Evaluation of the real-time fluorescence loop-mediated isothermal amplification assay for the detection of Ureaplasma urealyticum.

Ureaplasma urealyticum (UU) is commonly present in human reproductive tract, which frequently leads to genital tract infection. Hence, there is an urgent need to develop a rapid detection method for UU. In our study, a real-time fluorescence loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of UU. Two primers were specifically designed based on the highly conserved regions of ureaseB genes. The reaction was carried out for 60 min in a constant temperature system using Bst DNA polymerase, and the process was monitored by real-time fluorescence signal, while polymerase chain reaction (PCR) was performed simultaneously. In real-time fluorescence LAMP reaction system, positive result was only obtained for UU among 9 bacterial strains, with detection sensitivity of 42 pg/µL (4.2 × 105 CFU/mL), and all 16 clinical samples of UU could be detected. In conclusion, real-time fluorescence LAMP is a simple, sensitive, specific and effective method compared with conventional PCR, which shows great promise in the rapid detection of UU.

Biotransformation of cinobufagin by Cunninghamella elegans.

Cunninghamella elegans has been employed for the biotransformation of cinobufagin to afford 5 metabolites. The structures of the transformation products have been characterized as 12alpha-hydroxybufagin, 11alpha-hydroxybufagin, 12beta-hydroxy-desacetylcinobufagin, 3-oxo-12alpha-hydroxybufagin and 12beta-hydroxybufagin. Products 12alpha-hydroxybufagin and 11alpha-hydroxybufagin are new compounds. In vitro both the biotransformation products and cinobufagin all showed cytotoxic activities against HeLa cells.

[Molecular and Genetic Mechanism of Three Cases of Para-Bombay Blood Group].

OBJECTIVE: To explore molecular and genetic mechanism of 3 cases of para-Bombay blood group. METHODS: The bood samples of proband and family members were selected to identify their blood groups with conventional serologic methods, and salivary components carrying the ABH antigens were detected. The coding regions of FUT1 as well as exon 6 and 7 of the ABO gene were amplified using polymerase chain reaction(PCR), and the FUT1 gene was directly sequenced. RESULTS: All the 3 cases of proband were confirmed as para-Bombay blood group. Direct sequencing revealed h new2 (nt328G→A) and h1(nt 547 ΔAG) in FUT1 gene of the proband 1, and FUT1 genotype was h1/h new2. However, the genotypes of his parents were H/h1 and H/h new2, which were non-Bombay individuals. The FUT1 genotypes of proband 2 and 3 were h1h2 (nt 547 ΔAG) and h1h2 (nt 880 ΔTT), respectively. CONCLUSION: The technology of molecular biology can be used to detect the base deletion mutations in FUT1 gene, which contributes to the analysis of molecular and genetic mechanism of para-Bombay blood group.

[The inhibition of CYP2C9 isoenzyme in Cunninghamella blakesleeana AS 3. 910].

AIM: To investigate the variation of CYP2C9 isoenzyme activity in the microbial model in response to inhibitors of CYP2C9. METHODS: Using C. blakesleeana AS 3. 910 as a model strain, the impact of CYP2C9 inhibitors on the metabolites yields of CYP2C9 substrates was determined and the drug-drug interactions among CYP2C9 substrates were evaluated. Liquid chromatography-mass spectrometry was used to analyze biotransformation products. RESULTS: Benzbromarone decreased the yield of 4'-hydroxytolbutamide from 100% to 14.5%; sulfaphenazole decreased the yield of O-demethylindomethacin from 75.2% to 9.9%; valproic acid decreased the yield of 4'-hydroxydiclofenac from 98.6% to 2.7%, separately. Tolbutamide, indomethacin and diclofenac interacted with each other, resulting in the decreased formation of metabolites catalyzed by CYP2C9. CONCLUSION: Three CYP2C9 inhibitors inhibit the activity of CYP2C9 isoenzyme in C. blakesleeana AS 3. 910 differently, and there are drug-drug interactions among CYP2C9 substrates.

Biotransformation of metoprolol by the fungus Cunninghamella blakesleeana.

AIM: To investigate the biotransformation of metoprolol, a beta1-cardioselective adrenoceptor antagonist, by filamentous fungus, and to compare the parallels between microbial transformation and mammalian metabolism. METHODS: Five strains of Cunninghamella (C elegans AS 3.156, C elegans AS 3.2028, C echinulata AS 3.2004, C blakesleeana AS 3.153 and AS 3.910) were screened for the ability to transform metoprolol. The metabolites of metoprolol produced by C blakesleeana AS 3.153 were separated and assayed by liquid chromatography-tandem mass spectrometry (LC/MS(n)). The major metabolites were isolated by semipreparative HPLC and the structures were identified by a combination of LC/MS(n) and nuclear magnetic resonance analysis. RESULTS: Metoprolol was transformed to 7 metabolites; 2 were identified as new metabolites and 5 were known metabolites in mammals. CONCLUSION: The microbial transformation of metoprolol was similar to the metabolism in mammals. The fungi belonging to Cunninghamella species could be used as complementary models for predicting in vivo metabolism and producing quantities of metabolite references for drugs like metoprolol.

Biotransformation of indomethacin by the fungus Cunninghamella blakesleeana.

AIM: To investigate the biotransformation of indomethacin, the first of the newer nonsteroidal anti-inflammatory drugs, by filamentous fungus and to compare the similarities between microbial transformation and mammalian metabolism of indomethacin. METHODS: Five strains of Cunninghamella (C elegans AS 3.156, C elegans AS 3.2028, C blakesleeana AS 3.153, C blakesleeana AS 3.910 and C echinulata AS 3.2004) were screened for their ability to catalyze the biotransformation of indomethacin. Indomethacin was partially metabolized by five strains of Cunninghamella, and C blakesleeana AS 3.910 was selected for further investigation. Three metabolites produced by C blakesleeana AS 3.910 were isolated using semi-preparative HPLC, and their structures were identified by a combination analysis of LC/MS(n) and NMR spectra. These three metabolites were separated and quantitatively assayed by liquid chromatography-ion trap mass spectrometry. RESULTS: After 120 h of incubation with C blakesleeana AS 3.910, approximately 87.4% of indomethacin was metabolized to three metabolites: O-desmethylindomethacin (DMI, M1, 67.2%), N-deschlorobenzoylindomethacin (DBI, M2, 13.3%) and O-desmethyl-N-deschlorobenzoylindomethacin (DMBI, M3, 6.9%). Three phase I metabolites of indomethacin produced by C blakesleeana AS 3.910 were identical to those obtained in humans. CONCLUSION: C blakesleeana could be a useful tool for generating the mammalian phase I metabolites of indomethacin.