Dihydroorotate dehydrogenase is a flavin-dependent mitochondrial enzyme to catalyze the fourth step of the de novo synthesis of pyrimidine and to oxidize dihydroorotate to orotate. By selectively inhibiting dihydroorotate dehydrogenase, thereby inhibiting pyrimidine synthesis, the enzyme has been developed for the treatment of cancer, autoimmune diseases, bacterial or viral infections, parasitic diseases and so on. The development of inhibitory drugs requires a detailed understanding of the structural characteristics and catalytic cycle mechanism of dihydroorotate dehydrogenase. Therefore, this paper reviews these two aspects, and indicates perspectives of these inhibitors in clinical application.
Catalysis ; Mitochondria/metabolism* ; Oxidation-Reduction ; Oxidoreductases Acting on CH-CH Group Donors/metabolism*

Lignans are important defensive compounds in plants and have good biological activities protecting human health. In order to study the medicinal secondary metabolism of Fagopyrum cymosum (Trev.) Meisn, a traditional Chinese medicine with anti-tumor effect, a novel isoflavone reductase-like gene, FcIRL, was cloned using RACE strategy from a cDNA library of high flavonoids-producing callus. The full-length cDNA of the FcIRL was 1 217 bp (accession no. EU116032), which contained a 942 bp open reading frame (ORF) encoding a 313 amino acid protein. Two stop codons (TAG) and a putative polyadenylation signal ATAAA at 24 bp upstream from the polyadenylation site was found in 5' and 3' UTR, separately. And no intron was found in the genomic sequence yet. FcIRL contained a predicted N-terminal acetylation site (M1-K5) and a NADPH-binding motif (G10-G-T-G13-Y-I-G16) in the N-terminal region, a conserved NmrA (nitrogen metabolite repression regulator) domain (V6-N244), multi-phosphorylation sites and one conserved N-glycosylation site (N214). Sequence homology comparison, phylogenetic analysis and advanced structures prediction all suggested that FcIRL belonged to the class of pinoresinol-lariciresinol reductase (PLR), which is a key enzyme in synthetic pathway of 8-8'-linked lignans, with function in catalyzing reduction of pinoresinol and lariciresinol into secoisolariciresinol, and medicinal secondary metabolism and resistance in F. cymosum.
Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Fagopyrum ; enzymology ; genetics ; Flavonoids ; genetics ; Lignans ; metabolism ; Molecular Sequence Data ; Oxidoreductases ; genetics ; Oxidoreductases Acting on CH-CH Group Donors ; genetics ; Protein Structure, Tertiary ; Sequence Homology, Amino Acid

OBJECTIVE: To assay the changes of polyamine oxidase (PAO) activities and polyamine levels in the cortex and subcortex at different time of reperfusion following 2 h focal cerebral ischemia in rats in order to explore the regularity and signifiance ofh these changes. METHODS: Rats of 2 h reversible focal cerebral ischemia were produced by ameliorated method of Longa's intraluminal suture occlusion of middle cerebral artery (MCA). PAO activities and polyamine levels in the cortex and subcortex were measured by homovanillic acid fluorometry and high-performance liquid chromatograph (HPLC) after 2, 4, 8, and 24 h reperfusion following 2 h ischemia, respectively. RESULTS: PAO activity of the experimental group increased after 8 h reperfusion (P < 0.01). The peak value of PAO activity appeared after 24 h reperfusion (P < 0.01). Putrescine level of the experimental group was elevated after 4 h reperfusion (P < 0.05), and the peak value of putrescine appeared after 24 h reperfusion (P < 0.05). Spermidine and spermine levels of 8, 24 h reperfusion in the experiment group decreased significantly c eompared with the control group (P < 0.05). CONCLUSION PAO activities increased significantly after reperfusion following transient focal cerebral ischemia, which promoted the later peak production of putrescine. It may be contributed to the brain damage after cerebral ischemia.
Animals ; Cerebral Cortex ; metabolism ; Ischemic Attack, Transient ; metabolism ; Male ; Oxidoreductases Acting on CH-NH Group Donors ; metabolism ; Polyamines ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism

OBJECTIVETo study the effect of Guanxin No. 2 (GX2) on gene variant expression profile in rats after myocardial infarction (MI).

METHODSSix SD rats were equally randomized into the sham operated group, the model group and the GX2 group, and they received gastric perfusion with water and GX2 (10 g/kg) respectively. MI model was established by ligating the left-anterior descending branch of coronary artery after 10 days of perfusion, and rats' myocardial tissue in the junction zone was assessed 24 h later for gene chip detection with DNA microarray. Then a cluster analysis was conducted, and the different expressions of key genes were verified by real-time reverse transcription-polymerase chain reaction.

RESULTSThe up-regulating gene expressions in myocardial tissue in the junction zone increased after ischemia. After GX2 intervention, the up- or down-regulating gene expressions, especially the 2 genes, all-trans-13,14-dihydroretinol saturase (AF465614) and similar to expressed sequence AW413625 (AA799328) decreased significantly. In the common genes, more genes involving activity of signal transducer presented in the model group and the GX2 group and those in the latter showed a certain specificity.

CONCLUSIONGX2 could improve the characteristics of variant gene expression profile in MI rats to a certain extent.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression ; drug effects ; Gene Expression Profiling ; Myocardial Ischemia ; genetics ; metabolism ; Myocardium ; metabolism ; Oxidoreductases Acting on CH-CH Group Donors ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo analyze the impact of depletion of the twin arginine translocation (TAT) system on virulence and physiology of Yersinia enterocolitica for a better understanding of its pathogenicity.

METHODSWe constructed a DeltatatC::SpR mutant of Yersinia enterocolitica by P1 phage mediated transduction using Escherichia coli K-12 DeltatatC::SpR strain as a donor.

RESULTSA P1-mediated genetic material transfer was found between the two species of enterobacteria, indicating a great potential of acquisition of antibiotic resistance in emergency of a new threatening pathogen by genetic material exchanges. Periplasmic trimethylamine N-oxidase reductase activity was detected in the wild type Y. enterocolitica strain and translocation of this enzyme was completely abolished by the DeltatatC::SpR mutation. In addition, the DeltatatC::SpR mutation showed a pleiotropic effect on the metabolism of Y. enterocolitica. However, the tat mutation did not seem to affect the mobility and virulence of Y. enterocolitica under the conditions used.

CONCLUSIONUnlike other pathogenic bacteria studied, the TAT system of Y. enterocolitica might play an important role in the pathogenic process, which is distinct from other pathogens, such as Pseudomonas aeruginosa and enterohemorrhagic E. coli O157:H7.

Drug Resistance, Microbial ; genetics ; Genes, Bacterial ; Mutation ; Oxidoreductases Acting on CH-NH Group Donors ; metabolism ; Transduction, Genetic ; Virulence ; Yersinia enterocolitica ; enzymology ; genetics ; metabolism ; pathogenicity

Deoxyhypusine is a modified lysine and formed posttranslationally to be the eukaryotic initiation factor eIF5A by deoxyhypusine synthase, employing spermidine as butylamine donor. Subsequent hydroxylation of this deoxyhypusine-containing intermediate completes the maturation of eIF5A. The previous report showed that deoxyhypusine synthase was phosphorylated by PKC in vivo and the association of deoxyhypusine synthase with PKC in CHO cells was PMA-, and Ca(2+)/phospholipid-dependent. We have extended study on the phosphorylation of deoxyhypusine synthase by protein kinase CK2 in order to define its role on the regulation of eIF5A in the cell. The results showed that deoxyhypusine synthase was phosphorylated by CK2 in vivo as well as in vitro. Endogenous CK2 in HeLa cells and the cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced or decreased by the addition of CK2 effectors such as polylysine, heparin, and poly(Glu, Tyr) 4:1. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mainly phosphorylated on threonine residue and less intensely on serine. These results suggest that phosphorylation of deoxyhypusine synthase is CK2-dependent cellular event as well as PKC-mediated effect. However, there were no observable changes in enzyme activity between the phosphorylated and unphosphorylated forms of deoxyhypusine synthase. Taken together, besides its established function in hypusine modification involving eIF5A substrate, deoxyhypusine synthase and its phosphorylation modification may have other independent cellular functions because of versatile roles of deoxyhypusine synthase.
Animals ; Casein Kinase II ; Cell Line ; Cricetinae ; Hela Cells ; Humans ; Mice ; Oxidoreductases Acting on CH-NH Group Donors/genetics/*metabolism ; Phosphoamino Acids/metabolism ; Phosphorylation ; Protein Binding ; Protein-Serine-Threonine Kinases/*metabolism ; Recombinant Proteins/genetics/metabolism

OBJECTIVE: To detect potential variation in an ethnic Han Chinese family affected with late-onset lipid storage myopathy. METHODS: Next generation sequencing (NGS) was used to screen disease-related genes in the proband. Suspected mutation was validated with PCR and Sanger sequencing in two patients, their father, and 100 healthy controls. RESULTS: Heterozygous c.770A>G (p.Tyr257Cys) and c.1395dupT (p.Gly466Tryfs) mutation were detected in the two patients. Their father was found to be heterozygous for the c.770A>G (p.Tyr257Cys) mutation, while the c.1395dupT (p.Gly466Tryfs) variation was not reported previously and not found among the healthy controls. CONCLUSION Mutations of the ETFDH gene probably underlie the pathogenesis in this family. The novel c.1395dupT (p.Gly466Tryfs) has enriched the mutation spectrum of EDFDH gene.
Asian Continental Ancestry Group ; Electron-Transferring Flavoproteins ; genetics ; Heterozygote ; High-Throughput Nucleotide Sequencing ; Humans ; Iron-Sulfur Proteins ; genetics ; Lipid Metabolism, Inborn Errors ; genetics ; Muscular Dystrophies ; genetics ; Mutation ; Oxidoreductases Acting on CH-NH Group Donors ; genetics

The methanol poisoning by oral intake or skin contact occurs occasionally, which may have serious consequences including blindness and/or death. Methanol and its metabolites, formaldehyde and formic acid, are associated with metabolic acidosis, visual dysfunction and neurological symptoms. At present, the mechanism of methanol poisoning primarily focuses on the cell hypoxia, the alteration of structure and biological activity induced by free radical and lactic acid. Meanwhile, methanol poisoning causes changes in the balance between the production of free radicals and antioxidant capacity and in the proteases-protease inhibitors system, which lead to a series of disturbances.
Acidosis/chemically induced* ; Animals ; Formaldehyde/poisoning* ; Formates/poisoning* ; Free Radicals/metabolism* ; Humans ; Methanol/poisoning* ; Nervous System/pathology* ; Oxidoreductases Acting on CH-NH Group Donors/metabolism* ; Proteins/metabolism* ; Vision Disorders/pathology*

BACKGROUNDThe critical roles of polyamines in cell growth and differentiation have made polyamine metabolic pathway a promising target for antitumor therapy. Recent studies have demonstrated in vitro that some antitumor polyamine analogues could be used as substrates and oxidized by purified recombinant human N(1)-acetylpolyamine oxidase (APAO, an enzyme that catabolizes natural polyamines), indicating a potential role of APAO in determining the sensitivity of cancer cells to specific antitumor analogues. This study evaluated, in vivo, the effect of APAO on cytotoxicity of antitumor polyamine analogue, N(1)-cyclopropylmethyl-N(11)-ethylnorspermine (CPENS) and its mechanism when highly expressed in human lung cancer line A549.

METHODSA clone with high expression of APAO was obtained by transfecting A549 lung cancer cell line with pcDNA3.1/APAO plasmid and selecting with quantitative realtime PCR and APAO activity assay. Cell proliferation was determined by MTT method and apoptosis related events were evaluated by DNA fragmentation, sub-G1/flow cytometric assay, western blotting (for cytochrome C and Bax) and colorimetric assay (for casapse-3 activity).

RESULTSA clone highly expressing APAO was obtained. High expression of APAO in A549 cells inhibited accumulation of CPENS, decreased their sensitivity to the toxicity of CPENS and prevented CPENS induced apoptosis.

CONCLUSIONThese results indicate a new drug resisting, mechanism in the tumor cells. High expression of APAO can greatly decrease the sensitivity of tumor cells to the specific polyamine analogues by detoxifying those analogues and prevent analogue induced apoptosis.

Apoptosis ; drug effects ; Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Flow Cytometry ; Humans ; Oxidoreductases Acting on CH-NH Group Donors ; genetics ; metabolism ; Polyamines ; metabolism ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the effect of 7- dehydrocholesterol reductas (Dhcr-7) gene silencing on the palatal development by sonic hedgehog (Shh)-bone morphogenetic protein2(BMP-2) signal pathway in vitro.

METHODSA total of 60 pairs of palatal shelves fromgestation day (GD) 13.5 mouse embryos were divided into three groups (A, B, C) of 20 randomly. In group A (control), palatal shelves were cultured with medium containing no cholesterol.In group B (Dhcr-7-siRNA), palatal shelves were cultured without cholesterol medium but containing Dhcr-7 siRNA adenovirus. After 48h, the culture medium of groups A and B were changed with medium without cholesterol. In group C (cholesterol), palatal shelves were cultured without cholesterol medium but containing Dhcr-7 siRNA adenovirus. After 48h, the culture medium of group C was changed with medium containing 600 mg/L cholesterol. After 72h again, tissues dyeing and scanning electron microscope (SEM) technique were used to observe morphological changes of palates. Both RT-PCR and Western blottingtechniques were used to measure mRNA and protein expressions for Dhcr-7, Shh, and BMP-2, respectively.

RESULTSThe tissues dyeing and SEM showedthat the palates fusedin groups A and Candthe palates did not fuse in group B eventually. The expression of both mRNA and proteins for Shh and BMP-2 in group B wasdecreased with the Dhcr-7 reduction. In group B, the mRNA and protein expression of Shh was separately 0.063±0.018 and 0.092±0.065;the mRNA and protein expression quantity of BMP- 2 was separately 0.054±0.018 and 0.049±0.021. In group A, the mRNA and protein expression of Shh was separately 0.667±0.093 and 0.639±0.078;the mRNA and protein expression of BMP-2 was separately 0.591 ± 0.043 and 0.569 ± 0.081. The difference of Shh and BMP- 2 mRNA and protein expression between A and B group were statistically significant separately (P < 0.05). The expression of both mRNA and protein for Dhcr-7 (0.074±0.034 and 0.075±0.028) did not changebasicallyin group C, compared with the Dhcr- 7expression of mRNA and protein (0.083±0.045; 0.067±0.065) in group B, the difference wasnot statistically significant(P > 0.05). In group C, the mRNA and protein expressionof Shh (0.649±0.085 and 0.608±0.092) and BMP-2 (0.578±0.062 and 0.548±0.065) were significantly increased. The difference of Shh and BMP-2 mRNA and protein expression between B and C group were statistically significant separately (P < 0.05).

CONCLUSIONSDhcr-7 could influence the expression of Shh and BMP-2. Dhcr-7 reductase regulated the palatal development by the Shh-BMP-2 signal pathway.

Animals ; Bone Morphogenetic Protein 2 ; genetics ; metabolism ; Cholesterol ; Culture Media, Conditioned ; chemistry ; pharmacology ; Hedgehog Proteins ; genetics ; metabolism ; Mice ; Oxidoreductases Acting on CH-CH Group Donors ; metabolism ; Palate ; growth & development ; metabolism ; RNA, Messenger ; analysis ; metabolism ; RNA, Small Interfering ; metabolism ; Random Allocation ; Signal Transduction