Substrate Specificity and Stereoselectivity of Rat Brain Microsomal Anandamide Amidohydrolase.
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Journal Article↗
Biomedical subjects
Z Meng
Publications and source records attributed to Z Meng.
Substrate specificity and stereoselectivity of rat brain microsomal anandamide amidohydrolase.
Anandamide amidohydrolase (AAH) catalyzes the hydrolysis of arachidonylethanolamide (anandamide), an endogenous cannabinoid receptor ligand. To delineate the structural requirements of AAH substrates, rat brain microsomal AAH hydrolysis of a series of anandamide congeners was studied using two reverse-phase high-performance liquid chromatography (RP-HPLC) assays developed in our laboratory. Arachidonamide (1) was found to be the best substrate with an apparent Km of 2.34 mM and a Vmax of 2.89 nmol/min/mg of protein. Although anandamide (2) has a similar Km value, its Vmax is approximately one-half that of arachidonamide. N, N-Bis(2-hydroxyethyl)arachidonamide (3) was not hydrolyzed, suggesting specificity for unsubstituted or mono-N-substituted arachidonamides. Analogues with a methyl group at the 1'-position of the ethanolamido headgroup were also found to have greater resistance to enzymatic turnover and therefore increased metabolic stability. The enzyme exhibited high stereoselectivity as the rate of hydrolysis of (R)-alpha-methanandamide (2.4%) (anandamide = 100%) was about 10-fold lower than that of its (S)-enantiomer (23%). In contrast, (R)-beta-methanandamide was 6-times more susceptible (121%) than the (S)-beta-enantiomer (21%). Interestingly, an inverse correlation was shown between AAH stereoselectivity and the brain cannabinoid receptor affinity as the enantiomers with high receptor affinity displayed low susceptibility to hydrolysis by AAH. Metabolic stability is also imparted to analogues with a short hydrocarbon headgroup as well as to those possessing 2-monomethyl or 2,2-dimethyl substituents. 2-Arachidonylglycerol and racemic 1-arachidonylglycerol were shown to be excellent AAH substrates. To identify AAH inhibitors, hydrolysis of anandamide was also studied in the presence of a select group of cannabimimetics. Of these, (-)-Delta8-THC and SR141716A, a biarylpyrazole CB1 antagonist, were found to inhibit enzymatic activity. These newly defined enzyme recognition parameters should provide a foundation for the rational development of stable, therapeutically useful anandamide analogues with high receptor affinity.
Amidohydrolases↗
Polymerase chain reaction-based deletion screening of bisulfite (sulfur dioxide)-enhanced gpt-mutants in CHO-AS52 cells.
In this study, we have examined the mutagenicity of bisulfite (sulfur dioxide) at the xathine-guanine phosphoribosyl transferase locus (gpt) in the pSV2 gpt-transformed CHO cell line, AS52. Our results provide evidence for bisulfite as a weak gene mutagen because the chemical at high doses and at high cytotoxicity causes a 4-fold increase in mutant frequency (MF) and less than a doubling of the gpt gene deletion frequency compared to control. We suggest that the increase of MF in bisulfite-treated cells results from bisulfite activity,as a comutagen, enhancing the induction effect of unknown endogenous or exogenous factors on spontaneous mutagenesis of AS52 cells. For the spontaneous, 5 mM bisulfite- and 10 mM bisulfite-enhanced spontaneous mutants in AS52 cells, the percentage of total deletion mutations of the gpt gene is 36%, 44% and 65%, respectively
Animals↗
Molecule imprinting chiral stationary phase.
Molecule imprinting polymers (MIPs) with high chiral selectivity for N(alpha)-protected amino acids were synthesized in polar solvent using acrylamide (AM) or combined functional monomer methacrylic acid (MAA) + vinyl pyridine (VP). Factors that influence the chiral selectivity of MIPs and mechanisms of the chiral recognition process were investigated. It was found that the rigid structure and the polar functional group of the print molecule were very important for the chiral selectivity of MIPs. For MIPs made using a combined functional monomer, ionic and hydrophobic interaction may be responsible for the chiral recognition process in aqueous media. The number of binding sites on MIPs and dissociation constants for the complex of enantiomers and MIPs were determined by frontal chromatography analysis.
Acrylamide↗
Effect of six steroidal saponins isolated from anemarrhenae rhizoma on platelet aggregation and hemolysis in human blood.
Six steroidal saponins were isolated from Anemarrhena asphodeloides Bunge (Liliaceae), a traditional chinese medicine, and named anemarrhenasaponin I (An-I), anemarrhenasaponin Ia (An-Ia), timosaponin B-I (TB-I), timosaponin B-II (TB-II), timosaponin B-III (TB-III), and timosaponin A-III (TA-III). The effects of these six compounds on platelet aggregation and hemolysis in human blood were studied. All these compounds provoked remarkable inhibiting effect on platelet aggregation, and activated partial thromboplastin times (APTT) are sensitive to the presence of these six compounds. Using an in vitro system, APTT was delayed with the increment of the concentrations of these six compounds. In these six compounds, only timosaponin A-III appeared a strong effect on hemolysis, and anemarrhenasaponin Ia had a slight effect on hemolysis, other had no effect on hemolysis. These results suggested that these steroidal saponins isolated from Anemarrhena asphodeloides Bunge (Liliaceae) might be used as a novel antithrombotic therapeutic agents in post-myocardial infarction.
Antithrombins↗
Hyperhomocysteinemia and hyperlipidemia in coronary heart disease.
OBJECTIVE: To examine the relationship between coronary heart disease (CHD) and serum lipid, plasma homocysteine (HCY) as well as the factors related to HCY metabolisms. METHODS: The mutation of the 677C-->T transition of 5, 10-methylenetetrahydrofolate reductase (MTHFR) was determined by PCR-based assay. Whole-blood and plasma folate and plasma vitamin B12, as cofactors of MTHFR, were determined by radio-immunologic assay. Plasma HCY was determined by HPLC. RESULTS: Patients with CHD had elevated plasma HCY concentrations (17.38 +/- 1.94 mumol/L vs 10.25 +/- 1.57 mumol/L, P < 0.01). In patients with myocardial infarction (MI) and family history (FH) of CHD, plasma HCY were elevated even higher (P < 0.05). Plasma HCY concentrations had significant non-linear inverse correlation with plasma folate and B12 concentrations, i.e. the lower the serum folate or B12 concentrations, the higher the plasma HCY concentrations (P < 0.01). Patients with homozygous mutants had higher plasma HCY concentrations. Patients with CHD had increased serum Chol and LDL-C and Apo-B levels (P < 0.01, P < 0.05 and P < 0.05 respectively). But plasma HCY concentrations had no correlation with serum lipid levels. 24.1% of the patients had high lipid and high HCY level, 25.9% had high lipid level and normal HCY level, 20.4% had normal lipid and high HCY level, and 29.6% had normal lipid and HCY level. CONCLUSIONS: HCY may have strong association with the genesis of CHD. Low plasma folate and B12 concentrations may induce Hyperhomocysteinemia [HH(e)]. Plasma HCY concentrations have no correlation with serum lipid levels, so HCY may be an independent risk factor. CHD may be induced by different mechanisms and can be classified into hyperlipidemia, HH (e) and normolipidemia, and normohomocysteinemia.
5,10-Methylenetetrahydrofolate Reductase (FADH2)↗
[Polylactic acid combined with recombinant human bone morphogenetic protein and basic fibroblast growth factor to repair the mandibular defects in rabbits].
OBJECTIVE: To study the effect of basic fibroblast growth factor(bFGF) on bone formation induced by recombinant human bone morphogenetic protein-2(rhBMP-2) in repair of mandibular defects in rabbits. METHODS: 56 adult domestic rabbits were classified into five groups. The mandiblar three-wall defects(1.5 cm x 0.5 cm, full thick) were created in right sides. The defects were implanted with various grafts: PLA/rhBMP-2/bFGF, PLA/rhBMP-2, PLA/bFGF, PLA and no implant as control group. At 2, 4 and 8 weeks after operation, the effectivenesses of defect repaire were observed by histological and roentgenographic analysis. RESULTS: The composite consisting of three materials stimulated more bone formation than of two materials, and the latter was more than single PLA(at 4 weeks postoperation, P < 0.05). All experimental groups, which had one or two growth factors, were significantly higher compared with control group in every stage of postimplantation. CONCLUSION: PLA has good biocompatibility, absorbability and osteoconductibility, rhBMP-2 and bFGF are cooperated in bone healing process, and PLA/rhBMP-2/bFGF is a promising bone substitute in clinical uses.
Animals↗
[A study on homozygous deletion, hypermethylation, mutation and expression of p16 gene in human breast cancer].
OBJECTIVE: To investigate the homozygous deletion (HZD), hypermethylation and mutation of p16 gene in human breast cancer and the relationship between the structural alterations of p16 gene and its expression. METHODS: PCR and PCR-methylation assay with silver staining (PCR-MASS) were used to detect HZD and hypermethylation of p16 gene exon 1 in 60 fresh breast cancers and 24 normal breast tissues adjacent to cancer (as control tissues). PCR-SSCP and DNA sequencing were used for the analysis of p16 gene mutation. Moreover, p16 gene and mRNA were also detected in the 60 cases. RESULTS: Of the 60 breast cancers, HZD was found in 7 cases, hypermethylation and mutation were found in 16 and 4 cases respectively, and the difference was statistically significant. The positive rates of p16 protein and mRNA in breast cancer were 28/60 and 39/60 respectively. CONCLUSION: The results demonstrate that several kinds of p16 gene structural changes exist in breast cancer. The structural changes of p16 gene cause abnormal p16 expression, the main mechanisms are hypermethylation, while HZD or mutation are the secondary causes. Abnormal expression of p16 gene then becomes involved in the development and metastasis of breast cancer.
Breast Neoplasms↗
[Sequencing and analysis of 16S rDNA sequences for P. cocovenenans subsp farinofermentans].
In order to fix the phylogenetic taxonomic position of P. cocovenenans subsp. farinofermentans and describe its relationships with other closed species, seven primers were designed to amplify and sequence the 16S rDNA of 4 strains of it by using a clonal sequencing or direct sequencing method. The 16S rDNA sequence of the 4 strains were aligned with the 16S rDNA sequences of other species in genus Burkholderia, and the polygenetic tree was produced by using a Clustal V and treeview software. The results showed that P. cocovenenans subsp. farinofermentans was in a high homology with Burkholderia gladioli and Burkholderia cocovenenans, and they formed an independent phylogenetic clustal of genus Burkholderia.
DNA, Recombinant↗
[Effects of HCY-2 gene transfer on the development of chick neural tube].
In order to explore the relationship between novel gene HCY-2 and neural tube defects (NTDs) and to investigate whether HCY-2 gene was genotoxic, a mammalian expression vector containing HCY-2 cDNA (2014 bp) was transferred into chick fertilized ovum by using lipofectin reagent, then the biological effects of HCY-2 gene on embryos in culture for 3.5 days were observed. Western blotting and immunohistochemistry were used to analyse the expression and distribution of HCY-2 after gene being transferred. HCY-2 gene (5-20 micrograms/chick ovum) could induce NTDs in organogenesis period. Western blotting and immunohistochemical staining showed that HCY-2 protein could be greatly expressed by day 3.5, the time which NTDs developed, with stronger staining at the site of embryonic brain as compared with controls. Under electron microscope, it was observed that HCY-2 gene could damage embryonic cellular ultrastructures and cause excessive apoptosis of neurocytes. These findings suggested that HCY-2 gene with its product was dysmorphogenic to the neural tube, might be a genotoxic factor or teratogenic agent and should be responsible for chick NTDs and probably human NTDs as well.
Animals↗
[Treatment of chronic prostatitis with computerized herbal drug penetrator].
A computerized herbal drug penetrator for chronic prostatitis was designed and developed on the basis of a combination of the authors' clinical practice with modern Traditional Chinese Medicinal theories. The device has integrated many hi-techs of current engineering science. It works by applying special herbal medicine, electro-magnetic effects, thermal moxibustion, perfusion and pulsated massage with water-capsule. The result of using this technique in the treatment of 100 cases has demonstrated its satisfying effectiveness.
Acupuncture Therapy↗
[Beaded molecule imprinted polymer for stereo isomer separation].
Beaded molecule imprinted polymer (MIP) was made by suspension polymerization. Particles with the size of 50-70 microns in diameter were collected and evaluated in HPLC mode to separate stereo isomers. Stereo isomers cinchonine and cinchonidine were successfully discriminated with selectivity factor of 2.89 and resolution factor of 0.76. Stereo selectivity of the MIP was found to come from both the interaction between the analyte and carboxyl group on the MIP and the similarity between the stereo structure of imprinted molecule and the MIP. The thermal analysis results showed that the MIP had high thermal stability with initial thermal decomposition temperature of 320 degrees C. The pore volume of the MIP was 0.1849 mL/g, the specific surface area was 126.84 sqm/g and the average pore diameter was 5.8 nanometer. Scanning electron microscopy showed that MIP had perfect spherical morphology.
Chromatography, High Pressure Liquid↗
Coral as a carrier for recombinant human bone morphogenetic protein-2.
By combining coral with recombinant human bone morphogenetic protein-2 (rhBMP-2), rhBMP-2/coral composite was obtained in this study. Following implantation of the composite into the muscle pouches of mice, cartilage growth was induced in the pores or on the surface of the implants at one week, woven bone at three week and lamellar bone with bone marrow at six week, and coral was absorbed partially. The induced formation of endochondral bone was time-related and rhBMP-2 dose-related. The results of this study indicate that the composite possesses a superior ability of osteogenesis, and coral acts as one of the most suitable rhBMP-2 slowrelease carriers currently available. The composite will be a new type of bone substitute to be used in orthopaedics and maxillofacial surgery.
Animals↗
Age- and sex-related differences in zinc and lead levels in human hair.
The hair of 1518 healthy subjects between newborn and 70 yr old living in Linfen city and the surrounding rural area in Shanxi province, North China, was analyzed by means of a flame atomic absorption spectrophotometer method in order to determine the concentrations of zinc (Zn) and lead (Pb). The collected samples were subdivided on the basis of age, sex, and place of residence (urban and rural). In female hair, the contents of Zn were higher than in male, but the hair Pb concentrations of males were slightly higher than those of females. Age seems to have a different influence on the two elements. The maximum amount of hair Zn was found generally in newborns. From the baby group (<1 yr) to the puberty group (15-19 yr), Zn levels in hair increased with age, and then Zn levels decreased with age from the puberty group to elderly group (61-70 yr). The hair Pb levels were the lowest in newborns, increased with age from newborn to infant (1-2 yr), and then decreased with age from infant to the elderly. It was shown that the hair Pb and Zn levels were inversely related in various age groups, suggesting that there is possibly antagonism between Pb and Zn in the human body. The results also showed the hair Pb levels of subjects living in the urban area, except for newborns, were higher than those of subjects residing in the rural area. However, no difference between the hair Zn contents of the populations residing in urban and rural areas.
Adolescent↗
Production of emotional facial expressions in European American, Japanese, and Chinese infants.
European American, Japanese, and Chinese 11-month-olds participated in emotion-inducing laboratory procedures. Facial responses were scored with BabyFACS, an anatomically based coding system. Overall, Chinese infants were less expressive than European American and Japanese infants. On measures of smiling and crying, Chinese infants scored lower than European American infants, whereas Japanese infants were similar to the European American infants or fell between the two other groups. Results suggest that differences in expressivity between European American and Chinese infants are more robust than those between European American and Japanese infants and that Chinese and Japanese infants can differ significantly. Cross-cultural differences were also found for some specific brow, cheek, and midface facial actions (e.g., brows lowered). These are discussed in terms of current controversies about infant affective facial expressions.
Child, Preschool↗
[Immunogenicity and efficacy trials of live attenuated hepatitis A vaccines].
OBJECTIVE: To study two live vaccines, H2 and LA-1 strains, both attenuated in growth on human diploid cell culture at lower temperature (32 degrees C). METHODS: Randomized, controlled trials were performed among half million children. 135,340 children were divided into vaccine or control groups by individual and 360,012 by cluster. Those susceptibles with negative anti-HAV were bled at 2-6 months after the vaccination and blood test for anti-HAV. RESULTS: 37 hepatitis A cases were found in the control group. The protective efficacy of the two vaccines was 100%, and the 95% lower confidence limit (one sided) of combined efficacy of the two vaccines was 92.1%, that was comparable with the two inactivated vaccines produced by SmithKline beecham or Merck Sharp Dhome. CONCLUSION: The vaccines are safe, and the seroconversion to H2 strain (10(7.0) TCID50) and LA-1 strain (10(6.75) TCID50) is 94.87% and 83.16% respectively.
Child↗
[Expression and mutation of p53 and H-ras genes in the carcinogenesis and development of gastric adenocarcinoma induced by MNNG in rats].
OBJECTIVE: To investigate the effect of p53 and H-ras genes in the carcinogenesis of and development of gastric cancer induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Wistar rats. METHODS: The expression of p53 and H-ras genes in normal mucosa, precancerous lesions and induced-cancers were studied immunohistochemically. DNA isolated from the precancerous lesion and tumors were analysed by PCR-SSCP for exon 5-8 of p53 gene and exon 1 and 2 of H-ras gene. RESULTS: p53 protein were detected in 50% (20/40) of the gastric cancers studied, but not in the precancerous lesion, while p21 ras protein seen in 23% (9/40) of the gastric cancers and 44% (23/52) of the precancerous lesions. p53 gene mutations were detected in 45% (18/40) of the cancers and none in the precancerous lesions by PCR-SSCP. H-ras gene mutation was not found in either the cancers or the precancerous lesions. CONCLUSIONS: The activation of ras gene is an early event and may be involved in the carcinogenesis of gastric cancer induced by MNNG. Inactivation of p53 gene may influence the development of the cancer.
Adenocarcinoma↗
[Development and applications of high performance membrane chromatography].
Development and applications of high performance membrane chromatography (HPMC) are introduced. Base membranes, membrane geometries and separations of biomolecules using high performance affinity membrane chromatography (HPAMC), high performance ion-exchange membrane chromatography (HPIEMC) and high performance hydrophobic interaction membrane chromatography (HPHIMC) are reviewed. Enantiomeric separation using HPMC are prospected. Seventy-six references are cited.
Chromatography, Affinity↗