Transcriptase Polymerase Chain Reaction ; Salvia miltiorrhiza ; classification ; enzymology ; genetics

The aim of the present study was to investigate the role of peroxisome proliferator-activated receptor γ (PPARγ) signal transduction pathway in the expression of ATP binding cassette transporter A1 (ABCA1) and acyl-CoA:cholesterol acyltransferase 1 (ACAT1) induced by visfatin and to discuss the mechanism of foam cell formation induced by visfatin. THP-1 monocytes were induced into macrophages by 160 nmol/L phorbol myristate acetate (PMA) for 48 h, and then the macrophages were exposed to visfatin and PPARγ activator rosiglitazone, respectively. The expressions of PPARγ, ABCA1 and ACAT1 mRNA and protein were determined by RT-PCR and Western blot respectively. The contents of total cholesterol (TC) and free cholesterol (FC) were detected by enzyme fluorescence analysis. The content of cholesterol ester (CE) was calculated by the difference between TC and FC. The results showed that visfatin decreased the mRNA and protein expressions of PPARγ and ABCA1, increased the mRNA and protein expressions of ACAT1, and increased the contents of FC and CE in a concentration-dependent manner. These above effects of visfatin were inhibited by rosiglitazone in a concentration-dependent manner. These results suggest that visfatin may down-regulate the ABCA1 expression and up-regulate the ACAT1 expression via PPARγ signal transduction pathway, which decreases the outflow of FC, increases the content of CE, and then induces foam cell formation.
ATP Binding Cassette Transporter 1 ; ATP-Binding Cassette Transporters ; genetics ; metabolism ; Acetyl-CoA C-Acetyltransferase ; genetics ; metabolism ; Cell Differentiation ; Cell Line ; Cholesterol Esters ; metabolism ; Foam Cells ; cytology ; Humans ; Macrophages ; cytology ; Monocytes ; cytology ; Nicotinamide Phosphoribosyltransferase ; pharmacology ; PPAR gamma ; agonists ; physiology ; RNA, Messenger ; genetics ; metabolism ; Signal Transduction ; Thiazolidinediones ; pharmacology