[Mechanism of podocyte pyroptosis aggravated by up-regulation of phospholipase A2 receptor by hepatitis B virus X protein].

Objective: To explore the effect and underlying mechanism of increased expression of M-type phospholipase A2 receptor (PLA2R) on podocyte membrane induced by hepatitis B virus X protein (HBx) on podocyte pyroptosis in hepatitis B virus-associated glomerulonephritis (HBV-GN). Methods: Transfection of the HBx gene into human kidney podocytes was used to mimic the HBV-GN pathogenesis process. Subsequently, podocytes were divided into the following eight groups: normal control plus secretory phospholipase A2-ⅠB (sPLA2-ⅠB) group, empty plasmid plus sPLA2-ⅠB group, HBx group, HBx plus sPLA2-ⅠB group, HBx plus sPLA2-ⅠB plus PLA2R control siRNA group, HBx plus sPLA2-ⅠB plus PLA2R-siRNA group, HBx plus sPLA2-ⅠB plus ROS control siRNA group, and HBx plus sPLA2-ⅠB plus ROS-siRNA group. Podocyte morphology was observed under a transmission electron microscope, and PLA2R expression was detected under a fluorescence microscope. Podocyte pyroptosis and reactive oxygen species (ROS) expression were analyzed by flow cytometry, and the mRNA and protein expression of PLA2R, nucleotide-binding oligomerization domain-like receptor 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, interleukin (IL)-1ß and IL-18 were determined by real-time fluorescence quantitative PCR and Western blot. Results: Compared with the control group, the expression of PLA2R on podocyte membrane significantly increased after transfection with HBx plasmid in vitro (4.07±0.41 vs 1.01±0.17, P<0.001). Transmission electron microscope and fluorochrome-labeled inhibitor of caspases/propidium iodide (FLICA/PI) double staining suggested that overexpressed PLA2R combined with sPLA2-ⅠB caused aggravated podocyte injury and increased pyroptosis (20.22%±0.36% vs 7.86%±0.28%, P<0.001). Moreover, the expression levels of ROS (4 324 515±222 764 vs 12 920±46, P<0.001), NLRP3 (48.30±2.73 vs 1.00±0.11, P<0.001), ASC (4.02±0.84 vs 1.01±0.15, P<0.001), caspase-1 (3.99±0.42 vs 1.00±0.11, P<0.001), IL-1ß (9.08±0.75 vs 1.00±0.09, P<0.001) and IL-18 (19.20±0.70 vs 1.00±0.02, P<0.001) increased when PLA2R was overexpressed. In contrast, with the addition of PLA2R-siRNA or ROS-siRNA to knockdown the expression of related substances, podocyte injury was alleviated and the degree of pyroptosis decreased, and the expressions of genes related to the downstream signaling pathway (NLRP3, ASC, caspase-1, IL-1ß and IL-18) decreased (all P<0.01). Conclusion: HBx may promote podocyte pyroptosis in HBV-GN by targeting the ROS-NLRP3 signaling pathway via the upregulation of PLA2R.

[Hepatitis B virus X protein promotes podocyte pyroptosis in hepatitis B virus-associated glomerulonephritis by down-regulating microRNA -223 targeting NLRP3 inflammasome].

Objective: To investigate the potential function and related mechanism of microRNA-223 (miRNA-223) in the podocyte pyroptosis of hepatitis B virus (HBV)-associated glomerulonephritis induced by HBV X protein (HBx). Methods: HBx-overexpressing lentivirus was transfected into human renal podocytes to mimic the pathogenesis of HBV-GN. Real-time fluorescence quantitative PCR and Western blotting experiments were used to detect the mRNA and protein expression of pyroptosis-related proteins [nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1], and inflammatory factors (interleukin-1ß and interleukin-18), respectively.TUNEL staining and flow cytometry were used to detect the number of pyroptosis cells. Immunofluorescence staining was used to detect the expression of podocytes biomarkers desmin and nephrin; Hoechst 33342 staining was used to observe the morphological and quantitative changes of podocyte nuclei. Enzyme-linked immunosorbent assay was used to measure caspase-1 activity. The dual luciferase reporter gene assay was used to verify the downstream target of miRNA-223. Podocytes were divided into the following nine groups: control group (no special treatment), empty plasmid group (transfected with empty plasmid), HBx overexpression group (transfected with HBx overexpression lentivirus), HBx overexpression+miRNA-223 mimic group (transfected with HBx overexpression lentivirus and miRNA-223 mimic), HBx overexpression+miRNA-223 inhibitor group (transfected with HBx overexpression lentivirus and miRNA-223 inhibitor), HBx overexpression+miRNA-223 mimic+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 mimic+ NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 siRNA), HBx overexpression+miRNA-223 inhibitor+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 inhibitor+NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 siRNA). Results: miRNA-223 was down-regulated in HBx overexpression group compared with the control group (P < 0.05). TUNEL and immunofluorescence staining showed that NLRP3 knockdown attenuated podocyte injury and pyroptosis induced by HBx overexpression (P < 0.05). Dual luciferase reporter gene assay demonstrated that NLRP3 was one of the downstream targets of miRNA-223. Rescue experiments revealed that NLRP3 overexpression weakened the protective effect of miRNA-223 in podocyte injury (P < 0.05). The addition of miRNA-223 mimic and NLRP3 siRNA decreased the expression of NLRP3 inflammasome and cytokines, and reduced the number of pyroptosis cells induced by HBx overexpression (all P < 0.05); The addition of miRNA-223 inhibitor and NLRP3 overexpression plasmid significantly increased the expression of NLRP3 inflammasome and cytokines, caspase-1 activity, and the number of pyroptosis cells (all P < 0.05). Conclusion: HBx may promote podocyte pyroptosis of HBV-GN via downregulating miRNA-223 targeting NLRP3 inflammasome, suggesting that miRNA-223 is expected to be a potential target for the treatment of HBV-GN.

Emerging salt marshes as a source of Trichoderma arenarium sp. nov. and other fungal bioeffectors for biosaline agriculture.

AIMS: Sustainable agriculture requires effective and safe biofertilizers and biofungicides with low environmental impact. Natural ecosystems that closely resemble the conditions of biosaline agriculture may present a reservoir for fungal strains that can be used as novel bioeffectors. METHODS AND RESULTS: We isolated a library of fungi from the rhizosphere of three natural halotolerant plants grown in the emerging tidal salt marshes on the south-east coast of China. DNA barcoding of 116 isolates based on the rRNA ITS1 and 2 and other markers (tef1 or rpb2) revealed 38 fungal species, including plant pathogenic (41%), saprotrophic (24%) and mycoparasitic (28%) taxa. The mycoparasitic fungi were mainly species from the hypocrealean genus Trichoderma, including at least four novel phylotypes. Two of them, representing the taxa Trichoderma arenarium sp. nov. (described here) and T. asperelloides, showed antagonistic activity against five phytopathogenic fungi, and significant growth promotion on tomato seedlings under the conditions of saline agriculture. CONCLUSIONS: Trichoderma spp. of salt marshes play the role of natural biological control in young soil ecosystems with a putatively premature microbiome. SIGNIFICANCE AND IMPACT OF THE STUDY: The saline soil microbiome is a rich source of halotolerant bioeffectors that can be used in biosaline agriculture.

Modulation of rumen fermentation and microbial community through increasing dietary cation-anion difference in Chinese Holstein dairy cows under heat stress conditions.

AIMS: The effect of increasing dietary cation-anion difference (DCAD) on rumen fermentation and ruminal microbial community in dairy cows under heat stress (HS) conditions were evaluated. METHODS AND RESULTS: This study was performed as a two-period cross-over design during the summer season, with eight lactating dairy cows randomly distributed to either a control DCAD diet (CON: 33·5 mEq/100 g DM) or high DCAD diet (HDCAD: 50·8 mEq/100 g DM). Throughout the present study, the temperature and humidity index (THI; 80·2 ± 4·29) was generally elevated above the threshold (THI = 72) that is reported to cause HS in lactating dairy cows. Rumen liquid samples were collected on 15 and 21 d during each 21 d-period. The absolute concentration of ruminal total volatile fatty acid (TVFA) in HDCAD treatment was significantly (P < 0·05) higher than those in the control, whilst the ruminal pH, NH3 -N, and VFA molar percentages were unaffected through increasing DCAD. Furthermore, the copy numbers of the cellulolytic bacteria Ruminococcus albus and Ruminococcus flavefaciens in rumen fluid significantly (P < 0·05) rose along with the increment of DCAD. Although the Alpha diversity indexes and the bacterial microbiota structure were unaffected, increasing DCAD significantly (P < 0·05) enriched the phylum Fibrobacteres and genus Fibrobacter in the microflora of rumen fluid, whilst the genera Flexilinea and Dubosiella were the most differentially abundant taxa in the control. CONCLUSIONS: Increasing DCAD under HS conditions resulted in a greater concentration of total VFA without affecting rumen bacteria diversity or structure, although the enrichment of some cellulolytic/hemicellulolytic bacteria was observed. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides information on the modulation of rumen fermentation and microbial community through the increment of DCAD in Holstein dairy cows under HS conditions.

[Change and relationship between Gli1 and ß-catenin on rats' bone formation with chronic fluorosis].

Objective: To investigate the change and association of glioma-associated oncogene homolog 1 (Gli1) and ß-catenin on bone formation in rats with chronic fluorosis which were inhibited by cyclopamine (Cycl). Methods: Forty-eight Sprague-Dawley rats were evenly divided to four groups, including control, F, F+Cycl and F+DMSO groups. The control group were fed with tap water (NaF<1 ppm). The F, F+Cycl and F+DMSO groups were exposed to NaF (50 ppm) in drinking water as the chronic fluorosis model. Then the rats in F+Cycl or F+DMSO groups were injected by Cycl or DMSO after 6 months, respectively. Urine fluoride concentration was detected using fluorine ion selective electrode. The enzyme-linked immunosorbent assay (ELISA) was used to detect bone alkaline phosphatase (BALP). Bone tissues were stained with hematoxylin-eosin. The mRNA and protein expression of Gli1 and ß-catenin in bone tissue were detected using real-time PCR, immunohistochemistry and Western blot. Results: Compared with the controls, the urine fluoride concentration and the width and volume of bone trabeculae were increased in the F, F+Cycl and F+DMSO groups, but no statistical difference among the 3 fluorosis groups. The concentration of BALP was increased in the F group and decreased in F+Cycl group (P<0.05). The expression of Gli1 and ß-catenin mRNA and protein was higher in the F and F+Cycl groups than controls, but lower in the F+Cycl group than in the F group. There was positive correlation between the expression of Gli1 and ß-catenin (r=0.476, P<0.05). The expression of Gli1 and ß-catenin was also associated with BALP concentration and volume of bone trabeculae, respectively (r(1)=0.457, r(2)=0.466, r(3)=0.581, r(4)=0.554, respectively, P<0.05 for all). Conclusions: The expression of Gli1 can be inhibited by Cycl. It may be involved in the bone formation of rats with chronic fluorosis. It may also affect the expression of ß-catenin, which is an osteogenesis factor.

[Effect of GSK-3ß inhibitor on the expression of RANK-RANKL in rats kidney tissue with diabetic nephropathy].

Objective: To investigate the effect and significance of GSK-3ß inhibitor(LiCl)and RANK-RANKL on the renal tissue of diabetic nephropathy(DN) rats. Methods: SD rats were divided into normal control group (NC), DN model group (DN) and GSK-3ß inhibitor intervention group (LiCl). Twenty-four hour urine protein of rats were determined by Coomassie brilliant blue. Kidney tissue sections were stained by HE. The expression of GSK-3ß, RANK and RANKL protein were determined by immunohistochemistry staining. The mRNA of GSK-3ß, RANK, RANKL was detected by RT-qPCR. Results: Compared with NC group[(14.72±3.37)g], the level of 24-hour urinary protein[(154.17±20.65)g] increased significantly in DN group; compared with DN Group, the level of 24-hour urinary protein [(107.22±31.15)g]decreased in LiCl group(P<0.05). Compared with NC group(2.10±0.60, 1.10±0.20, 1.21±0.20; 19.52±3.20, 1.80±1.10, 1.81±0.50), the pathological changes of renal tissues of DN group aggravated, the mRNA and expression of protein of GSK-3ß, RANK and RANKL increased(9.10±2.15, 8.95±2.40, 9.90±2.60; 32.70±7.20, 19.20±4.32, 20.92±5.90); compared with DN group, the pathological changes of renal tissues of LiCl group alleviated, mRNA and the expression of protein of factors above declined(2.70±0.80, 2.32±0.65, 3.58±1.10; 22.35±3.25, 4.20±2.42, 5.90±2.36; P<0.05). Conclusion: RANK and RANKL play an important role in the development of DN, LiCl influence Wnt and NF-κB signal pathway down-regulating RANK and RANKL to suspend development of diabetic nephropathy.

Middle Cerebral Artery Plaque Hyperintensity on T2-Weighted Vessel Wall Imaging Is Associated with Ischemic Stroke.

BACKGROUND AND PURPOSE: Vessel wall imaging can identify intracranial atherosclerotic plaque and give clues about its components. We aimed to investigate whether the plaque hyperintensity in the middle cerebral artery on T2-weighted vessel wall imaging is associated with ischemic stroke. MATERIALS AND METHODS: We retrospectively reviewed our institutional vessel wall MR imaging data base. Patients with an acute ischemic stroke within 7-day onset in the MCA territory were enrolled. Patients with stroke and stenotic MCA plaque (stenosis degree, ≥50%) were included for analysis. Ipsilateral MCA plaque was defined as symptomatic, and contralateral plaque, as asymptomatic. Plaque was manually delineated on T2-weighted vessel wall imaging. The plaque signal was normalized to the ipsilateral muscle signal. The thresholds and volume of normalized plaque signal were investigated using logistic regression and receiver operating characteristic analysis to determine the association between normalized plaque signal and stroke. RESULTS: One hundred eight stenotic MCAs were analyzed (from 88 patients, 66 men; mean age, 58 ± 15 years), including 72 symptomatic and 36 asymptomatic MCA plaques. Symptomatic MCA plaque showed larger plaque hyperintensity volume compared with asymptomatic MCA plaque. The logistic regression model incorporating stenosis degree, remodeling ratio, and normalized plaque signal 1.3-1.4 (OR, 6.25; 95% CI, 1.90-20.57) had a higher area under curve in differentiating symptomatic/asymptomatic MCA plaque, compared with a model with only stenosis degree and remodeling ratio (area under curve, 0.884 versus 0.806; P =.008). CONCLUSIONS: The MCA plaque hyperintensity on T2-weighted vessel wall imaging is independently associated with ischemic stroke and adds value to symptomatic MCA plaque classification. Measuring the normalized signal intensity may serve as a practical and integrative approach to the analysis of intracranial atherosclerotic plaque.

Quantitative Identification of Compound-Dependent On-Modules and Differential Allosteric Modules From Homologous Ischemic Networks.

Module-based methods have made much progress in deconstructing biological networks. However, it is a great challenge to quantitatively compare the topological structural variations of modules (allosteric modules [AMs]) under different situations. A total of 23, 42, and 15 coexpression modules were identified in baicalin (BA), jasminoidin (JA), and ursodeoxycholic acid (UA) in a global anti-ischemic mice network, respectively. Then, we integrated the methods of module-based consensus ratio (MCR) and modified Zsummary module statistic to validate 12 BA, 22 JA, and 8 UA on-modules based on comparing with vehicle. The MCRs for pairwise comparisons were 1.55% (BA vs. JA), 1.45% (BA vs. UA), and 1.27% (JA vs. UA), respectively. Five conserved allosteric modules (CAMs) and 17 unique allosteric modules (UAMs) were identified among these groups. In conclusion, module-centric analysis may provide us a unique approach to understand multiple pharmacological mechanisms associated with differential phenotypes in the era of modular pharmacology.

A test using cultured cells with induced mixed-function oxygenase in the unscheduled DNA synthesis assay for detecting promutagens/procarcinogens.

The human FL cell line contains very low levels of constitutive AHH activity, but it could be greatly induced by NE, beta-NF and 3-MC, and induced slightly by PB. When two different types of inducer, for example, 3-MC and PB or 3-MC and NE were given in combination, an additive inductive effect was not observed. Both the constitutive and induced AHH in FL cells have characteristics of MFO, namely, NADPH-dependence and CO-sensitivity. The fact that the constitutive and induced AHH in FL cells could be inhibited by a known hydroxylase inhibitor 7,8-BF indicated that the AHH in FL cells belongs to the cytochrome P-448 dependent MFO type. After removal of inducer from the medium, the induced AHH activity remained at a high level for at least 24-36 h. By using AHH-induced FL cells in the UDS assay system for the detection of promutagens/procarcinogens, we found that AFB1 and 3-MC did not induce a UDS reaction in uninduced FL cells, while in beta-NF induced cells, 10(-6)-10(-4) M AFB1 and 10(-7)-10(-6) M 3-MC elicited a very significant UDS reaction, which was concordant with the results obtained in the UDS assay system using HeLa cells or FL cells supplemented with liver microsomes or using primary cultured hepatocytes as indicator cells. B(a)P elicited the UDS reaction at concentrations of 10(-6)-10(-3) M in beta-NF induced cells, whereas 10(-4)-10(-3) M was required in uninduced cells. The results above indicate that this new design is feasible, but further study is needed to assure its accuracy.

Cell cycle effects on the basal and DNA-damaging-agent-stimulated ADPRT activity in cultured mammalian cells.

ADP-ribosyl transferase (ADPRT) is a DNA-dependent chromatin-associated enzyme which covalently attaches ADP-ribose moieties derived from NAD+ to protein acceptors to form poly(ADP-ribose). ADPRT activity is strongly stimulated by breaks in DNA, and it is suggested that its activity is required for efficient DNA excision repair. In this paper, a cell-cycle-dependent fluctuation of basal ADPRT activity was demonstrated by measuring it in permeabilized FL cells. The cell used was subjected to arginine starvation for 48 h before being released from the block by replacement of deficient medium with complete medium and cells in different proliferating stages were traced by [3H]TdR pulse labelling and obtained at different intervals after block release. The peak basal ADPRT activity appeared 4-6 h after the appearance of the peak of DNA synthesis. After treating the cells with MNNG (10(-4) M), MMS (10(-3)-10(-4) M) and 4NQO (10(-5) M) for 90 min just after release of the block, the ADPRT activity was markedly stimulated. It was further demonstrated that the effects of MNNG/4NQO and cell cycle influence on the level of poly(ADP-ribose) synthesis appear to be additive. While concerning MMS, quite a different pattern of ADPRT stimulation in the cell cycle was demonstrated, i.e., the activity of ADPRT stimulation of 10(-3) M MMS was found to be completely dependent on the basal ADPRT activity. In the cells with the highest basal ADPRT activity 12 h after block release, the MMS-induced ADPRT stimulation could not be observed. It was suggested that more than one pathway might be present in ADPRT stimulation induced by DNA-damaging chemicals, and the cells synchronized in late G1 stage might be the most suitable for demonstrating poly(ADP-ribose) synthesis after DNA damage.

Induction of cytochrome P450 isozymes in human amnion FL cells.

The human epithelial FL cell line contains low levels of constitutive ethoxyresorufin O-deethylase, ethoxycoumarin O-deethylase and aminopyrine N-demethylase activities, of which ethoxyresorufin O-deethylase could be induced by norepinephrine, beta-naphthoflavone and 3-methylcholanthrene, but ethoxy-couarin O-deethylase and aminopyrine N-demethylase activities could be induced by norepinephrine, beta-naphthoflavone and 3-methylcholanthrene as well as phenobarbital. Inducibility of ethoxyresorufin O-deethylase, ethoxycoumarin O-deethylase and aminopyrine N-demethylase activities was 3.1-6.7-, 1.8-3.1- and 1.4-2.0-fold respectively. Co-treatment with 3-methylcholanthrene and norepinephrine resulted in higher ethoxyresorufin O-deethylase and aminopyrine N-demethylase activities than treatment with 3-methylcholanthrene alone. The induced ethoxyresorufin O-deethylase and ethoxycoumarin O-deethylase activities remained at a high level for 24-36 h after removal of the inducer from the medium. The metabolizing ability of induced FL cells is stronger than that of non-induced ones in a given time following induction. The results further confirm that it is feasible to use the cytochrome P450 isozyme-induced FL cell as a biological indicator in short-term tests for screening promutagens/procarcinogens.

A modified method of UDS detection in vitro suitable for screening the DNA-damaging effects of chemicals.

The UDS induced in cultured FL cells by exposure to chemicals was measured as hydroxyurea-resistant incorporation of 3H-TdR in the acid-insoluble fraction of the 14C-TdR-prelabelled cells synchronized by the combination of arginine starvation and pretreatment with hydroxyurea. The level of UDS is represented by the ratios of 3H/14C radioactivities which are measures of specific activities of 3H. Two direct-acting alkylating agents, MMS and MNNG, a cross-linking agent, mitomycin C, and 3 procarcinogens, B(a)P, AFB1 and cyclophosphamide elicited UDS in the absence or presence of the liver-metabolizing system. Three chemicals of unknown carcinogenicity were also able to induce UDS in this assay system, i.e., bis-(O,O-diethylphosphinothioyl)-disulphide, 4-chlorophenoxy acetic acid (sodium salt) and caramelized malt sugar. With the exception of 4-chlorophenoxy acetic acid, they were also active in the Ames test.

Cloning of UGT1A9 cDNA from liver tissues and its expression in CHL cells.

AIM: To clone the cDNA of UGT1A9 from a Chinese human liver and establish the Chinese hamster lung (CHL) cell line expressing human UGT1A9. METHODS: cDNA of UGT1 A9 was transcripted from mRNA by reverse transcriptase-ploymerase chain reaction, and was cloned into the pGEM-T vector which was amplified in the host bacteric E.Coli DH5(alpha). The inserted fragment, verified by DNA sequencing, was subcloned into the Hind III /Not I site of a mammalian expression vector pREP9 to construct the plasmid termed pREP9-UGT1A9. CHL cells were transfected with the resultant recombinants, pREP9-UGT1A9, and selected by G418 (400 mg x L(-1)) for one month. The surviving clone (CHL-UGT1A9) was harvested as a pool and sub-cultured in medium containing G418 to obtain samples forUGT1A9 assays. The enzyme activity of CHL-UGT1A9 towards propranolol in S9 protein of the cell was determined by HPLC. RESULTS: The sequence of the cDNA segment cloned, which was 1666 bp in length, was identical to that released by Gene Bank (GenBank accession number: AF056188) in coding region. The recombinant constructed, pREP9-UGT1A9, contains the entire coding region, along with 18 bp of the 5' and 55 bp of the 3' untranslated region of theUGT1A9 cDNA, respectively. The cell lines established expressed the protein of UGT1A9, and the enzyme activity towards propranolol in S9 protein was found to be 101+/- 24 pmol x min(-1) x mg(-1) protein (n=3), but was not detectable in parental CHL cells. CONCLUSION: The cDNA of UGT1A9 was successfully cloned from a Chinese human liver and transfected into CHL cells. The CHL-UGT1 A9 cell lines established efficiently expressed the protein ofUGT1A9 for the further enzyme study of drug glucuronidation.

Antimutagenic effect of garlic (Allium sativum L.) on 4NQO-induced mutagenesis in Escherichia coli WP2.

Aqueous extract prepared from garlic bulbs markedly suppressed the mutagenesis in both E. coli WP2 trp- and E. coli WP2 trp- uvrA- induced by 4-nitroquinoline 1-oxide (4NQO), but not that induced by UV. Cellular toxicity, inhibition of the expression of the Trp+ phenotype and delay of the first cell division after 4NQO treatment were not observed in the presence of the extract. Since the extract showed identical antimutagenic effects against 4NQO in both test strains but no effect on the mutagenesis of UV, it seems that the extract might act by inactivating the electrophilic group(s) of 4NQO or inhibiting its metabolic activation.

The cell-cycle dependent and the DNA-damaging agent-induced changes of cellular NAD content and their significance.

NAD is the substrate of a novel chromatin-associated enzyme-ADP-ribosyl transferase (ADPRT). In this study, the cell-cycle dependent change in cellular NAD content was observed in a line of human amnion FL cells. It was found that the cellular NAD content of FL cells was highest in G1 and lowest in S/G2-G2. 3AB, a potent ADPRT inhibitor, can inhibit the cell cycle dependent change in cellular NAD content and also inhibit DNA synthesis in the S phase and extend the S phase. The results indicate that ADP-ribosylation may be involved in DNA replication and cell cycle progression. It was also found that the DNA-damaging agents, MNNG, MMS and 4NQO could lower cellular NAD content in a dose-dependent way. This DNA-damage-induced NAD lowering could be partially or completely prevented by the ADPRT inhibitors, 3AB or nicotinamide, which were shown to exert no influence on either the cellular NAD content of normal quiescent FL cells or the metabolic blocking agent, 2,4-DNP-induced cellular NAD lowering. The possibility of establishing a simple and specific method to detect DNA-damaging agents by measuring cellular NAD content in the presence or absence of ADPRT inhibitor is explored.

Genotoxic activity of caramel on Salmonella and cultured mammalian cells.

The genetic activity of 2 commercial caramel preparations, manufactured either by heating the malt sugar solution directly (non-ammoniated caramel) or by heating it with ammonia (ammoniated caramel) was studied in the Salmonella mutagenicity test and UDS assay in cultured mammalian cells. The non-ammoniated caramel was found to be mutagenic to S. typhimurium TA100, while the ammoniated one was genetically active in all the tester strains used, namely TA100, TA97 and TA98. It was also demonstrated that non-ammoniated caramel was capable of inducing UDS in cultured human amnion FL cells, but for the ammoniated one, no such activity was observed. Furthermore, based on the results obtained in the DNA synthesis inhibition assay, it was suggested that the DNA synthesis inhibition seen in our experiments with the ammoniated caramel was probably not of DNA damage in origin. These data indicate that the mutagenic fractions formed during ammoniated and non-ammoniated caramelization were quite different.

Difference in Protein Expression in Vero Cells after Antisense-blocking Genes Involved in the Suppression of Non-targeted Mutagenesis.

A cDNA fragment (fragment 9) has been isolated by mRNA differential display and antisense technology in this lab, and its relevant gene (fragment 9 related gene, FNR gene) might be involved in the inhibition of non-targeted mutagenesis induced by N -methyl- N ' -nitro-N-nitrosoguanidine(MNNG) in mammalian cells. In order to elucidate the functional mechanism of the FNR gene, the protein expression was compared between MNNG-exposed Vero cells transfected with antisense RNA expression plasmid (Vero-pM-amp(-)-9(-)) and those with vector DNA (Vero-pM-amp(-)), by using two-dimensional gel electrophoresis followed by 2D image software analysis. Our analysis indicated that 12 proteins were specifically expressed only in Vero-pM-amp(-)-9(-), and 2 proteins in Vero-pM-amp(-). In addition,there were 24 proteins expressed in higher level in Vero-pM-amp(-)-9(-) as compared with Vero-pM-amp(-)( P <0.05), among them the expression of 7 proteins were enhanced by greater than 5 folds. These results suggest that antisense blocking the FNR gene expression triggered a series of alteration of other gene expression and the FNR gene might be a regulatory factor. This study will also facilitate the identification and characterization of these proteins and corresponding genes involved in the non-targeted mutagenesis.

Study of SOS-inducing kinetics and inhibitory effect on induction of Escherichia coli.

It was found that modified pulse induction could produce SOS responses of the sfi gene in 15 minutes. The responses peaked at 30-60 minutes and were twice as strong as those by persistent induction. The modified pulse induction could therefore more truly reflect the inducing effect. However, the SOS-inducing kinetics of MMC and 4NQO was not identical suggesting distinct kinetic patterns of induction by different mutagens. A suppressive effect on 4NQO-induced SOS response by cinnamic aldehyde and diallyl trisulfate was also observed. The effect might be due to an interference with the metabolic activation of 4NQO or breakdown of 4NQO by the bacteria.

Concentration dependent stereoselectivity of propafenone N-depropylation metabolism with human hepatic recombinant CYP1A2.

Concentration dependency of stereoselective N-depropylation metabolism of propafenone was studied by using transgenic cell line expressing human CYP1A2. Enantiomers of propafenone and N-depropylpropafenone were separated and assayed simultaneously by RP-HPLC with precolumn GITC chiral derivatization. The experimental results showed that CYP1A2 was involved in enantioselective N-depropylation of propafenone and that the metabolic stereoselectivity depends on substrate concentration. For racemic propafenone, stereoselectivity was observed at low substrate concentration and was not seen at high substrate concentration. For individual isomers, S-(+)-propafenone was metabolized faster than its antipode at higher enantiomer concentrations and R-(-)-propafenone was eliminated faster than its antipode at lower enantiomer concentrations. There is interaction between S- and R-propafenone. R-(-)-propafenone inhibited the metabolism of S-(+)-propafenone with IC50 0.225 mmol/L for human CYP1A2.