Positive selection sites in tertiary structure of Leguminosae chalcone isomerase 1.

Isoflavonoids and the related synthesis enzyme, chalcone isomerase 1 (CHI1), are unique in the Leguminosae, with diverse biological functions. Among the Leguminosae, the soybean is an important oil, protein crop, and model plant. In this study, we aimed to detect the generation pattern of Leguminosae CHI1. Genome-wide sequence analysis of CHI in 3 Leguminosae and 3 other closely related model plants was performed; the expression levels of soybean chalcone isomerases were also analyzed. By comparing positively selected sites and their protein structures, we retrieved the evolution patterns for Leguminosae CHI1. A total of 28 CHI and 7 FAP3 (CHI4) genes were identified and separated into 4 clades: CHI1, CHI2, CHI3, and FAP3. Soybean genes belonging to the same chalcone isomerase subfamily had similar expression patterns. CHI1, the unique chalcone isomerase subfamily in Leguminosae, showed signs of significant positive selection as well as special expression characteristics, indicating an accelerated evolution throughout its divergence. Eight sites were identified as undergoing positive selection with high confidence. When mapped onto the tertiary structure of CHI1, these 8 sites were observed surrounding the enzyme substrate only; some of them connected to the catalytic core of CHI. Thus, we inferred that the generation of Leguminosae CHI1 is dependent on the positively selected amino acids surrounding its catalytic substrate. In other words, the evolution of CHI1 was driven by specific selection or processing conditions within the substrate.

Functional analysis of DNA methylation in lung cancer.

OBJECTIVES: We aimed to explore the DNA methylation difference between lung cancer samples and non-cancer lung samples, and to investigate the role of DNA methylation in the mechanism of lung cancer development. Besides, we analyzed the transcriptional regulation network of DNA methylation and the miRNAs regulated by DNA methylation. This study provides a framework for DNA methylation in other tumors or diseases. MATERIALS AND METHODS: DNA methylation and gene expression profiles used were obtained from Gene Expression Omnibus. Firstly, we identified differentially methylated genes (DMGs) by Student's t-test. Then we detected the biological processes and pathways changed in lung cancer by Gene Ontology (GO) and KEGG pathway enrichment analysis. The transcriptional factors in differential genes were identified and the microRNAs regulated by them were also obtained in TransmiR. RESULTS: We obtained 108 DMGs between lung cancer samples and non-cancer samples. Besides development related biological processes and pathways were dramatically disordered. For the DMGs, we identified 11 transcriptional factors regulating them. Moreover, we screened out 21 relationships between DMGs and their transcriptional targets. Five microRNAs are reported to be regulated by DNA methylation genes. Finally a regulation network of DNA methylation was constructed. CONCLUSIONS: DNA methylation participates in carcinogenesis at the transcriptional and post-transcriptional level. Aberrant DNA methylation will prevent its binding with the upstream regulatory proteins, inhibit the function of downstream target genes and regulate the expression of downstream miRNA, and consequently affect cell development, immunoresponse and apoptosis.

Molecular evolution of two consecutive carotenoid cleavage dioxygenase genes in strigolactone biosynthesis in plants.

Strigolactones are newly discovered plant hormones that perform various functions, from signaling in symbiotic interactions with arbuscular mycorrhizal fungi to controlling outgrowth of axillary buds. We examined the phylogenetic relationships of two carotenoid cleavage dioxygenase genes (CCD7 and CCD8) that are involved in consecutive upstream steps of the proposed strigolactone biosynthesis pathway. The CCD7 and CCD8 sequences from 11 model species, divided into two clades, correspond to sequences from monocotyledons and dicotyledons. However, the sequences from the primitive moss, Physcomitrella patens, appeared to be evolutionarily distinct from those of the angiosperms. CCD7 and CCD8 are much conserved, since no significant positive selection was detected among these plants. Ks values indicated that CCD7 and CCD8 diverged about 290 to 430 million years ago. As essential genes in the strigolactone pathway, the divergence timing of the conserved CCD7 and CCD8 genes reflects the approximate time of generation of strigolactone as a regulatory substance. This timing calculation also coincides with initiation of symbiosis between plants and microorganisms, inferred from the fossil record. Molecular evolution analyses of genes in metabolic pathways can provide insight concerning gene evolution.

[Relationship between C genotype HBV mutation and intrauterine transmission].

Objective: To analyze the virus genome mutation of mothers with C genotype HBV and explore its relationship with HBV intrauterine transmission. Methods: A total of 399 mothers carrying HBV and their newborns hospitalized in the obstetrics department of the Third People's Hospital of Taiyuan from 2011 to 2013 were selected. Necessary information about mothers and children was obtained through a questionnaire survey and medical records. HBV DNA and HBV serological markers were detected by quantitative fluorescence PCR and electrochemiluminescence. Within 24 hours after birth and before active/passive immunization, those with positive HBsAg and/or HBV DNA in femoral venous blood were determined as HBV intrauterine transmission. According to the requirements of cloning and sequencing, mothers' HBV DNA load should be ≥106 IU/ml. Among 54 cases of HBV intrauterine transmission, 22 pairs of mothers and their newborns meeting the requirements of cloning and sequencing were used as the intrauterine transmission group. The same number of mothers and their newborns without intrauterine transmission was selected as the random seed method's control group. After PCR amplification of HBV DNA, gene cloning, and sequencing, the gene mutation analysis of mothers with C genotype HBV was performed. Results: Among the 44 samples, 39 (88.63%, 39/44) were genotype C, 2 were genotype B, and 3 were mixed genotype B, and C. A total of 406 clone beads from 42 mothers with C genotype HBV were analyzed for gene mutation, including 204 in the intrauterine transmission group and 202 in the control group. The base substitution mutation rate of PreS1, S, C, and P regions in the HBV intrauterine transmission group were significantly lower than those in the control group (χ2 ranged from 8.67 to 40.73, P<0.05). The mutation rate of base deletion in PreC and X regions in the HBV intrauterine transmission group was lower than that in the control group (χ2 values were 17.82 and 34.78, P<0.001). Two clones in the X region had 31 bp insertion mutations between nt1644 and nt1645, and two clones had 27 bp insertion mutations between nt1649 and nt1650, all of which took place in the control group. Conclusions: The base substitution mutations in the PreS1, S, C, and P segments of the HBV genome in mothers with C genotype HBV were associated with the occurrence of intrauterine transmission of HBV. Deletion mutations in the PreC region, insertion and deletion mutations in the X region may reduce intrauterine transmission risk.

[Relationship between mutations of HBV basal core promoter region in HBsAg-positive mothers and intrauterine transmission].

Objective: To analyze the relationship between maternal mutations in basal core promoter region of hepatitis B virus (HBV) genotype C and intrauterine transmission. Methods: We collected information on general demographic characteristics and process of delivery among 399 pairs of consecutive HBsAg-positive mothers and their neonates, from the Third People's Hospital of Taiyuan in Shanxi province, China. Fluorescence quantitative polymerase chain reaction (FQ-PCR) and Electro-chemiluminescence immuno-assay (ECLIA) kits were used to detect both maternal and neonatal HBV DNA and serological markers in the peripheral blood. From 113 mothers with HBV DNA load ≥10(6) IU/ml, we selected 22 mothers whose neonates were with intrauterine transmission and randomly selected the same number of mothers whose neonates were without intrauterine transmission, as controls. The whole-length HBV DNA were extracted, amplified, cloned, sequenced and genotyped. Finally, a total of 39 mothers with genotype C of HBV were selected for mutation analysis. Results: Thirty-nine cases of genotype C (88.63%) were finally included in the study, with 19 cases in the intrauterine transmission group and 20 cases as controls. Rates of A1762T/G1764A double mutations were significantly different between the intrauterine transmission group and the control group (7.53% vs. 27.72%, P<0.001). Results from the multivariate analysis showed that the A1762T/G1764A double mutations had reduced the risk of intrauterine transmission (aOR=0.065, 95%CI: 0.006-0.746, P=0.028). Maternal A1762T/G1764A double mutations appeared to be possibly associated with neonatal HBeAg (P=0.050). Conclusion: A1762T/G1764A double mutations of HBV DNA from the genotype C of those HBsAg-positive mothers could reduced the risk of HBV intrauterine transmission during pregnancy.

Subunit- and brain region-specific reduction of GABAA receptor subunit mRNAs during chronic treatment of rats with diazepam.

The mRNA levels for several GABAA receptor subunits were measured by Northern blot analysis. Rats were treated for 3 wk by continuous release of diazepam (DZP) from subcutaneous reservoirs, and then sacrificed immediately or 48 h after removing the reservoirs. Poly(A)+ RNAs, isolated from cerebral cortex, cerebellum, and hippocampus, were hybridized with oligonucleotide probes for GABAA receptor subunits and a cDNA probe for beta-actin. Subunit mRNAs were expressed relative to the corresponding beta-actin mRNA. DZP treatment decreased the alpha 1 subunit mRNA level 40% in hippocampus, but it was not changed in cortex or cerebellum. The alpha 5 subunit mRNA level was decreased in cerebral cortex (28%) and hippocampus (15%). The gamma 2 subunit mRNA level was decreased (40%) only in cortex. DZP treatment did not affect alpha 2, alpha 3, alpha 4, beta 2, or beta 3 subunit mRNA levels. Decreases in mRNA levels had reversed within 48 h after stopping chronic treatment. Acute DZP did not change alpha 1, alpha 5, or gamma 2 subunit mRNA levels. The decreases in GABAA receptor subunit mRNA levels were specific to subunit and brain region. These results, coupled with those after chronic flurazepam treatment, also indicated that the effects on GABAA receptor subunit mRNA levels are specific to the benzodiazepine (BZ) used for chronic treatment.

Decreased expression of gamma-aminobutyric acid type A/benzodiazepine receptor beta subunit mRNAs in brain of flurazepam-tolerant rats.

The expression of GABAA/benzodiazepine beta subunit mRNAs was studied in cerebral cortex, hippocampus, and cerebellum of flurazepam-treated rats. Immediately following 4 wk of treatment, beta 2 and beta 3 subunit mRNAs were significantly reduced in cerebellum and hippocampus, whereas only beta 2 was decreased in cortex. These decreases had largely reversed 48 h following flurazepam treatment. After 2 wk of treatment, both beta 2 and beta 3 mRNAs were reduced in cerebellum, and beta 3 mRNA was reduced in hippocampus, but neither was changed in cortex. Four hours after an acute flurazepam treatment, the only change was a decrease in beta 3 mRNA in hippocampus. These results indicate that the expression of GABAA receptor beta subunit mRNAs in different brain regions is differentially regulated during chronic flurazepam treatment, and some changes occur within hours after a single large dose.

Treatment with an antisense oligodeoxynucleotide to the GABAA receptor gamma 2 subunit increases convulsive threshold for beta-CCM, a benzodiazepine "inverse agonist', in rats.

The gamma 2 subunit of the gamma-aminobutyric acid type-A (GABAA) receptor is associated with the actions of benzodiazepines and related drugs. A phosphorothioate-modified antisense oligodeoxynucleotide directed against the gamma 2 subunit was given by i.c.v. injection (18 micrograms in 2 microliters saline) to male Sprague-Dawley rats every 12 h for 3 days. Controls received the corresponding sense oligodeoxynucleotide. 4-6 h after the last i.c.v. treatment, rats were given methyl-beta-carboline-3-carboxylate (beta-CCM), a benzodiazepine "inverse agonist', by slow i.v. infusion. Compared to naive rats, the beta-CCM threshold dose was not affected by the sense oligodeoxynucleotide, but was increased 87% in antisense oligodeoxynucleotide-treated rats. The treatment had no effect on the seizure threshold for picrotoxin. Both antisense and sense oligodeoxynucleotide treatments slightly increased the threshold for strychnine seizures. The results suggest that antisense oligodeoxynucleotide treatment altered GABAA receptor composition and interfered with the actions of a benzodiazepine receptor ligand in vivo, and may provide a tool for studying regulation of receptor structure and function.

Treatment of ovarian malignant germ cell tumors with preservation of fertility.

Conservative operation and postoperative chemotherapy were given to 15 patients with malignant germ cell tumors of the ovary with the preservation of fertility and ovarian functions. Four patients, one with endodermal sinus tumor and three immature teratoma, had full term deliveries after the operation. The possibility was discussed to preserve young women's fertility and ovarian function in treating their malignant germ cell tumors.

Surviving starvation: essential role of the ghrelin-growth hormone axis.

After brief starvation, vertebrates maintain blood glucose by releasing fatty acids from adipose tissue. The fatty acids provide energy for gluconeogenesis in liver and are taken up by muscle, sparing glucose. After prolonged starvation, fat stores are depleted, yet blood glucose can be maintained at levels sufficient to preserve life. Using a new mouse model, we demonstrate that survival after prolonged starvation requires ghrelin, an octanoylated peptide hormone that stimulates growth hormone (GH) secretion. We studied wild-type mice and mice lacking ghrelin as a result of knockout of GOAT, the enzyme that attaches octanoate to ghrelin. Mice were fed 40% of their normal intake for 7 d. Fat stores in both lines of mice became depleted after 4 d. On day 7, mice were fasted for 23 h. In wild-type mice, ghrelin and GH rose massively, and blood sugar was maintained at ~60 mg/dL. In Goat(-/-) mice, ghrelin was undetectable and GH failed to rise appropriately. Blood sugar declined to ~20 mg/dL, and the animals were moribund. Infusion of ghrelin or GH prevented hypoglycemia. Our results support the following sequence: (1) Starvation lowers blood glucose; (2) glucose-sensing neurons respond by activating sympathetic neurons; (3) norepinephrine, released in the stomach, stimulates ghrelin secretion; (4) ghrelin releases GH, which maintains blood glucose. Thus, ghrelin lies at the center of a hormonal response that permits mice to survive an acute fast superimposed on chronic starvation.

Reduced expression of gamma-aminobutyric acid type A/benzodiazepine receptor gamma 2 and alpha 5 subunit mRNAs in brain regions of flurazepam-treated rats.

Previous studies showed that chronic benzodiazepine administration in rats affected the gamma-aminobutyric acid (GABA)A/benzodiazepine receptor. The present experiment investigated the effects of chronic flurazepam treatment on the mRNA levels for alpha 1, alpha 5, gamma 2, and gamma 2L (an alternatively spliced product of the gamma 2 gene) subunits of the GABAA/benzodiazepine receptor in rat cerebral cortex, cerebellum, and hippocampus. Rats were treated with flurazepam for 2 or 4 weeks, and the mRNA levels were measured while rats were still receiving drug or 48 hr after 4-week flurazepam treatment had been stopped. The level of alpha 5 mRNA was also measured in other rats 4 hr after a single injection of flurazepam or diazepam. The levels of mRNAs were analyzed by Northern blotting using digoxigenin-labeled oligonucleotide probes. Compared with the pair-handled controls, the levels of gamma 2 subunit mRNA in cortex and hippocampus were not changed after flurazepam treatment for 2 weeks. However, with rats treated with flurazepam for 4 weeks the levels of gamma 2 subunit mRNA were significantly reduced in cortex (31%) and hippocampus (39%) but not in cerebellum. The values returned to control levels by 48 hr after termination of the treatment. The regional distribution and time course of reduced gamma 2 levels matched the decrease in benzodiazepine binding produced by the same chronic flurazepam treatment. The amounts of alpha 5 mRNA were reduced in cortex (23%) and hippocampus (18%) 4 hr after a single dose of flurazepam but not diazepam. The levels of alpha 5 mRNA remained reduced in cerebral cortex and hippocampus (about 50%) after 2 weeks but returned to control after 4 weeks of chronic treatment with flurazepam. No change in alpha 1 or gamma 2L subunit mRNAs was observed in any of the three brain regions examined after 4 weeks of flurazepam treatment. These results suggest that benzodiazepine receptor down-regulation after chronic benzodiazepine treatment may be related to the reduced expression of gamma 2 subunit mRNA, and they also suggest differential temporal and regional regulation of alpha 5 and gamma 2 subunit mRNAs in rat brain.

Decreased benzodiazepine binding with little effect on gamma-aminobutyric acid binding in rat brain after treatment with antisense oligodeoxynucleotide to the gamma-aminobutyric acidA receptor gamma-2 subunit.

Benzodiazepine potentiation of gamma-aminobutyric acid (GABA) neurotransmission is associated with the presence of a gamma-2 subunit in the GABAA receptor. A method was developed to modify the gamma-2 subunit expression in adult rat brain. Unilateral intracerebroventricular (i.c.v.) infusion of a 17-base phosphorothioate-modified antisense oligodeoxynucleotide (ASO) was performed every 12 hr for 3 days. Controls were treated with a sense oligodeoxynucleotide. Parasagittal brain sections were used for quantitative autoradiographic analysis of radioligand binding. ASO treatment caused a 15% to 25% decrease of specific [3H]flunitrazepam binding in most brain areas, with statistically significant decreases in frontal cortex, cerebellar molecular layer, zona reticulata of substantia nigra and CA3 of hippocampus. In contrast, [3H]muscimol binding was not changed. [3H]GABA binding was also unchanged, except for a 10% decrease in cerebellar granule cell layer. The effect on the chloride channel of the GABAA receptor complex was examined by 4'-ethynyl-4-n-[2, 3-3H2]propylbicycloorthobenzoate binding; most brain areas showed small decreases in 4'-ethynyl-4-n-[2, 3-3H2]propylbicycloorthobenzoate binding. However, hippocampal regions showed much larger decreases. Binding of the adenosine A1 receptor antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine was used to examine possible secondary effects of the ASO. There was a decrease in [3H]8-cyclopentyl-1,3-dipropylxanthine binding, but this was much smaller than the change in [3H]flunitrazepam binding, and no area showed a significant effect. Quantitative immunoblotting with a monoclonal antibody that recognizes GABAA receptor beta-2 and beta-3 subunits showed no change in immunoreactivity in cerebellar tissue after ASO treatment. The results indicate a selective effect on benzodiazepine binding to GABAA receptors and a possible change in receptor subunit composition.

Acute pentylenetetrazol injection reduces rat GABAA receptor mRNA levels and GABA stimulation of benzodiazepine binding with No effect on benzodiazepine binding site density.

The effects of a single convulsive dose of pentylenetetrazol (PTZ, 45 mg/kg i.p.) on rat brain gamma-aminobutyric acid type A (GABAA) receptors were studied. Selected GABAA receptor subunit mRNAs were measured by Northern blot analysis (with beta-actin mRNA as a standard). Four hours after PTZ, the GABAA receptor gamma2-mRNA was decreased in hippocampus, cerebral cortex, and cerebellum; alpha1-mRNA was decreased in cerebellum; and beta2 subunit mRNA was decreased in cortex and cerebellum. The alpha5 subunit mRNA level was not altered. Those mRNAs that had been reduced were increased in some brain regions at the 24-h time point, and these changes reverted to control levels by 48 h. PTZ effect on GABAA receptors was also studied by autoradiographic binding assay with the benzodiazepine agonist [3H]flunitrazepam (FNP), the GABAA agonist [3H]muscimol, and the benzodiazepine antagonist [3H]flumazenil. There was an overall decrease in [3H]FNP binding 12 but not 24 h after PTZ treatment. In contrast, [3H]muscimol binding was minimally affected, and [3H]flumazenil binding was unchanged after PTZ treatment. Additional binding studies were performed with well-washed cerebral cortical homogenates to minimize the amount of endogenous GABA. There was no PTZ effect on specific [3H]FNP binding. However, there was a significant reduction in the stimulation of [3H]FNP binding by GABA. The results showed that an acute injection of PTZ caused transient changes in GABAA receptor mRNA levels without altering receptor number but affected the coupling mechanism between the GABA and benzodiazepine sites of the GABAA receptor.