The pre-diagnostic test for preimplantation genetic diagnosis (PGD) of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency was performed by polymerase chain reaction (PCR) and direct sequencing for hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (HADHA) gene. We obtained unexpected genotyping results of HADHA gene by allele drop-out in the analysis of patients' genomic DNA samples with a referred PCR primer set. Upon further analysis with a re-designed primer set, we found a novel single nucleotide polymorphism (SNP) at the referred primer-binding site in the normal allele of HADHA gene (NT_022184, 5233296 a>t). We found that the frequency of this novel SNP was 0.064 in Korean population. Pre-diagnostic test using single lymphocytes and clinical PGD were successfully performed with the re-designed primer set. Nineteen embryos (95.0%) among 20 were successfully diagnosed to 5 homozygous mutated, 8 heterozygous carrier and 6 wild type. Among 6 normal embryos, well developed and selected 4 embryos were transferred into the mother's uterus, but a pregnancy was not achieved. We proposed that an unknown SNP at primer-binding sites would be a major cause of allele drop-out in the PGD for single gene dis-order.
*Preimplantation Diagnosis ; *Polymorphism, Single Nucleotide ; Polymerase Chain Reaction ; Mutation ; Multienzyme Complexes/*genetics ; Male ; Humans ; Female ; Binding Sites ; Adult ; 3-Hydroxyacyl CoA Dehydrogenases/deficiency

We expressed 17-hydroxysteroid dehydrogenase10 (17β-hsd10) recombinant protein, prepared anti-17β- hsd10 polyclonal antibodies and established sandwich enzyme linked immunosorbent assay (ELISA) test for detection of 17β-hsd10. RT-PCR was used to get the gene of 17β-hsd10 of mouse liver, and a prokaryotic protein expression system pET 15b-17β-hsd10/Escherichia coli BL21 (DE3) which induced with isopropyl-1-thio-β-galactopyranoside (IPTG) for recombinant protein expression was constructed subsequently. The target protein purified using His-Binding-resin column was used to immunize BALB/c mice and rabbits, serum total IgGs from immunized animals were purified by ammonium sulfate precipitation method. We established a Double-antibody Sandwich enzyme linked immunosorbent assay about 17β-hsd10 using the two antibodies we prepared. We got the concentration of 1.5 mg/mL of 17β-hsd10 protein with molecular weight of 29.5 kDa, and polyclonal antibodies from mouse and rabbit with the tite 1.25 x 10(4) and 2.5 x 10(4) respectively. The concentration of 0.1 g/mL of 17β-hsd10 can be detected by the Double-antibody Sandwich ELISA we established, and the assay was sensitive and specific. It can be widely used in clinical and experimental study.
3-Hydroxyacyl CoA Dehydrogenases ; genetics ; immunology ; Animals ; Antibodies ; immunology ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; Immunization ; Mice ; Mice, Inbred BALB C ; Rabbits ; Recombinant Proteins ; genetics ; immunology

OBJECTIVETo explore action mechanism of acupuncture and moxibustion for Alzheimer's disease (AD) to provide evidence for prevention and treatment with acupuncture and moxibustion on AD in clinic.

METHODSEighty SPF-grade male Wistar rats, (200 +/- 20) g, were randomly divided into a normal group, a sham-operation group, a model group and a treatment group, 20 cases in each one. The model was duplicated with injection of Abeta1-42 in rats' hippocampus. Expect the treatment group, the rest groups were treated with regular feeding after respective intervention. The treatment group was treated with acupuncture and moxibustion at "Baihui" (GV 20) and "Shenshu" (BL 23), once a day, seven days as a treatment course and totally for two courses. There was one day of interval between the courses. The immunohistochemistry and quantitative RT-PCR methods were applied to test level of Abeta-binding alcohol dehydrogense (ABAD) and cytochrome oxidase IV (COX IV) in hippocampal neurons mitochondria.

RESULTSAcupuncture and moxibustion could reduce effectively level of ABAD and improve activity of COX IV in hippocampal neurons mitochondria in the treatment group, which has statistical significance compared with that in the model group (P < 0.01) and no statistical significance compared with that in the normal group and sham-operation group (P > 0.05). This indicated that acupuncture and moxibustion could effectively suppress overexpression of ABAD, improve activity of COX IV and reduce leak of reactive oxygen species, which could improve metabolic disturbance of mitochondria energy to achieve the goal of prevention and treatment of AD.

CONCLUSIONThe prevention and treatment of AD with acupuncture and moxibustion could be related with suppressing overexpression of ABAD and improving activity of COX IV in hippocampal neurons mitochondria to improve mitochondria energy metabolism.

3-Hydroxyacyl CoA Dehydrogenases ; genetics ; metabolism ; Acupuncture Therapy ; Alzheimer Disease ; enzymology ; metabolism ; therapy ; Animals ; Electron Transport Complex IV ; genetics ; metabolism ; Energy Metabolism ; Hippocampus ; cytology ; enzymology ; metabolism ; Humans ; Male ; Mitochondria ; enzymology ; metabolism ; Moxibustion ; Neurons ; enzymology ; metabolism ; Rats ; Rats, Wistar

To research the correlation between accumulation of triterpenoids and expression of key enzymes genes in triterpenoid biosynthesis of Alisma orientale,the study utilized UPLC-MS/MS method to detect eight triterpenoids content in the tuber of A. orientale from different growth stages,including alisol A,alisol A 24 acetate,alisol B,alisol B 23 acetate,alisol C 23 acetate,alisol F,alisol F 24 acetate and alisol G,and then the Real time quantitative PCR was used to analyze the expression of key enzymes genes HMGR and FPPS in triterpenoid biosynthesis. Correlation analysis showed that there was a significant positive relation between the total growth of these eight triterpenoids and the average relative expression of HMGR and FPPS(HMGR: r = 0. 998,P<0. 01; FPPS: r = 0. 957,P<0. 05),respectively. Therefore,the study preliminarily determined that HMGR and FPPS genes could regulate the biosynthesis of triterpenoids in A. orientale,which laid a foundation for further research on the biosynthesis and regulation mechanism of triterpenoids in A. orientale.
Alisma ; chemistry ; genetics ; Chromatography, Liquid ; Geranyltranstransferase ; genetics ; Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent ; genetics ; Phytochemicals ; analysis ; Plant Extracts ; Plant Proteins ; genetics ; Plant Tubers ; chemistry ; Tandem Mass Spectrometry ; Triterpenes ; analysis

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

OBJECTIVETo study the distribution of ScrF1 restriction polymorphism in intron 2 of the 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA) reductase gene in Chinese Han population and the association of the polymorphism with coronary heart disease(CHD).

METHODSHMG-CoA reductase genotyping was performed using polymerase chain reaction-restriction fragment polymorphism.

RESULTSHMG-CoA reductase allelic frequencies of A, a were 0.519, 0.481; 0.440, 0.560 in CHD group and control group respectively. There was no significant difference in frequencies of allele and genotype in ScrF1 polymorphism between CHD group and control group(P>0.05). However, the levels of plasma very low density lipoprotein (VLDL) and TG in CHD patients with AA genotype were higher than those in CHD patients with other genotypes(P<0.05). The frequencies of A, a alleles at ScrF1 polymorphic site were significantly different from those reported in European Caucasians (0.44 vs 0.55, 0.56 vs 0.45, P<0.05).

CONCLUSIONNo direct association was found between the ScrF1 polymorphism and CHD, but there is a significant correlation between the AA genotype of the HMG-CoA reductase gene and the levels of plasma VLDL and TG in CHD group.

Adult ; Aged ; Aged, 80 and over ; Asian Continental Ancestry Group ; genetics ; Blood Chemical Analysis ; Cholesterol, VLDL ; blood ; Female ; Genetic Predisposition to Disease ; Humans ; Hydroxymethylglutaryl CoA Reductases ; genetics ; Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent ; genetics ; Lipid Metabolism ; genetics ; Male ; Middle Aged ; Polymorphism, Genetic ; Triglycerides ; blood

BACKGROUNDPreeclampsia (PE) is associated with abnormal fatty acid beta-oxidation (FAO), especially metabolic disorders of long-chain fatty acid oxidation. The role of FAO dysfunction in inadequate invasion is unclear. The aim of this study was to explore the influence of various lengths fatty acids oxidation on invasiveness of trophoblasts.

METHODSPrimary human trophoblast cells and HTR8/SVneo cells were treated with fatty acids of various lengths. Morphological changes, lipid deposition and ultrastructure changes of trophoblast cells were detected. Cells invasiveness was determined by transwell insert. CPT1, CPT2 and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) protein expression were analyzed. The correlation between intracellular lipid droplets deposition and cells invasiveness was evaluated.

RESULTSCells treated with long-chain fatty acids showed significant increased lipid droplets deposition, severe mitochondrial damage, decreased CPT2 and LCHAD protein expression (P < 0.05) but no significant difference in CPT1 protein expression (P > 0.05). Invasiveness of the trophoblast cells of the LC-FFA group significantly decreased (P < 0.05). Intracellular lipid droplets deposition was negatively correlated with invasivenss (R = -0.745, P < 0.05).

CONCLUSIONTrophoblast cells after stimulation with long chain fatty acids exist fatty acid oxidation disorders, and reduce the ability of trophoblastic invasion.

Cell Line ; Cells, Cultured ; Fatty Acids, Nonesterified ; pharmacology ; Humans ; Lipid Metabolism ; Long-Chain-3-Hydroxyacyl-CoA Dehydrogenase ; metabolism ; Trophoblasts ; drug effects

OBJECTIVE: To explore the driving gene of hepatocellular carcinoma (HCC) occurrence and progression and its potential as new therapeutic target of HCC. METHODS: The transcriptome and genomic data of 858 HCC tissues and 493 adjacent tissues were obtained from TCGA, GEO, and ICGC databases. Gene Set Enrichment Analysis (GSEA) identified EHHADH (encoding enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase) as the hub gene in the significantly enriched differential pathways in HCC. The downregulation of EHHADH expression at the transcriptome level was found to correlate with TP53 mutation based on analysis of the TCGA- HCC dataset, and the mechanism by which TP53 mutation caused EHHADH downregulation was explored through correlation analysis. Analysis of the data from the Metascape database suggested that EHHADH was strongly correlated with the ferroptosis signaling pathway in HCC progression, and to verify this result, immunohistochemical staining was used to examine EHHADH expression in 30 HCC tissues and paired adjacent tissues. RESULTS: All the 3 HCC datasets showed signficnatly lowered EHHADH expression in HCC tissues as compared with the adjacent tissues (P < 0.05) with a close correlation with the degree of hepatocyte de-differentiation (P < 0.01). The somatic landscape of HCC cohort in TCGA dataset showed that HCC patients had the highest genomic TP53 mutation rate. The transcriptomic level of PPARGC1A, the upstream gene of EHHADH, was significantly downregulated in HCC patients with TP53 mutation as compared with those without the mutation (P < 0.05), and was significantly correlated with EHHADH expression level. GO and KEGG enrichment analyses showed that EHHADH expression was significantly correlated with abnormal fatty acid metabolism in HCC. The immunohistochemical results showd that the expression level of EHHADH in HCC tissues was down-regulated, and its expression level was related to the degree of hepatocytes de-differentiation and the process of ferroptosis. CONCLUSION TP53 mutations may induce abnormal expression of PPARGC1A to cause downregulation of EHHADH expression in HCC. The low expression of EHHADH is closely associated with aggravation of de-differentiation and ferroptosis escape in HCC tissues, suggesting the potential of EHHADH as a therapeutic target for HCC.
Humans ; Carcinoma, Hepatocellular/genetics* ; Transcriptome ; Liver Neoplasms/genetics* ; Gene Expression Profiling ; Fatty Acids ; Peroxisomal Bifunctional Enzyme

There exists many kinds and a huge number of terpenoid in medicinal plants, which show a wide range of pharmacological activities. 3-Hydroxy-3-metllylglutaryl coenzyme A reductase(HMGR) is a key rate-limiting enzyme in terpenoid biosynthetic pathway . HMGR plays an important role in the regulation of secondary metabolism of the terpenoid. The paper summarized the biological function and the catalytic mechanism of HMGR, the cloning and the structure of the gene as well as its research progress in some medicinal plants.
Hydroxymethylglutaryl CoA Reductases ; metabolism ; Plants, Medicinal ; enzymology ; Terpenes ; metabolism

OBJECTIVETo study the hypolipidemic active compounds from Crataegus pinnatifida and mechanism of action of those.

METHODGuided by the inhibitory activity to HMG-CoA reductase, the active compounds were separated and purified with macroporous resin and silica gel.

RESULTFour active compounds were obtained, which were quercetin, hyperoside, rutin and chlorogenic acid, the sum of their inhibitory rate was 50.01%, and the total inhibitory rate of the mixture of four active compounds matched was 79.48%.

CONCLUSIONQuercetin and hyperoside were the principle active components inhibiting HMG-CoA reductase in Hawthorn fruit, and there were synergistic action among them.

Crataegus ; chemistry ; Fruit ; chemistry ; Hydroxymethylglutaryl CoA Reductases ; analysis ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Plant Extracts ; pharmacology