The present study analyzed the correlations between curcumin(Cur), nuclear factor E2 related factor 2(NRF2)-dimethylarginine dimethylaminohydrolase(DDAH)-asymmetric dimethylarginine(ADMA)-nitric oxide(NO) pathway, and endothelial-mesenchymal transition(EndMT) based on SD rats with cardiac fibrosis, and explored the effect and mechanism of Cur in resisting cardiac fibrosis to provide an in-depth theoretical basis for its clinical application in the treatment of heart failure. The cardiac fibrosis model was induced by subcutaneous injection of isoprenaline(Iso) in rats. Thirty-two rats were randomly divided into a control group, a model group, a low-dose Cur group(100 mg·kg~(-1)·d~(-1)), and a high-dose Cur group(200 mg·kg~(-1)·d~(-1)), with eight in each group. After 21 days of treatment, cardiac function was detected by echocardiography, degree of cardiac fibrosis by Masson staining, expression of CD31 and α-SMA by pathological staining, expression of VE-cadherin, vimentin, NRF2, and DDAH by Western blot, and ADMA level by HPLC. Compared with the model group, the Cur groups showed alleviated cardiac fibrosis, accompanied by increased CD31 and VE-cadherin expression and decreased α-SMA and vimentin expression, indicating relieved EndMT. Additionally, DDAH and NRF2 levels were elevated and ADMA and NO expression declined. Cur improves cardiac fibrosis by inhibiting EndMT presumedly through the NRF2-DDAH-ADMA-NO pathway.
Amidohydrolases/metabolism* ; Animals ; Curcumin ; Fibrosis ; NF-E2-Related Factor 2/genetics* ; Nitric Oxide/metabolism* ; Rats ; Rats, Sprague-Dawley

A stable Zr-based metal-organic framework (MOF, UiO-66-NH2) synthesized via micro-water solvothermal method was used to immobilize amidase by using the glutaraldehyde crosslinking method. The effect of immoblization conditions on enzyme immoblization efficiency was studied. An activity recovery rate of 86.4% and an enzyme loading of 115.3 mg/g were achieved under the optimal conditions: glutaraldehyde concentration of 1.0%, cross-linking time of 180 min, and the weight ratio of MOF to enzyme of 8:1. The optimal temperature and optimal pH of the immobilized amidase were determined to be 40 °C and 9.0, respectively, and the Km, Vmax and kcat of the immoblized amidase were 58.32 mmol/L, 16.23 μmol/(min·mg), and 1 670 s⁻¹, respectively. The immobilized enzyme was used for (S)-4-fluorophenylglycine synthesis and the optimal reaction conditions were 300 mmol/L of N-phenylacetyl-4-fluorophenylglycine, 10 g/L of immobilized enzyme loading, and reacting for 180 min at pH 9.0 and 40 °C. A conversion rate of 49.9% was achieved under the optimal conditions, and the conversion rate can be increased to 99.9% under the conditions of enantiomeric excess. The immobilized enzyme can be repeatedly used, 95.8% of its original activity can be retained after 20 cycles.
Amidohydrolases ; Enzyme Stability ; Enzymes, Immobilized/metabolism* ; Glycine/analogs & derivatives* ; Hydrogen-Ion Concentration ; Metal-Organic Frameworks ; Temperature

OBJECTIVE: To analyze the molecular etiology of a Chinese child affected with dihydropyrimidinase deficiency. METHODS: Genomic DNA was extracted from peripheral blood samples of the family members. Pathogenic variant was determined by whole exome sequencing and verified by Sanger sequencing. RESULTS: The child was found to harbor homozygous c.905G>A (p.Arg302Gln) variants in exon 5 of the DPYS gene, for which her parents were both heterozygous carriers. CONCLUSION The homozygous c.905G>A (p.Arg302Gln) variants of the DPYS gene probably underlies the dihydropyrimidinase deficiency in the child. Above result has enabled genetic counseling and prenatal diagnosis for this family.
Amidohydrolases/genetics* ; Asian Continental Ancestry Group/genetics* ; Child ; Exons ; Female ; Humans ; Metabolism, Inborn Errors/genetics* ; Mutation ; Pedigree

OBJECTIVE: To explore the molecular etiology for a Chinese family affected with beta-ureidopropinoase deficiency. METHODS: Genomic DNA was extracted from the peripheral blood samples of family members. All exons and flanking intron regions of the UPB1 gene were amplified by PCR and detected by direct sequencing. The pathogenicity of identified mutation was analyzed using Polyphen2 and SIFT software. RESULTS: Compound heterozygous mutations of the UPB1 gene, including c.853G>A (p.A285T) and c.917-1G>A, were discovered in the proband, which were inherited respectively from his mother and father. Bioinformatics analysis suggested that this novel mutation was damaging. CONCLUSION The compound heterozygous mutations of the UPB1 gene probably underlie the beta-ureidopropinoase deficiency in the infant. Discovery of c.853G>A also enriched the mutation spectrum of the UPB1 gene.
Abnormalities, Multiple ; genetics ; Amidohydrolases ; deficiency ; genetics ; Asian Continental Ancestry Group ; Brain Diseases ; genetics ; China ; Exons ; Humans ; Infant ; Introns ; Movement Disorders ; genetics ; Mutation ; Pedigree ; Purine-Pyrimidine Metabolism, Inborn Errors ; genetics

OBJECTIVE To detect potential mutation in a Chinese family affected with beta-ureidopropinoase deficiency. METHODS Genomic DNA was extracted from peripheral blood samples. All exons and flanking intron regions of the UPB1 gene were amplified by PCR and detected by direct sequencing. RESULTS A homozygous mutation c.977G>A was identified in exon 9 of the UPB1 gene in the proband. Both parents of the proband had heterozygous change of the same site. CONCLUSION The c.977G>A mutation of the UPB1 gene is responsible for the pathogenesis of the disease in the infant.
Abnormalities, Multiple ; genetics ; Amidohydrolases ; deficiency ; genetics ; Brain Diseases ; genetics ; Exons ; Humans ; Infant ; Male ; Movement Disorders ; genetics ; Mutation ; Purine-Pyrimidine Metabolism, Inborn Errors ; genetics

OBJECTIVETo investigate the protective effects of oxymatrine on chronic heart failure induced by isoproterenol (ISO) and to observe its effects on ADMA metabolism pathway in ISO-induced chronic heart failure in rats.

METHODMale Sprague-Dawley rats were given oxymatrine (100,50 mg kg-1) orally for 14 days. Heart failure was induced in rats by subcutaneous injection of isoproterenol (5 mg kg-1 d-1 ) at the 8th day for 1 week. Serum parameters, haemodynamic parameters, Heart weight, and histopathological variables were analysed. Expression of protein levels were measured by Western blot.

RESULTOxymatrine (100,50 mg kg-1) significantly attenuated serum content of cTn I, improved left ventricle systolic and diastolic function and left ventricular remodeling, reduced the ISO-induced myocardial pathological changes compared with ISO group. In addition, oxymatrine (100,50 mg kg-1) significantly reduced serum level of ADMA (P <0. 01), normalize the reduced dimethylarginine dimethylaminohydrolase 2 (DDAH2) expression (P <0. 01) , but had no effect on the isoproterenol-induced upregulated protein arginine methyltransferases 1 expression.

CONCLUSIONOxymatrine could ameliorate the experimental ventricular remodeling in ISO-induced chronic heart failure in rats and the mechanism involved in reducing serum content of ADMA and increased DDAH2 expression.

Alkaloids ; pharmacology ; therapeutic use ; Amidohydrolases ; metabolism ; Animals ; Arginine ; analogs & derivatives ; blood ; metabolism ; Chronic Disease ; Gene Expression Regulation, Enzymologic ; drug effects ; Heart Failure ; drug therapy ; metabolism ; pathology ; physiopathology ; Hemodynamics ; drug effects ; Isoproterenol ; adverse effects ; Male ; Organ Size ; drug effects ; Quinolizines ; pharmacology ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Troponin I ; metabolism

OBJECTIVESTo identify a NFB responsive element within the dimethylarginine dimethylaminohydrolase 2 gene (DDAH) promoter and demonstrate its role in DDAH2 transactivation.

METHODSDDAH2 promoter was analyzed with software to identify potential binding sites of transcription factors. A series of truncated DDAH2 promoter luciferase reporter plasmids were constructed and transfected into human embryonic kidney derived HEK293 cells. Luciferase assays were carried out to analyze the activity of the promoter. Electrophoresis mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were used to identify the NFB responsive element in vitro and in vivo. DDAH2 promoter luciferase reporter plasmid with mutated NFB site was constructed and transfected into cells, and its activity was compared with that of the wild-type plasmid.

RESULTSPotential bindings sites of many transcription factors were found within the DDAH2 promoter. The transcription activity of the DDAH2 promoter was high, and -530 to -437 was a positive regulating region. -476 to -469 of the DDAH2 promoter was a NFB responsive element, to which NFB can specifically bind. Mutation of the NFB element could significantly decrease the DDAH2 promoter activity.

CONCLUSION-476 to -469 of the DDAH2 promoter was a NFB responsive element and is important for the transactivation of DDAH2.

Amidohydrolases ; genetics ; metabolism ; Base Sequence ; Gene Expression Regulation, Enzymologic ; Humans ; Molecular Sequence Data ; NF-kappa B ; metabolism ; Promoter Regions, Genetic ; Protein Binding ; Response Elements

In order to analyze the correlation between critical residues in the catalytic centre of BSH and the enzyme substrate specificity, seven mutants of Lactobacillus salivarius bile salt hydrolase (BSH1) were constructed by using the Escherichia coli pET-20b(+) gene expression system, rational design and site-directed mutagenesis. These BSH1 mutants exhibited different hydrolytic activities against various conjugated bile salts through substrate specificities comparison. Among the residues being tested, Cys2 and Thr264 were deduced as key sites for BSH1 to catalyze taurocholic acid and glycocholic acid, respectively. Moreover, Cys2 and Thr264 were important for keeping the catalytic activity of BSH1. The high conservative Cys2 was not the only active site, other mutant amino acid sites were possibly involved in substrate binding. These mutant residues might influence the space and shape of the substrate-binding pockets or the channel size for substrate passing through and entering active site of BSH1, thus, the hydrolytic activity of BSH1 was changed to different conjugated bile salt.
Amidohydrolases ; genetics ; metabolism ; Bile Acids and Salts ; metabolism ; Escherichia coli ; metabolism ; Gene Expression ; Lactobacillus ; enzymology ; genetics ; Substrate Specificity

Developing a high-throughput screening method is of great importance for directed evolution of atrazine chlorohydrolase. A mutagenesis library of atzA from Pseudomonas sp. ADP and Arthrobacter sp. AD1 was constructed using error-prone PCR and DNA shuffling. Candidate mutants were screened through Haematococcus pluvialis expression system, using atrazine as selection pressure. Sequence analysis showed that mutations in the obtained 12 mutants with enhanced activity were all point-substitutions and scattered throughout the gene. Enzymatic activity analysis showed that the mutants all had higher activities than that of the wild type. The activities were 1.8-3.6 fold of the wild-type enzyme when cultured in BBM medium with 1 mg/L atrazine, whereas 1.8-2.6 fold with 2 mg/L atrazine. These results indicated that Haematococcus pluvialis expression system is an ideal high throughput screening system for directed evolution of atrazine chlorohydrolase.
Amidohydrolases ; genetics ; Atrazine ; metabolism ; Bacterial Proteins ; genetics ; Biodegradation, Environmental ; Chlorophyta ; genetics ; metabolism ; Herbicides ; metabolism ; High-Throughput Screening Assays ; Hydrolases ; biosynthesis ; genetics ; Mutagenesis, Insertional ; Pseudomonas ; enzymology ; genetics

Aging ; metabolism ; Amidohydrolases ; metabolism ; Animals ; Caenorhabditis elegans ; cytology ; drug effects ; growth & development ; metabolism ; Caenorhabditis elegans Proteins ; genetics ; metabolism ; Caloric Restriction ; Ethanolamines ; metabolism ; pharmacology ; Forkhead Transcription Factors ; Gene Expression Regulation, Developmental ; Isoenzymes ; genetics ; metabolism ; Signal Transduction ; Superoxide Dismutase ; genetics ; metabolism ; Trans-Activators ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism