[Mechanism of cleft palate in C57BL/6N mice induced by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin].

Objective: To explore the molecular mechanism of cleft palate in mice induced by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). Methods: The pregnant mice were randomly divided into TCDD-treated group (n=42) and control group (n=42). TCDD-treated group was given by gavage a single dose of TCDD (64 µg/kg) at 8: 00 AM on gestation day 10 (GD10) and the control group was given by gavage the isopyknic corn oil. At GD13-GD15, the fetal mice palate development was observed by HE staining. The mouse embryonic palatal mesenchymal cell proliferation was detected by 5-bromo-2-deoxyuridine (BrdU) immunofluorescence. The localization and expression of maternally expressed gene3 (MEG3) in mouse embryonic palatal mesenchymal cells was detected by situ hybridization and real-time PCR (RT-PCR). The key protein expressions of transforming growth factor-ß (TGF-ß)/Smad signaling pathway in mouse embryonic palatal mesenchyme were analyzed by Western blotting. The interaction of MEG3 and TGF-ß receptor Ⅰ (TGF-ßRⅠ) was examined by RNA binding protein immunoprecipitation (RIP). Results: At GD13 and GD14, compared with the control group, the ratio of BrdU-positive cells in the palatal mesenchyme of TCDD-treated fetuses decreased significantly (GD13, t=6.66, P=0.003; GD14, t=6.56, P=0.003). However, at GD15, the ratio of BrdU-positive cells was significantly increased (t=-5.98, P=0.004). MEG3 was mainly expressed in the nuclei of fetal mouse palatal mesenchymal cells, and the expression of MEG3 in TCDD group was significantly increased at GD13, GD14 and GD15(GD13, t=39.28, P=0.012; GD14, t=18.75, P=0.042; GD15, t=28.36, P=0.045). At GD14, TCDD decreased the levels of p-Smad2 and Smad4 in embryonic palate mesenchymal cells (p-Smad2, t=9.48, P=0.001;Smad4, t=63.10, P=0.001), whereas the expression of Smad7 was significantly increased at GD14 (t=30.77, P<0.001). The results of the RIP experiment showed that the amount of TGF-ßRⅠ-bound MEG3 in mouse embryonic palatal mesenchymal cells in the TCDD group (23.940±1.301) was higher than that in the control group (8.537±1.523)(t=24.55, P<0.001). Conclusions: MEG3 is involved in the suppression of mouse embryonic palatal mesenchymal cell proliferation, functioning at least in part via interacting with the TGF-ßRⅠ protein and thereby suppressing Smad signaling in the context of TCDD induced cleft palate.

Molecular, biochemical and enzymatic characterization of photorespiratory 2-phosphoglycolate phosphatase (PGLP1) in rice.

Phosphoglycolate phosphatase (PGLP, EC3.1.3.18) is a key enzyme in photorespiration. However, genes encoding the rice photorespiratory PGLP have not yet been identified or characterized. Here, PGLP for photorespiration in rice was identified and its enzymatic properties were investigated. In order to define the function of PGLP homologs, rice PGLP mutants were constructed using CRISPR/Cas9, the transcriptional expressions were analyzed by RT-qPCR, and subcellular localizations were detected via rice protoplast transient expression analysis. Based on sequence alignment, proteins encoded by genes OsPGLP1, OsPGLP2, and OsPGLP3 in the rice genome were predicted to have PGLP activity. Subsequent experimentation showed that OsPGLP1 and OsPGLP3 are chloroplast proteins, while OsPGLP2 is localized in the cytoplasm. In rice leaves, levels of PGLP1 transcript were substantially higher than those of PGLP2 and PGLP3, whereas in roots, levels of PGLP2 transcript were higher than those of PGLP1 and PGLP3. There was no detectable PGLP activity in leaves of the OsPGLP1 mutant, which was non-viable in ambient air condition (400 ppm CO2 ) and high CO2 (4000 ppm) was unable to restore normal growth. In contrast, mutations of PGLP2 or PGLP3 did not result in visible phenotypes and the leaf PGLP activities were also unaffected It is suggested that PGLP1, encoded by Os04g0490800, is responsible for photorespiration. Furthermore, PGLP1 is a dimer with an apparent molecular mass of ca.65 kDa, and its Km is 272 µM, with a higher broad optimum pH (7.5 to 10.0) for PGLP activity than that in other higher plants.

[Serum exosomal miRNAs profiling and functional study in patients with non-alcoholic fatty liver disease].

Objective: Differential expression of serum exosomal miRNAs were detected for NAFLD patients and healthy controls, thereby determining the role of serum exosomal miRNAs in the pathogenesis, diagnosis, and treatment of NAFLD. Methods: Four patients with S2-3 NAFLD who shared similar demographic features and personal histories, and matched healthy controls were recruited for high-throughput sequencing of serum exosomal miRNAs. Four miRNAs with the most significant differential expression were verified by qRT-PCR in three groups (S1, S2-3, and control groups) with 20 cases in each group. Target gene prediction was performed for these differentially-expressed miRNAs, along with GO and KEGG enrichment analyses for the target genes. T-test or ANOVA were used for normally distributed data. Wilcoxon rank sum test was used for ranked data and non-normally distributed data. The count data used Pearson chi-square test or Fisher's exact test. Results: There were 19 serum exosomal miRNAs with significantly different levels of expression (P < 0.05) and a fold-change > 2. The expression of hsa-miR-122-5p, hsa-miR-146b-5p, and hsa-miR-197-3P was highest in the S2-3 group, followed by the S1 and control groups (in order); hsa-miR-483-3p expression was higher in the NAFLD group (S1 or S2-3) than the control group. There were 84 pathways significantly enriched in target genes. From 20 pathways closely related to NAFLD, at least 5 target genes which were simultaneously correlated to all 10 pathways were screened (PIK3R2, AKT2, AKT3, MAPK1, and NFKB1). Conclusion: Differential expression of serum exosomal miRNAs was detected in NAFLD patients and healthy controls. Four miRNAs with the greatest fold-changes were assessed to judge the severity of fatty degeneration of the liver. The research findings provide reference for non-invasive identification of new biomarkers and specific targets for NAFLD treatment.

[Study on the influence of sleeve height and implant length on accuracy of static computer-assisted implant surgery].

Objective: To evaluate the influence of the sleeve lengths and implant lengths on accuracy of static computer-assisted implant surgery (sCAIS). Methods: Twenty-eight models of bilateral mandibular single tooth loss were included. Fifty-five implants were placed under the guidance of sCAIS (Straumann Bone Level 4.1 mm×10 mm). According to the height of metal sleeve of static guide plate, 55 implants were divided into 11 groups (free hand group, 1 mm group, 2 mm group, 3 mm group, 4 mm group, 5 mm group, 6 mm group, 7 mm group, 8 mm group, 9 mm group, 10 mm group), with 5 implants in each group. Eight research models were included. Group with 5 mm sleeve guides were used to place implants of different length, (Straumann Bone Level width 4.1 mm, height was 8 mm, 10 mm and 14 mm), 5 implants in each group. Eighteen patients with mandibular single tooth loss were included in the Department of Oral Implantology, Tianjin Stomatological Hospital from October 2018 to June 2019. There were 10 males and 8 females, 18-46(33.7±7.9) years old. A total of 18 implants were implanted and divided into 3 groups (free hand group, 3 mm group and 5 mm group) with 6 implants in each group. Digital software was used to compare the implant positions before and after implantation. Non-parametric Kruskal-Wallis test or one-way ANOVA were used to analyze the results. Results: There was no significant difference in implant vertical deviation between different sleeve height groups (1-10 mm) and free hand group, but the neck deviation in free hand group[(1.04±0.13) mm] was significantly higher than that in different sleeve height groups (1-10 mm) (P<0.05). The tip deviations of free hand group, 1 mm group and 2 mm group [(1.32±0.43), (0.83±0.10) and (0.78±0.11) mm, respectively] was significantly higher than that of 10 mm group [(0.31±0.14) mm](P<0.05). The angle deviation of free hand group and 1 mm group (3.99°±0.85° and 2.59°±0.69°), respectively] was significantly higher than that of 10 mm group (0.61°±0.03°) (P<0.05). The tip deviations of implants in the 14 mm group [(0.83±0.22) mm] was significantly higher than that in the 8 mm and 10 mm groups [(0.44±0.07) and (0.49±0.06) mm, respectively]. Clinical studies showed that there was no significant difference in neck deviation, tip deviation and angle deviation between 3 mm group and 5 mm group (P>0.05), but deviations were significantly lower than those in free hand group (P<0.05). Conclusions: The length of the sleeves has significant influence on the accuracy of the surgical guide. There was no significant difference in accuracy of the implant guide with 3 mm or 5 mm metal sleeves. The vitro study has some limitations and needs further systematic research.

In vitro evaluation of secoiridoid glucosides from the fruits of Ligustrum lucidum as antiviral agents.

Six secoiridoid glucosides, lucidumoside C (1), oleoside dimethylester (2), neonuezhenide (3), oleuropein (4), ligustroside (5) and lucidumoside A (6), isolated from the fruits of Ligustrum lucidum (Oleaceae), were examined in vitro for their activities against four strains of pathogenic viruses, namely herpes simplex type I virus (HSV-1), influenza type A virus (Flu A), respiratory syncytial virus (RSV) and parainfluenza type 3 virus (Para 3). Antiviral activities were evaluated by the cytopathic effect (CPE) inhibitory assay. The purpose was to check if the antioxidative potency of these glucosides correlated with their antiviral potency. Results showed that none of the glucosides had any significant activity against HSV-1 and Flu A. Oleuropein, however, showed significant antiviral activities against RSV and Para 3 with IC50 value of 23.4 and 11.7 microg/ml, respectively. Lucidumoside C, oleoside dimethylester and ligustroside showed potent or moderate antiviral activities against Para 3 with IC50 values of 15.6-20.8 microg/ml. These results also documented that the anti-oxidative potency of these secoiriodoid glucosides was not directly related to their antiviral effects.

A novel 1:1 complex of potassium mikanin-3-O-sulfate with methanol.

Mikanin-3-O-sulfate (1), in the form of its potassium salt, together with mikanin (2) and alpinetin (3) were isolated from Mikania micrantha. The crystal structures of K(1) x CH3OH, 2 and 3 x H2O were established by X-ray crystallography. The potassium ions in K(1) x CHO3H are bridged by O5, O7 and O8 to form a chain of face-sharing KO8 coordination polyhedra, from which the aglycon units are outstretched to form a polymeric molecular column. Adjacent molecular columns are linked by pi-pi stacking between parallel, intercalating aglycon units to form layers matching the (101) family of planes, which are further interconnected into a three-dimensional supramolecular assembly. Sulfation at 3-OH induced better co-planarity and conjugation of the rings.

Purification of phenylethanoids from Brandisia hancei and the antiproliferative effects on aortic smooth muscle.

The present study describes the isolation and purification of acteoside, 2'-acetylacteoside, poliumoside and brandioside, four phenylethanoid glycosides from Brandisia hancei. We examined their effects on the proliferation of cultured A7r5 rat aortic smooth muscle cells. The proliferative response was measured from the [(3)H]-thymidine incorporation into DNA. All four glycosides suppressed the proliferative response in the presence of 2 % or 5 % fetal bovine serum in a concentration-dependent manner. The rank order of effectiveness for inhibition of cell proliferation was: brandioside > or = poliumoside > 2'-acetylacteoside > or = acteoside. The acetyl group at position 2' of glucose does not seem necessary for the anti-proliferative effects of acteoside and 2'-acetylacteoside, while the hydroxy groups of the aromatic rings appear to play a role. Inhibition of smooth muscle cell proliferation by phenylethanoids indicates that these compounds may have preventative effects on arteriosclerosis.

Relaxing effects of Ligstrum purpurascens extract and purified acteoside in rat aortic rings.

The present study describes the effects of an extract obtained from the leaves of Ligstrum purpurascens and acteoside purified from the extract on the contractile response to various agonists in rat isolated aortic rings. L. purpurascens extract relaxed 9,11-dideoxy-11 alpha,9 alpha-epoxymethanoprostaglandin F2 alpha (U46619)-preconstricted rings in a concentration-dependent manner (IC50: 0.14 +/- 0.01 mg/ml with endothelium and 0.16 +/- 0.01 mg/ml without endothelium). The extract also reduced contraction induced by 35 mM K+ or by 1 microM phorbol 12,13-diacetate (PDA) in endothelium-intact rings. The extract (0.1-0.3 mg/ml) reduced the concentration-response to U46619 in normal Krebs solution or to CaCl2 in 35 mM K(+)-containing solution. Acteoside accounts for 2.05% of total L. purpurascens extract in weight. Acteoside induced relaxation of rings preconstricted by U46619 (IC50: 0.22 +/- 0.01 mg/ml) but it caused an increase in 35 mM K(+)-induced tone. Removal of endothelium enhanced the relaxing effect of acteoside. Besides, pretreatment with acteoside inhibited endothelium/nitric oxide-mediated relaxation induced by acetylcholine. These results indicate that acteoside is unlikely the major ingredient responsible for the vasodilator effect of L. purpurascens extract. The extract relaxed the preconstricted aortic rings probably through multiple mechanisms by acting on smooth muscle cells. The inhibitory effect on endothelial nitric oxide-mediated relaxation suggests that acteoside could also act on the endothelial cells to reduce nitric oxide release.

Antioxidative glucosides from the fruits of Ligustrum lucidum.

The ethanol extract of the fruits of Ligustrum lucidum was shown to have inhibitory effects on the hemolysis of red blood cells induced by 2,2'-azo-bis-(2-amidinopropane) dihydrochloride. Bioassay-guided analysis led to the isolation of ten secoiridoid glucosides. Two of them were new, lucidumosides C and D. Their structures were elucidated by spectroscopic methods. The other eight compounds were identified as oleoside dimethyl ester, ligustroside, oleuropein, nuezhenide, isonuezhenide, neonuezhenide, lucidumoside A and lucidumoside B. Five compounds, oleoside dimethyl ester, oleuropein, neonuezhenide, lucidumoside B and lucidumoside C, exhibited strong antioxidant effect against hemolysis of red blood cells induced by free radicals.

Antioxidative activities of phenylethanoid glycosides from Ligustrum purpurascens.

Tea and kudingcha (bitter tea) are the two most popular beverages consumed in China. Tea derived from the leaves of Camellia sinensis has been well studied for its various health benefits, but there are very limited data on the biological activities of bitter tea derived from the leaves of Ligustrum purpurascens (LP). The present study was carried out to characterize the antioxidants present in the bitter tea brewed from the leaves of LP. It was found that the crude glycoside fraction possessed strong protection against oxidation of human low-density lipoprotein (LDL). The column chromatographic separation led to the isolatation of five phenylethanoid glycosides, namely, acteoside, ligupurpuroside A, cis-ligupurpuroside B, trans-ligupurpuroside B, and osmanthuside B. When acteoside was heated in the boiling water, it was isomerized to form isoacteoside. Acteoside, isoacteoside, and ligupurpuroside A purified from LP were protective, whereas cis-ligupurpuroside B, trans-ligupurpuroside B, and osmanthuside B exhibited no protection to human LDL from Cu(2+)-medicated oxidation. Acteoside, isoacteoside, and ligupurpuroside A were also effective in preventing the peroxyl free radical-induced oxidation of alpha-tocopherol in human LDL. The antioxidant activities of acteoside, isoacteoside, and ligupurpuroside A were comparable to that observed for a green tea antioxidant, (-)-epicatechin gallate. The inhibitory effect of these three phenylethanoid glycosides on oxidation of human LDL and alpha-tocopherol was dose-dependent at concentrations of 5-40 microM. The present results suggest that the bitter tea beverage derived from LP contains effective antioxidants that may have an equal benefit as a green tea beverage.

Vasorelaxant effects of cardamonin and alpinetin from Alpinia henryi K. Schum.

The vascular effects of cardamonin and alpinetin from Alpinia henryi K. Schum. were examined in the rat isolated mesenteric arteries. 1H and 13C nuclear magnetic resonance spectra showed that cardamonin is present in trans-form, and single-crystal radiographic structure revealed that alpinetin is present in S configuration. Both cardamonin and alpinetin produced a rightward shift in the concentration-response curve for phenylephrine in a noncompetitive manner, and they induced relaxation of phenylephrine-preconstricted arteries with respective mean inhibitory concentrations (IC50) of 9.3+/-0.6 microM and 27.5+/-2.8 microM. Both compounds also relaxed arteries preconstricted by endothelin I or U46619. Their relaxant effects were decreased in endothelium-removed rings. Pretreatment with N(G)-nitro-L-arginine methyl ester or methylene blue inhibited relaxation induced by both agents, and pretreatment with L-arginine reversed the effect of N(G)-nitro-L-arginine methyl ester on cardamonin-induced endothelium-dependent relaxation. The relaxant effects of cardamonin and alpinetin were unaffected by indomethacin (3 microM). Cardamonin and alpinetin inhibited 60 mM K+-induced contraction with respective IC50 of 11.5+/-0.3 microM and 37.9+/-3.6 microM. In addition, both agents inhibited the transient contraction induced by 3 microM phenylephrine or by 10 mM caffeine in Ca2+-free Krebs solution. Finally, these two agents also concentration dependently relax the arteries preconstricted by 1 microM phorbol 12,13-diacetate in Ca2+-free Krebs solution. These results indicate that purified cardamonin and alpinetin from A. henryi K. Schum. relaxed rat mesenteric arteries through multiple mechanisms. They induced both endothelium-dependent and -independent relaxation; the former is likely mediated by nitric oxide whereas the latter is probably mediated through nonselective inhibition of Ca2+ influx and intracellular Ca2+ release and inhibition of the protein kinase C-dependent contractile mechanism.

Secoiridoid constituents from the fruits of Ligustrum lucidum.

Two secoiridoid glucosides, lucidumosides A and B, as well as six known glucosides, oleoside dimethyl ester, ligustroside, oleuropein, nuezhenide, isonuezhenide, and neonuezhenide, were isolated from the fruits of Ligustrum lucidum. Their structures were elucidated by spectroscopic methods.

Antioxidant activity of phenylethanoid glycosides from Brandisia hancei.

Brandisia hancei is a medicinal herb in China. The ethanol extract of this plant and four phenylethanoid glycosides, acteoside (1), 2'-acetylacteoside (2), poliumoside (3) and brandioside (4), isolated from it were shown to have inhibitory effects on free radical-induced hemolysis of red blood cells and free radical scavenging activities in vitro. Brandioside (4) and poliumoside (3) showed stronger antioxidant effect than acteoside (1), 2'-acetylacteoside (2) and trolox.

An A-type proanthocyanidin from Prunus armeniaca.

Roots of Prunus armeniaca yielded a new A-type proanthocyanidin whose structure was assigned as ent-epiafzelechin-3-O-p-hydroxybenzoate-(4alpha-->8, 2alpha-->O-->7)-epiafzelechin (1). The structure of 1 was determined through extensive 1D and 2D NMR studies.

Monoterpenoid and phenylethanoid glycosides from Ligustrum pedunculare.

Two new phenylethanoid glycosides, lipedosides A-I and A-II as well as six new monoterpene glycosides, lipedosides B-I-B-VI were isolated together with three known constituents, osmanthuside B, anatolioside and linalool from Ligustrum pedunculare. Their structures have been elucidated by chemical and spectroscopic methods.

Brandioside, a phenylpropanoid glycoside from Brandisia hancei.

A new phenylpropanoid glycoside, brandioside, was isolated from Brandisia hancei. Its structure, [beta-(3',4'-dihydroxylphenyl)-ethyl]-(2-O-acetyl)-(3,6-O-di-alpha -L-rhamnopyranosyl)-(4-O-caffeoyl)-beta-D-glucopyranoside, was established by chemical and spectroscopic methods.