Oct4 is epigenetically regulated by methylation in normal placenta and gestational trophoblastic disease.

Oct4 is a transcription factor that plays a crucial role in maintaining pluripotency of embryonic stem cells. Down-regulation of Oct4 is associated with the differentiation of trophectoderm cell lineage, from which the normal placenta derives. We investigated the methylation and expression status of Oct4 in normal placenta and gestational trophoblastic disease (GTD) as attempts to investigate the role of Oct4 in the pathogenesis of GTD. By methylation-specific PCR, we observed both methylated and unmethylated Oct4 alleles in all 25 first trimester and 10 term placentas while 33% (18/54) of hydatidiform moles, and two choriocarcinoma cell line (JEG3 and JAR), only displayed methylated Oct4 allele. By quantitative TaqMan real-time PCR, Oct4 mRNA was significantly reduced in hydatidiform moles (P=0.04), JEG3 and JAR (P=0.024) when compared with normal placentas. Oct4 methylation was significantly correlated with Oct4 mRNA expression in placenta and GTD (P=0.012). Hypermethylation in minimal promoter and exon 1 region of Oct4 were confirmed in JEG3 and JAR by bisulfite genomic sequencing. The Oct4 mRNA expression in JEG3 and JAR increased after treatment with 5-aza-2'-deoxycytidine and/or trichostatin A. Our findings suggest that Oct4 is down-regulated by hypermethylation in normal placenta and GTD and such process is important in pathogenesis of GTD.

DNA breakage induced by 1,2,4-benzenetriol: relative contributions of oxygen-derived active species and transition metal ions.

We report here the relative roles of metals and selected reactive oxygen species in DNA damage by the genotoxic benzene metabolite 1,2,4-benzenetriol, and the interactions of antioxidants in affording protection. 1,2,4-Benzenetriol induces scission in supercoiled phage DNA in neutral aqueous solution with an effective dose (ED(50)) of 6.7 microM for 50% cleavage of 2.05 microg/ml supercoiled PM2 DNA. In decreasing order of effectiveness: catalase (20 U/ml), formate (25 mM), superoxide dismutase (20 U/ml), and mannitol (50 mM) protected, from 85 to 28%. Evidently, H(2)O(2) is the dominant active species, with O(2)(*)(-) and *OH playing subordinate roles. Desferrioxamine or EDTA inhibited DNA breakage by 81-85%, despite accelerating 1,2,4-benzenetriol autoxidation. Consistent with this suggestion of a crucial role for metals, addition of cupric, cuprous, ferric, or ferrous ions enhanced DNA breakage, with copper being more active than iron. Combinations of scavengers protected more effectively than any single scavenger alone, with implications for antioxidants acting in concert in living cells. Synergistic combinations were superoxide dismutase with *OH scavengers, superoxide dismutase with desferrioxamine, and catalase with desferrioxamine. Antagonistic (preemptive) combinations were catalase with superoxide dismutase, desferrioxamine with *OH scavengers, and catalase with *OH scavengers. The most striking aspect of synergism was the extent to which metal chelation (desferrioxamine) acted synergistically with either catalase or superoxide dismutase to provide virtually complete protection. Concluding, 1,2,4-benzenetriol-induced DNA damage occurs mainly by site-specific, Fenton-type mechanisms, involving synergism between several reactive intermediates. Multiple antioxidant actions are needed for effective protection.

DNA-breaking versus DNA-protecting activity of four phenolic compounds in vitro.

Given the paradoxical effects of phenolics in oxidative stress, we evaluated the relative pro-oxidant and antioxidant properties of four natural phenolic compounds in DNA nicking. The phenolic compounds differed dramatically in their ability to nick purified supercoiled DNA, with the relative DNA nicking activity in the order: 1,2,4-benzenetriol (100% nicking) > gallic acid > caffeic acid > gossypol (20% nicking). Desferrioxamine (0.02 mM) decreased DNA strand breakage by each phenolic, most markedly with gallate (85% protection) and least with caffeic acid (26% protection). Addition of metals accelerated DNA nicking, with copper more effective (approximately 5-fold increase in damage) than iron with all four phenolics. Scavengers revealed the participation of specific oxygen-derived active species in DNA breakage. Hydrogen peroxide participated in all cases (23-90%). Hydroxyl radicals were involved (32-85%), except with 1,2,4-benzenetriol. Superoxide participated (81-86%) with gallic acid and gossypol, but not with caffeic acid or 1,2,4-benzenetriol. With 1,2,4-benzenetriol, scavengers failed to protect significantly except in combination. Thus, in the presence of desferrioxamine, catalase or superoxide dismutase inhibited almost completely. When DNA breakage was induced by Fenton's reagent (ascorbate plus iron) the two catechols (caffeic acid and gossypol) were protective, whereas the two triols (1,2,4-benzenetriol and gallic acid) exacerbated damage.

The NoH value in EPR spin trapping: a new parameter for the identification of 5,5-dimethyl-1-pyrroline-N-oxide spin adducts.

The ratio of the nitrogen to hydrogen hyperfine splittings (aN/aH) of spin adducts derived from the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) has been found to be a useful parameter for adduct identification. For example, this parameter makes it possible to distinguish between the superoxide (aN/aH = 1.22-1.26) and peroxyl (aN/aH = 1.33-1.40) radical adducts of DMPO in aqueous solution. Since the aN to aH ratio corrects for minor differences in EPR spectrometer calibration, it is a more reproducible parameter than the aN and aH values themselves.

Effect of non-steroidal anti-inflammatory drugs on gastric and duodenal prostaglandin concentrations in patients with Helicobacter pylori infection.

BACKGROUND/AIMS: Both Helicobacter pylori and non-steroidal anti-inflammatory drugs are reported to affect gastroduodenal prostaglandin synthesis. However, their influence on gastric mucosal prostaglandins remains unclear. The aim of this study was to investigate the influence of nonsteroidal anti-inflammatory drugs on mucosal prostaglandin synthesis in patients with Helicobacter pylori infection. METHODOLOGY: We enrolled 87 Helicobacter pylori-infected patients in this study (gastric ulcer: 33, duodenal ulcer: 41, and non-ulcer dyspepsia: 13). Of them, 27 patients received non-steroidal anti-inflammatory drugs. Endoscopy was performed and biospy specimens from gastric body, antrum and duodenal bulb were assessed for Helicobacter pylori and prostaglandin concentration. RESULTS: A significantly lower mucosal prostaglandin E2 level at gastric body (142.2 +/- 28.1 ng/mg vs. 222.0 +/- 12.4 ng/mg, mean +/- SEM) and antrum (131.3 +/- 26.4 ng/mg vs. 226.0 +/- 19.0 ng/mg) was noted in Helicobacter pylori-infected gastric ulcer patients with non-steroidal anti-inflammatory drugs ingestion than in that of patients without non-steroidal anti-inflammatory drugs ingestion (p < 0.05). Using a multivariate analysis, we found that non-steroidal anti-inflammatory drug was an independent variable affecting gastric and duodenal mucosal prostaglandin E2 synthesis in patients with Helicobacter pylori-infected gastric ulcer. CONCLUSIONS: Non-steroidal anti-inflammatory drugs decrease gastroduodenal mucosal prostaglandin E2 synthesis in gastric ulcer patients with Helicobacter pylori infection.

[The chromosome-specific PCR marker's screening and identification of barley 6H chromosome].

Two barley 6H chromosome specific RAPD markers were obtained by screening DNA of barley Hordeum vulgare (Betzes) and wheat-barley 6H addition line with 200 primers, then the RAPD markers were changed into specific PCR markers. Checking different plant materials by the PCR markers, it revealed that there was a specific band in those materials containing 6H chromosome such as Betzes, Igri, CS6H, and there was no specific band if the material did not contain 6H chromosome, such as Triticum aestivum, Secale cereale, Agropyron intermedium, Haynaldia villosa, Thinopyrum elongatum. Therefore, those PCR markers specific to chromosome 6H of barley are established. Southern hybridization indicated that the two cloned DNA fragments belong to barley genomic specific high-copy repeat sequence and low-copy sequence in wheat and barley genomes respectively.

Genetic transformation of Lycium barbarum L. via A. tumefaciens.

A system for transformation and regeneration of Lycium barbarum L., an important Chinese medical plant, has been established. Young stem segments from Lycium barbarum L. were infected with Agrobacterium tumefaciens C58cl(pGV3850::neo1103), and the transformed calli selected from the callus induction medium containing 50 micrograms/ml kanamycin could regenerate buds on differentiation medium containing 25 micrograms/ml kanamycin. 30% of the regenerated buds were normal in morphology. The normal buds could develop into whole plantlets after they were transferred to the rooting medium to induce roots. Nopaline detection, NPT-II enzyme activity assay and Southern blotting hybridization indicated that the foreign genes had been integrated into the genome of Lycium barbarum L. and expressed in the plant. In the processes of experiments, it was found that (i) after the pre-processes, the explants which formed callus quickly were easy to transform; (ii) the rate of normal regenerated plants from transgenic calli was higher than that from the untransgenic ones.

[Clinical and experimental studies on preventing and treating anaphylactic asthma with Zusanli point immunotherapy].

We have studied on preventing and treating anaphylactic asthma with Zusanli (S36) point immunotherapy (ZPIT). Sixty-nine patients were observed. The results showed that the clinical curative effect of ZPIT was not only much higher than that of conventional desensitization therapy, but also the patients' total IgE level was reduced, anti-acarid IgE was lowered, SIgA level was raised, the absolute eosinophilic granulocyte level dropped and pulmonary function recovered. Animal experiment results showed that the ZPIT could more effectively suppress the guinea pigs' anaphylactic asthma allergized by albumin and more obviously resist the guinea pigs' bronchial spasm induced by histamine and acetylcholine than the conventional desensitization therapy and injected normal saline. The immunomodulating action of the ZPIT are elucidated from clinical study and animal experiment in the paper.

The methylation pattern of p16INK4a gene promoter in psoriatic epidermis and its clinical significance.

BACKGROUND: Alteration of the p16INK4a gene by epigenetic changes has been described in some hyperproliferative skin diseases, but its importance in psoriasis has not yet been established. OBJECTIVES: To investigate the methylation status of the p16INK4a gene in psoriatic epidermis, its clinical significance and the possible epigenetic mechanisms of psoriasis. METHODS: DNA and RNA specimens were obtained from the lesional epidermis of 56 patients with plaque psoriasis. Methylation-specific polymerase chain reaction (PCR) and DNA sequencing were used to detect the density and sites of methylation in the p16INK4a promoter region. The reverse transcription-PCR technique was applied to detect the mRNA expression of p16INK4a. RESULTS: p16INK4a gene promoter methylation was shown in 17 of 56 (30%) patients with psoriasis. Psoriasis Area and Severity Index scores in patients showing methylation were higher than in those who did not (P<0.05). The mRNA expression level of p16INK4a in the methylated group was significantly lower than in the unmethylated group (t=2.515, P=0.015). In the methylated group, about 50% of the CpG islands were methylated in the promoter region. CONCLUSIONS: Overall, methylation of the p16INK4a gene promoter is found in psoriatic epidermis, which is associated with the mRNA level of p16INK4a expression and activity of the disease. These data indicate that methylation of the p16INK4a promoter may play a potential role in the pathogenesis of psoriasis.

FWCW: a personal view.

PIP: The personal experiences at the Beijing Fourth World Conference on Women and the Nongovernmental Organization (NGO) Forum were described by a Chinese American veterinarian. The author became aware that women were primarily the persons living in poverty in the world. Business and government appeared more concerned with economic growth. As a consequence, developed countries with 20% of world population consume 80% of the world's resources. The privilege of money secures a position of power in the world, power to buy up the resources of the world. This process is unsustainable and inequitable. Women are viewed as key players in shifting the balance in favor of a higher quality of life. The NGO workshops on Women in Livestock Development (WILD) was run by women veterinarians, who worked at the local level with women in poor areas. WILD operates out of offices in Arkansas in the USA. WILD women work in Sichuan, China, among the Han and Tibetans in increasing family income through heifer and livestock production. Conference participants from WILD programs talked about their experiences with increased income and loans to other women. The World Women's Veterinary Association members, who attended the conference, visited a small animal clinic in Beijing run by the Agriculture Ministry, and Beijing Agricultural University and its Veterinary Teaching Hospital. Demonstrations were given of small animal acupuncture. The author found the conference to be a success and found that press reports misrepresented the energy generated by the meeting.^ieng

O2- photogenerated from aqueous solutions of tetracycline antibiotics (pH 7.3) as evidenced by DMPO spin trapping and cytochrome c reduction.

UV-irradiation of several tetracycline antibiotics in aqueous buffer (pH 7.3) resulted in the generation of the superoxide anion radical (O2-) which was detected by cytochrome c reduction and by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide and was inhibited by superoxide dismutase. A comparison of the O2- yields from the tetracyclines examined showed the trend chlortetracycline (CTC) greater than oxytetracycline (OXY) greater than demeclocycline (DEM) much greater than (doxycycline (DOXY) = tetracycline (TC) = minocycline (MINO) = 0). This trend is in reasonable agreement with clinical reports that CTC, OXY and DEM are potent photosensitizers, TC is only weakly phototoxic whereas MINO is not. These findings suggest that the O2- production may be involved in tetracycline-induced phototoxicity. While the two methods for O2- detection gave comparable results for most of the tetracyclines, the spin trapping technique was clearly superior for DOXY which reduced cytochrome c in the dark.

Superoxide dismutase amplifies dye photosensitized production of desferal free radical: an electron spin resonance study.

Desferal free radical (DFFR) photogenerated from dye sensitization was studied by electron spin resonance. When irradiated at the visible maximum in the presence of O2, both rose bengal and riboflavin sensitized the oxidation of Desferal (DF) and generated the DFFR. The yield of DFFR was amplified by superoxide dismutase (SOD). The SOD enhancement was attributed to the inhibition of superoxide-induced DFFR destruction. Similar SOD enhancement was observed with dyes Rhodamine 123 and Gentian Violet. Our studies suggest that when Desferal is used as a chelating agent in the presence of SOD, systems involving O2- could face interference from DFFR even at concentrations as low as 10 microM DF. DFFR may interfere with the chain reaction of lipid peroxidation resulting in an apparent protective action which, in fact, has very little to do with chelating the catalytic iron.

In vitro pollen plant induction and embryoid clone establishment of Panax ginseng.

In anther culture of Panax ginseng, its callus formation showed a wide adaptability to culture media. Large numbers of calli were induced on media exhibiting better effects of induction. Supplements of 5 mg 2,4-D/1 and 1 mg KT/1 to the media proved to be much effective. Regeneration of the whole plantlets from anther culture of Panax ginseng is usually quite difficult. During the past three years, however, sixteen of the 100 medium formulae tested were proved to be suitable. The formulae of MS + 0.5 mg BA/1 + 2 mg GA/1 + 1000 mg LH/1 + 3% sucrose were considered good and effective. A visibly differentiated body, which was light-milky white and later turned into a light green spot, was formed 40 days after the callus was transferred to the differentiation media. This body differentiated subsequently into buds, roots and, eventually, seedlings. The embryoid clones have been established in order to maintain its ability of continual differentiation into plantlets through successive culturings of many generations. The test-tube ginseng thus formed were transferred to regular flowerpots and grew well. Based upon chromosome examination of the callus cells and the root tips, we tentatively affirmed that the majority of these regenerated from anther of Panax ginseng were originated from pollen cells.