Construction and characterization of ribonuclease H2 C subunit-knockout NIH3T3 cells.

Mammalian ribonuclease (RNase) H2 is a trimer consisting of catalytic A and accessory B and C subunits. RNase H2 is involved in the removal of misincorporated ribonucleotides from genomic DNA. In humans, mutations in RNase H2 gene cause a severe neuroinflammatory disorder, Aicardi-Goutières syndrome (AGS). Here, we constructed RNase H2 C subunit (RH2C)-knockout mouse fibroblast NIH3T3 cells. Compared with the wild-type NIH3T3 cells, the knockout cells exhibited a decreased single ribonucleotide-hydrolyzing activity and an increased accumulation of ribonucleotides in genomic DNA. Transient expression of wild-type RH2C in the knockout cells increased this activity and decreased this ribonucleotide accumulation. Same events were observed when RH2C variants with an AGS-causing mutation, R69W or K145I, were expressed. These results corresponded with our previous results on the RNase H2 A subunit (RH2A)-knockout NIH3T3 cells and the expression of wild-type RH2A or RH2A variants with an AGS-causing mutation, N213I and R293H, in the RH2A-knockout cells.

Comparative multi-omics analysis reveals diverse latex-based defense strategies against pests among latex-producing organs of the fig tree (Ficus carica).

MAIN CONCLUSION: Latexes in immature fruit, young petioles and lignified trunks of fig trees protect the plant using toxic proteins and metabolites in various organ-dependent ways. Latexes from plants contain high amounts of toxic proteins and metabolites, which attack microbes and herbivores after exudation at pest-induced wound sites. The protein and metabolite constituents of latexes are highly variable, depending on the plant species and organ. To determine the diversity of latex-based defense strategies in fig tree (Ficus carica) organs, we conducted comparative proteomic, transcriptomic and metabolomic analyses on latexes isolated from immature fruit, young petioles and lignified trunks of F. carica after constructing a unigene sequence library using RNA-seq data. Trypsin inhibitors were the most abundant proteins in petiole latex, while cysteine proteases ("ficins") were the most abundant in immature fruit and trunk latexes. Galloylglycerol, a possible defense-related metabolite, appeared to be highly accumulated in all three latexes. The expression levels of pathogenesis-related proteins were highest in the latex of trunk, suggesting that this latex had adapted a defensive role against microbe attacks. Although young petioles and immature fruit are both unlignified soft organs, and potential food for herbivorous insects, unigenes for the sesquiterpenoid pathway, which likely produces defense-associated volatiles, and the phenylpropanoid pathway, which produces toxic furanocoumarins, were expressed less in immature fruit latex. This difference may indicate that while petioles and fruit protect the plant from attack by herbivores, the fruit must also attract insect pollinators at younger stages and animals after ripening. We also suggest possible candidate transcription factors and signal transduction proteins that are involved in the differential expression of the unigenes.

Use of human Caco-2 cells and HPAE-PAD for α-glucosidase assay.

To measure α-glucosidase activity, rat intestinal acetone powder is commonly used as a source of α-glucosidase, and the mutarotase-glucose oxidase (GOD) methods commonly used to quantitate glucose produced by enzymatic hydrolysis of the substrates. In this study, we compared human Caco-2 cell extracts with rat intestinal acetone powder extracts. We also compared high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) with the mutarotase-GOD method. The sensitivity of HPAE-PAD was higher than that of mutarotase-GOD. The glucose concentration quantified by HPAE-PAD was similar to that quantified using the mutarotase-GOD method. In the maltase reaction, 1-deoxynojirimycin (1-DNJ) exerted a more potent inhibitory effect on human enzymes than on rat enzymes. This order was reversed during the sucrase reaction. These results suggested that the combined use of Caco-2 cell extracts and HPAE-PAD is advantageous for use in α-glucosidase-related basic research.

Analysis of ribonucleotide content in the genomic DNA of ribonuclease H2 A subunit (RH2A)-knockout NIH3T3 cells after transient expression of wild-type RH2A or RH2A variants with an Aicardi-Goutières syndrome-causing mutation.

Ribonuclease (RNase) H2 is involved in the removal of ribonucleotides embedded in genomic DNA. Eukaryotic RNase H2 is a heterotrimer consisting of the catalytic A subunit (RH2A) and the accessory B and C subunits. This study aimed to compare the cellular activities of wild-type ribonuclease (RNase) H2 and its variants with a mutation causing neuroinflammatory autoimmune disease, Aicardi-Goutières syndrome (AGS). We first analyzed cellular RNase H2 activity and ribonucleotide content in the genomic DNA of RH2A-knockout (KO) mouse fibroblast NIH3T3 cells after transfection with a transient expression plasmid encoding mouse wild-type RH2A. From 4 h after transfection, the RNase H2 activity increased and the amount of ribonucleotides decreased, as compared with the corresponding non-transfected RH2A-KO cells. This demonstrated the rapidness of ribonucleotide turnover in mammalian genomic DNA and the importance of continuous expression of RNase H2 to maintain the ribonucleotide amount low. Next, we expressed mouse RH2A variants with a mutation corresponding to a human AGS-causing mutation in RH2A-KO NIH3T3 cells. Neither increase in RNase H2 activity nor decrease in ribonucleotide amount was observed for G37S; however, both conditions were observed for N213I and R293H. This corresponded with our previous results on the activity of recombinant human RNase H2 variants.

Synergetic effects of prednisolone and olopatadine on atopic dermatitis model of hairless mice.

We investigated the synergetic effects of glucocorticoid and histamine H1 receptor antagonists on an atopic dermatitis model. Hairless mice were used in this study and an atopic dermatitis model was made by repeated application of 2,4,6-trinitrochlorobenzene. The effects of glucocorticoid, histamine H1 receptor antagonists, and the simultaneous use of these drugs were investigated by measuring scratching behavior, skin symptoms and nerve growth factor (NGF) in the skin. Topical application of prednisolone significantly inhibited scratching behavior, skin symptoms and NGF contents in the skin by repeated application. Olopatadine also showed a significant effect on scratching behavior and NGF contents in the skin, whereas chlorpheniramine showed no significant inhibitory effect on these indices. Furthermore, the combined use of prednisolone and olopatadine potentiated the inhibition of scratching behavior, skin symptoms, and NGF in the skin. From these findings, olopatadine potentiated the inhibitory effect of prednisolone on the symptoms of atopic dermatitis by inhibiting NGF.

Effect of topical application of Brazilian propolis on scratching behavior induced by compound 48/80 in mice.

BACKGROUND AND AIM: We investigated the effect of topical application of Brazilian propolis on scratching behavior induced by compound 48/80 in mice. RESULTS: Propolis inhibited compound 48/80-induced scratching behavior when applied immediately after treatment with propolis at a dose of 3 mg/site. Dibucaine 0.3 mg/site also significantly inhibited compound 48/80-induced scratching behavior immediately after application. On the other hand, propolis inhibited compound 48/80-induced scratching behavior even 15, 30 and 60 min after application; however, dibucaine showed no significant inhibition of compound 48/80-induced scratching behavior 15, 30 and 60 min after application. In addition, propolis had no effect on increased vascular permeability just after application, but the drug had a significant effect 15, 30 and 60 min after application. On the contrary, histamine-induced scratching behavior was inhibited significantly by propolis just after application. On the other hand, propolis significantly inhibited histamine release from rat mast cells induced by compound 48/80 at a concentration of more than 10 microg/ml. CONCLUSION: From these results, it can be concluded that inhibition of scratching behavior induced by topical application occurred by both its local anesthetic and systemic action through inhibition of histamine release.

Effect of Brazilian propolis on sneezing and nasal rubbing in experimental allergic rhinitis of mice.

We studied the effect of Brazilian propolis on sneezing and nasal rubbing in experimental allergic rhinitis of mice. A single administration of propolis caused no significant effect on both antigen-induced nasal rubbing and sneezing at a dose of 1000 mg/kg, but a significant inhibition was observed after repeated administration for 2 weeks at this dose. Propolis caused no significant inhibitory effect on the production of total IgE level after repeated administration of 1000 mg/kg. The drug also caused no significant inhibition of histamine-induced nasal rubbing and sneezing at a dose of 1000 mg/kg. On the other hand, propolis significantly inhibited histamine release from rat mast cells induced by antigen and compound 48/80 at a concentration of more than 10 microg/ml. These results clearly demonstrated that propolis may be effective in the relief of symptoms of allergic rhinitis through inhibition of histamine release.