Discovery and Characterization of the Key Constituents in Ginkgo biloba Leaf Extract With Potent Inhibitory Effects on Human UDP-Glucuronosyltransferase 1A1.

Human UDP-glucuronosyltransferase 1A1 (hUGT1A1) is one of the most essential phase II enzymes in humans. Dysfunction or strong inhibition of hUGT1A1 may result in hyperbilirubinaemia and clinically relevant drug/herb-drug interactions (DDIs/HDIs). Recently, a high-throughput fluorescence-based assay was constructed by us to find the compounds/herbal extracts with strong inhibition against intracellular hUGT1A1. Following screening of over one hundred of herbal products, the extract of Ginkgo biloba leaves (GBL) displayed the most potent hUGT1A1 inhibition in HeLa-UGT1A1 cells (Hela cells overexpressed hUGT1A1). Further investigations demonstrated that four biflavones including bilobetin, isoginkgetin, sciadopitysin and ginkgetin, are key constituents responsible for hUGT1A1 inhibition in living cells. These biflavones potently inhibit hUGT1A1 in both human liver microsomes (HLM) and living cells, with the IC50 values ranging from 0.075 to 0.41 µM in living cells. Inhibition kinetic analyses and docking simulations suggested that four tested biflavones potently inhibit hUGT1A1-catalyzed NHPN-O-glucuronidation in HLM via a mixed inhibition manner, showing the K i values ranging from 0.07 to 0.74 µM. Collectively, our findings uncover the key constituents in GBL responsible for hUGT1A1 inhibition and decipher their inhibitory mechanisms against hUGT1A1, which will be very helpful for guiding the rational use of GBL-related herbal products in clinical settings.

Cytochrome P450s Are Essential for Insecticide Tolerance in the Endoparasitoid Wasp Meteorus pulchricornis (Hymenoptera: Braconidae).

With the widespread application of insecticides, parasitoid wasps may also be under risk when exposed to insecticides directly at their free-living stages. The endoparasitoid wasp Meteorus pulchricornis is the predominant natural enemy of many lepidopteran pests, such as Spodoptera litura and Helicoverpa armigera. The cytochrome P450 monooxygenases constitute a ubiquitous and complex superfamily of hydrophobic, haem-containing enzymes. P450s are involved in the detoxification of many xenobiotics. However, their exact roles in the tolerance mechanism in parasitoids toward insecticides has received less attention. Here, 28 P450 genes in M. pulchricornis were identified from a previously constructed transcriptome dataset. These P450 genes belonged to CYP2, -3, and -4, and mitochondrial clans. Subsequently, eight candidate MpulCYPs were selected from four CYP clans to validate their expression patterns under phoxim, cypermethrin, and chlorfenapyr exposure by qRT-PCR. The results showed that all three insecticides had significant effects on the expression of MpulCYPs. To further study the function of P450s, CYP369B3 was silenced, and its expression levels of CYP369B3 were significantly decreased. Survival analysis indicated that after dsRNA injection, the mortality rate of wasps was significantly increased when M. pulchricornis females were exposed to insecticides compared to control groups. Our findings provide a theoretical base for elucidating the mechanism of insecticide tolerance and promote functional research on P450 genes in parasitoid wasps.

Accurate and sensitive detection of Catechol-O-methyltransferase activity by liquid chromatography with fluorescence detection.

Catechol-O-methyltransferase (COMT) is a major drug metabolizing enzyme in humans. COMT expression is directedly associated with various mental diseases and cancers due to its essential role in catalyzing metabolic inactivation of endogenous catecholamines and catechol estrogens. However, a practical method to precisely measure COMT activities in biological samples is lacking. In the current study, we established a liquid chromatography-fluorescence detection (LC-FD) method based on fluorometric detection of the methylated product of 3-BTD, a fluorescent probe for COMT, to sensitively quantify COMT activities in human erythrocytes and cell homogenates. Assay validation of the established LC-FD based method was conducted for selectivity and sensitivity, range of linearity, precision and accuracy, recovery, biological matrices effect and stability. The limit of quantification for 3-BTMD (the methylated product of 3-BTD by COMT) of this method was 0.0083 nM, which is nearly 10-fold lower than that for previously published methods. The method was precise with intra- and inter-day relative standard deviation (RSD) lower than 5%. In addition, this method showed an excellent anti-interference ability with no effects of the endogenous substances on the fluorometric detection of 3-BTMD. The practical use of this method was established by its successful application for the measurement of COMT activities in individual human erythrocytes (n = 13), and in cell homogenates generated from four different human cell lines. Our results suggest that this method will be of great value in accurately determining the native activity of COMT in biological samples, which is beneficial for a complete understand of the role of COMT both in physiological and pathological conditions.

An ultra-sensitive and easy-to-use assay for sensing human UGT1A1 activities in biological systems.

The human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most essential conjugative enzymes, is responsible for the metabolism and detoxification of bilirubin and other endogenous substances, as well as many different xenobiotic compounds. Deciphering UGT1A1 relevance to human diseases and characterizing the effects of small molecules on the activities of UGT1A1 requires reliable tools for probing the function of this key enzyme in complex biological matrices. Herein, an easy-to-use assay for highly-selective and sensitive monitoring of UGT1A1 activities in various biological matrices, using liquid chromatography with fluorescence detection (LC-FD), has been developed and validated. The newly developed LC-FD based assay has been confirmed in terms of sensitivity, specificity, precision, quantitative linear range and stability. One of its main advantages is lowering the limits of detection and quantification by about 100-fold in comparison to the previous assay that used the same probe substrate, enabling reliable quantification of lower amounts of active enzyme than any other method. The precision test demonstrated that both intra- and inter-day variations for this assay were less than 5.5%. Furthermore, the newly developed assay has also been successfully used to screen and characterize the regulatory effects of small molecules on the expression level of UGT1A1 in living cells. Overall, an easy-to-use LC-FD based assay has been developed for ultra-sensitive UGT1A1 activities measurements in various biological systems, providing an inexpensive and practical approach for exploring the role of UGT1A1 in human diseases, interactions with xenobiotics, and characterization modulatory effects of small molecules on this conjugative enzyme.

Expression Analysis of mRNA Decay of Maternal Genes during Bombyx mori Maternal-to-Zygotic Transition.

Maternal genes play an important role in the early embryonic development of the silkworm. Early embryonic development without new transcription depends on maternal components stored in the egg during oocyte maturation. The maternal-to-zygotic transition (MZT) is a tightly regulated process that includes maternal mRNAs elimination and zygotic transcription initiation. This process has been extensively studied within model species. Each model organism has a unique pattern of maternal transcriptional clearance classes in MZT. In this study, we identified 66 maternal genes through bioinformatics analysis and expression analysis in the eggs of silkworm virgin moths (Bombyx mori). All 66 maternal genes were expressed in vitellogenesis in day eight female pupae. During MZT, the degradation of maternal gene mRNAs could be divided into three clusters. We found that eight maternal genes of cluster 1 remained stable from 0 to 3.0 h, 17 maternal genes of cluster 2 were significantly decayed from 0.5 to 1.0 h and 41 maternal genes of cluster 3 were significantly decayed after 1.5 h. Therefore, the initial time-point of degradation of cluster 2 was earlier than that of cluster 3. The maternal gene mRNAs decay of clusters 2 and 3 is first initiated by maternal degradation activity. Our study expands upon the identification of silkworm maternal genes and provides a perspective for further research of the embryo development in Bombyx mori.

Study on the Role of Cytc in Response to BmNPV Infection in Silkworm, Bombyx mori (Lepidoptera).

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the primary pathogens of the silkworm. Cytochrome c (cytc) showed a significant response to BmNPV infection in our previous transcriptome study. However, little is known about the role of Bombyx mori cytc (Bmcytc) in resistance to BmNPV infection. In this study, the expression levels analysis of Bmcytc showed stable expression levels in selected tissues of the resistant strain AN following BmNPV infection, while there was downregulation in the susceptible strain p50, except in the malpighian tubule. To further study the role of Bmcytc in viral infection, Bmcytc was knocked down with siRNA in vitro, resulting in significant downregulation of selected downstream genes of the mitochondrial pathway, including Bmapaf, Bmcaspase-Nc, and Bmcaspase-1; this was also confirmed by overexpression of Bmcytc using the pIZT/V5-His-mCherry insect vector, except Bmcaspase-1. Moreover, knockdown of Bmcytc significantly promoted the infection process of BmNPV in vitro, while the infection was inhibited by overexpression of Bmcytc at the early stage and subsequently increased rapidly. Based on these results, we concluded that Bmcytc plays a vital role in BmNPV infection by regulating the mitochondrial apoptosis pathway. Our work provides valuable data for the clarification of the mechanism of silkworm resistance to BmNPV infection.

Glucuronidation of D-Luciferin In Vitro: Isoform Selectivity and Kinetics Characterization.

BACKGROUND: D-luciferin is one of the most commonly used substrates in bioluminescence imaging for real-time monitoring of sophisticated biological processes in models of human biology or disease in vitro and in vivo. D-luciferin is rapidly cleared from blood circulation after being exogenously delivered in vivo and the presence of phenolic groups indicates that glucuronide conjugation is a possible metabolic pathway for the compound. OBJECTIVES: This study aimed to characterize the glucuronidation pathway of D-luciferin in human liver microsomes (HLM) and human intestine microsomes (HIM). METHODS: HLM and HIM were employed to catalyze the glucuronidation of D-luciferin in vitro. The activity of recombinant uridine-diphospho-glucuronosyltransferase (UGT) isoforms towards D-luciferin glucuronidation was screened. Chemical inhibition assay and kinetic analysis was combined to determine the UGT isoforms responsible for the formation of D-luciferin glucuronide in HLM and HIM. RESULTS: D-luciferin could be catalyzed to form one mono-glucuronide which was characterized as 6'-O-glucuronide in HLM and HIM. UGT1A1, 1A3, 1A6, 1A8, 1A9 and 1A10 participated in the formation of D-luciferin glucuronide, with UGT1A1 exhibiting the highest catalytic activity. Both chemical inhibition assays and kinetic analysis showed that UGT1A1 and UGT1A3 played important roles in D-luciferin-6'-O-glucuronidation in HLM and HIM, with UGT1A6 also giving a non-negligible contribution to this biotransformation in HLM. CONCLUSIONS: UGT1A1, UGT1A3 and UGT1A6 were responsible for 6'-O-glucuronidation of D-luciferin in HLM, while UGT1A1 and UGT1A3 were the major contributors to this biotransformation in HIM.

Interspecies comparison in the COMT-mediated methylation of 3-BTD.

Catechol-O-methyltransferase (COMT) is a druggable biological target and COMT modulators have been widely applied in the treatment of various central and peripheral nervous system disorders. The interspecies differences of COMT were carefully investigated using 3-BTD (a newly developed fluorescent probe of COMT) methylation as the probe reaction, and liver S9 from humans and seven experimental animals including monkeys, dogs, mice, rats, minipigs, guinea pigs and New Zealand rabbits as the enzyme source. Metabolite profiling demonstrated that all the tested liver S9 samples from the different animals could catalyse 3-BTD methylation but displayed significant differences in reaction rate. Also, the differential effects of tolcapone (a potent inhibitor against COMT) on 3-BTD methylation among various species were observed. The apparent kinetic parameters and the maximum intrinsic clearances (Clint) for 3-BTD methylation in liver S9 from the different animals were determined, and the order of the Clint values for the formation of 3-BTD was RLS9 > DLS9 ≈ PLS9 > MLS9 > CyLS9 > RaLS9 > GpLS9 > HLS9. These findings are helpful for further exploring COMT-associated biological processes in animal models, as well as for developing therapeutic molecules that target COMT.