OBJECTIVETo investigate the effect of Angelicae Pubescentis Radix (APR) on the activity of endocannabinoid hydrolase and N-acylethanolamine-hydrolyzing acid amidase (NAAA), and to demonstrate the mechanism of anti-inflammatory effect of APR by in vitro lipopolysaccharide (LPS)-induced inflammation model.

METHODAPR essential oil was extracted by steam distillation, and the chemical components were identified by GC-MS. Enzymatic activity was performed by using recombinant NAAA-overexpressing protein and detected by LC-MS. Lipids were extracted by methonal/chloroform mixure and analyzed by LC-MS. mRNA and protein expression levels of proinflammatory genes were examined by Real time-PCR and ELISA assay kit, respectively. The content of nitro oxide (NO) was detected by Griess reaction.

RESULTTwenty active components were identified from APR essential oil which inhibited NAAA activity in a dose-dependent manner. On the LPS-induced RAW264.7 cells, APR essential oil reversed LPS-suppressed N-palmitoylethanolamide (PEA) contents in a dose-dependent manner and reduced LPS-induced proinflammatory genes, TNF-alpha and IL-6. Moreover, APR essential oil reduced the mRNA expression of iNOS, subsequently reduced the release of NO, a classic inflammatory marker.

CONCLUSIONThe research demonstrated that the effect of APR on inflammation is mediated by the inhibition of NAAA activity, which increase the cellular endobioactor PEA levels and decrease proinflammatory factor. The results suggest that APR can serve as a nature NAAA inhibitor.

Amidohydrolases ; antagonists & inhibitors ; Angelica ; chemistry ; Animals ; Anti-Inflammatory Agents ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Lipopolysaccharides ; pharmacology ; Mice ; Oils, Volatile ; analysis ; pharmacology

Three model silkworms, highly resistant strain, highly susceptible strain and their near isogenic line were established by hybridization and backcross. The resistance of silkworm (Bombyx mori L.) to BmNPV was studied at proteomic level using two-dimensional gel electrophoresis and MALDI TOF/TOF MS. Differential expression profiles of proteome in resistant strain, susceptible strain and near isogenic line were obtained, and 180, 190 and 187 of protein spots were shown, respectively. Among them, 80% were concentrated in pI 5-9. Twelve differential protein spots in total were obtained from 3 gels. Using MALDI TOF/TOF MS, five proteins, including aminoacylase, were identified from these spots. The exclusive expression of aminoacylase in highly resistant strain and near isogenic line and its absence in susceptible strain suggest that it might be a BmNPV-resistance related protein.
Amidohydrolases ; analysis ; genetics ; Animals ; Bombyx ; chemistry ; genetics ; virology ; Electrophoresis, Gel, Two-Dimensional ; Hemolymph ; chemistry ; Host-Pathogen Interactions ; genetics ; Insect Proteins ; analysis ; Nucleopolyhedrovirus ; pathogenicity ; physiology ; Proteome ; analysis ; Proteomics ; methods ; Recombination, Genetic ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVE: To investigate the protective effect of losartan against on injury induced by ox-LDL in endothelial cells and the relationship with asymmetric dimethylarginine (ADMA). METHODS: Endothelial injury was induced by incubation with ox-LDL 100 mg/L in cultured HUVECs for 24 h, and the levels of ADMA, lactate dehydrogenase (LDH), nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) in the conditioned medium were measured. The activity of dimethylarginine dimethylaminohydrolase (DDAH) of cultured endothelial cells was also determined. RESULTS: Incubation of endothelial cells with ox-LDL 100 mg/L for 24 h induced a marked elevation of the levels of ADMA, LDH and TNF-alpha in the conditioned medium and a significant decrease in the activity of DDAH and the content of NO (P < 0.05). Pretreatment with losartan (10(-8) - 10(-6) mmol/L) significantly inhibited the increased levels of ADMA, LDH and TNF-alpha, attenuated the decreased levels of NO and the decreased activity of DDAH induced by ox-LDL (P < 0.05). CONCLUSION Losartan may preserve ox-LDL-induced endothelial cell injury by increasing the DDAH activity and decreasing the ADMA level.
Amidohydrolases ; metabolism ; Arginine ; analogs & derivatives ; analysis ; Cells, Cultured ; Endothelium, Vascular ; pathology ; Humans ; L-Lactate Dehydrogenase ; analysis ; Lipoproteins, LDL ; adverse effects ; Losartan ; pharmacology ; Nitric Oxide ; analysis ; Protective Agents ; pharmacology ; Umbilical Veins ; cytology

Renal cell carcinoma (RCC) is one of the most malignant tumors in urology, and due to its insidious onset patients frequently have advanced disease at the time of clinical presentation. Thus, early detection is crucial in management of RCC. To identify tumor specific proteins of RCC, we employed proteomic analysis. We prepared proteins from conventional RCC and the corresponding normal kidney tissues from seven patients with conventional RCC. The expression of proteins was determined by silver stain after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The overall protein expression patterns in the RCC and the normal kidney tissues were quite similar except some areas. Of 66 differentially expressed protein spots (p<0.05 by Student t-test), 8 different proteins from 11 spots were identified by MALDI-TOF-MS. The expression of the following proteins was repressed (p<0.05); aminoacylase-1, enoyl-CoA hydratase, aldehyde reductase, tropomyosin alpha-4 chain, agmatinase and ketohexokinase. Two proteins, vimentin and alpha-1 antitrypsin precursor, were dominantly expressed in RCC (p<0.05).
Aged ; Aldehyde Reductase/analysis ; Amidohydrolases/analysis ; Carcinoma, Renal Cell/*metabolism/pathology ; Comparative Study ; Electrophoresis, Gel, Two-Dimensional ; Enoyl-CoA Hydratase/analysis ; Female ; Fructokinases/analysis ; Humans ; Kidney Neoplasms/*metabolism/pathology ; Male ; Middle Aged ; Proteome/*analysis ; Proteomics/*methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Tropomyosin/analysis ; Ureohydrolases/analysis ; Vimentin/analysis ; alpha 1-Antitrypsin/analysis

A strain, MMR003, used for D-p-HPG production in industry was classified as Burkholderia pickettii by morphological observation and biochemical characterization. The gene encoding the D-hydantoinase enzyme was cloned, sequenced and expressed in Escherichia coli. The nucleotide sequence of the 5.0 kb insert of subclone pXZ-total was determined. One open reading frame of 1374 bp was found and predicted to encode a polypeptide consisting of 458 amino acids in size of 50 kD. The amino acid sequence alignment of D-hydantoinase from Burkholderia pickettii shows the 85% homologous with the corresponding enzyme from Agrobacterium radiobacter NRRL B11291. The D-hydantoinase gene (dha) harboured in the plasmid pXZPH2 in E. coli BL21(DE3) was highly expressed by IPTG induction. The D-hydantoinase activity for D, L-p-hydroxyphenylhydantion is 0.66 u/mL broth, which is 2-fold increase compared to the parent strain Burkholderia pickettii.
Amidohydrolases ; genetics ; metabolism ; Amino Acid Sequence ; Animals ; Burkholderia ; classification ; enzymology ; genetics ; Cloning, Molecular ; DNA Probes ; Escherichia coli ; Gene Expression ; Molecular Sequence Data ; Recombination, Genetic ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid

BACKGROUNDHyperhomocysteinemia (HHcy)-mediated dysfunction of endothelial NO system is an important mechanism for atherosclerotic pathogenesis. Dimethylarginine dimethylaminohydrolase (DDAH) is the key enzyme for degrading asymmetric dimethylarginine (ADMA), which is an endogenous inhibitor of endothelial nitric oxide (NO) synthase (eNOS). This study was designed to investigate whether the dysfunction of endothelial NO system originates from HHcy-mediated aberrant methylation modification in promotor region of DDAH2 gene.

METHODSHuman umbilical vein endothelial cells (HUVECs) were cultured to the third generation and treated with homocysteine (Hcy) at different concentrations (0, 10, 30, 100, and 300 micromol/L) for 72 hours. The methylation pattern in promoter region CpG island of DDAH2 gene was analyzed by nested methylation-specific PCR (nMSP). The mRNA expression of eNOS gene and DDAH2 gene was detected by semi-quantitative RT-PCR. The activity of DDAH2 and eNOS in cells, and the concentrations of ADMA and NO in culture medium were assayed respectively.

RESULTSMild increased concentration of Hcy (10 and 30 micromol/L) induced hypomethylation, while high concentration of Hcy (100 and 300 micromol/L) induced hypermethylation in the promoter CpG island of DDAH2 gene. The mRNA expression of DDAH2 increased in mild enhanced concentration of Hcy, and decreased in high concentration of Hcy correspondingly. The inhibition of DDAH2 activity, the increase of ADMA concentration, the reduction of eNOS activity and the decrease of NO production were all consistently relevant to the alteration of Hcy concentration.

CONCLUSIONThe increased concentration of Hcy induced aberrant methylation pattern in promotor region of DDAH2 gene and the successive alterations in DDAH/ADMA/NOS/NO pathway, which showed highly relevant and dose-effect relationship. The results suggested that the dysfunction of endothelial NO system induced by HHcy could be partially originated from Hcy-mediated aberrant methylation in DDAH2 gene.

Amidohydrolases ; genetics ; Arginine ; analogs & derivatives ; blood ; Cells, Cultured ; DNA Methylation ; drug effects ; Homocysteine ; pharmacology ; Humans ; Nitric Oxide ; analysis ; physiology ; Nitric Oxide Synthase Type III ; genetics ; physiology ; Promoter Regions, Genetic

Recently, a mitochondrial ceramidase has been identified and cloned, whose mitochondrial localization strongly suggests the existence of an unexpected mitochondrial pathway of ceramide metabolism that may play a key role in mitochondrial functions, especially in the regulation of apoptosis. To explore the biological effect of mitochondrial ceramidase on cells, pcDNA 3.1/His-CDase plasmid, containing mitochondrial ceramidase cDNA sequence, was transducted into K562 cells mediated by liposome, and G418 was used to screen for positive colonies. A stable transfected K562 cell line was established and named as 'K562TC'. The difference between K562 and K562TC cells in chemotheraputic cytotoxicity response and serum-withdrawal resistance and Bcl-2 protein expression were evaluated by MTT assay, annexin V/PI test, flow cytometry or Western blotting, respectively. The results showed that although survival was comparable between K562 and K562TC cells after exposed to adriamycin, etoposide or arsenious acid, K562TC cells with elevated Bcl-2 protein expression level as identified by FCM or Western blotting revealed stronger resistance to apoptosis induced by serum withdrawal than their parental cells. Inhibition of mitochondrial ceramidase expression in K562TC cells by its specific antisense oligodeoxynucleotide was correlated with a decrease in Bcl-2 protein level. N, N-dimethylsphingosine, a sphingosine kinase inhibitor, depleted intracellular sphingosine-1-phosphate production, also abrogated Bcl-2 protein expression in K562TC cells, while Bcl-2 protein level in K562 cells was up-regulated by exogenous sphingosine-1-phosphate. It is concluded that mitochondrial ceramidase overexpression in K562 cells leads to markedly elevated level of Bcl-2 protein and results in more resistance to serum withdrawal. This effect is initiated not by sphingosine, the direct metabolite of mitochondrial ceramidase, but via sphingosine-1-phosphate, its phosphorylated form. This is the first evidence that mitochondrial ceramidase, through its sphingoid metabolite sphingosine-1-phosphate, up-regulates Bcl-2 protein expression in K562 cells.
Amidohydrolases ; physiology ; Apoptosis ; Arsenites ; pharmacology ; Ceramidases ; Doxorubicin ; pharmacology ; Etoposide ; pharmacology ; Humans ; K562 Cells ; Lysophospholipids ; physiology ; Mitochondria ; enzymology ; Oligonucleotides, Antisense ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Sphingosine ; analogs & derivatives ; physiology ; Up-Regulation