Studies on interactions of carbazole derivatives with DNA,cell image,and cytotoxicity

DNA-binding agents have been considered as an established opportunity for the development of anticancer drugs and DNA fluorescence probes. This work reported the synthesis of two novel carbazole derivatives (1 and 2) and investigated their DNA binding properties, living cell image, and cytotoxicity. The results demonstrated that both compounds presented the higher binding affinity to G-quadruplex than to duplex DNA by means of UV–Vis absorption titration and fluorescent intercalator displacement. Continuous variation analysis indicated that their binding stoichiometries of the compound/G-quadruplex were near 2 except the compound/bcl-2. Circular dichroism spectra showed that both compounds had no influence on the conformation of G-quadruplexes. Fluorescence titrations indicated that 2 had the potential to be a G-quadruplex selective fluorescent probe, while 1 could be used as a fluorescent probe for duplex DNA. Confocal fluorescence images indicated that both compounds could enter the living HepG2 cells, and 1 mainly located in nucleus whereas 2 mainly distributed in cytoplasm. DNase and RNase digest tests indicated that both compounds could enter into the nucleus and interact with duplex DNA, especially, 2 could interact with RNA and/or G-quadruplex DNA. They also exhibited an obvious antiproliferative activity to HepG2 by using MTT assays, with IC₅₀ values of 2.7 and 9.5?μM for 1 and 2, respectively.     

Peripubertal vitamin D(3) deficiency delays puberty and disrupts the estrous cycle in adult female mice

The mechanism(s) by which vitamin D(3) regulates female reproduction is minimally understood. We tested the hypothesis that peripubertal vitamin D(3) deficiency disrupts hypothalamic-pituitary-ovarian physiology. To test this hypothesis, we used wild-type mice and Cyp27b1 (the rate-limiting enzyme in the synthesis of 1,25-dihydroxyvitamin D(3)) null mice to study the effect of vitamin D(3) deficiency on puberty and reproductive physiology. At the time of weaning, mice were randomized to a vitamin D(3)-replete or -deficient diet supplemented with calcium. We assessed the age of vaginal opening and first estrus (puberty markers), gonadotropin levels, ovarian histology, ovarian responsiveness to exogenous gonadotropins, and estrous cyclicity. Peripubertal vitamin D(3) deficiency significantly delayed vaginal opening without affecting the number of GnRH-immunopositive neurons or estradiol-negative feedback on gonadotropin levels during diestrus. Young adult females maintained on a vitamin D(3)-deficient diet after puberty had arrested follicular development and prolonged estrous cycles characterized by extended periods of diestrus. Ovaries of vitamin D(3)-deficient Cyp27b1 null mice responded to exogenous gonadotropins and deposited significantly more oocytes into the oviducts than mice maintained on a vitamin D(3)-replete diet. Estrous cycles were restored when vitamin D(3)-deficient Cyp27b1 null young adult females were transferred to a vitamin D(3)-replete diet. This study is the first to demonstrate that peripubertal vitamin D(3) sufficiency is important for an appropriately timed pubertal transition and maintenance of normal female reproductive physiology. These data suggest vitamin D(3) is a key regulator of neuroendocrine and ovarian physiology.     

Characterization of zebrafish mutants with defects in bone calcification during development

Using the fluorescent dyes calcein and alcian blue, we stained the F3 generation of chemically (ENU) mutagenized zebrafish embryos and larvae, and screened for mutants with defects in bone development. We identified a mutant line, bone calcification slow (bcs), which showed delayed axial vertebra calcification during development. Before 4–5 days post-fertilization (dpf), the bcs embryos did not display obvious abnormalities in bone development (i.e., normal number, size and shape of cartilage and vertebrae). At 5–6 dpf, when vertebrae calcification starts, bcs embryos began to show defects. At 7 dpf, for example, in most of the bcs embryos examined, calcein staining revealed no signals of vertebrae mineralization, whereas during the same developmental stages, 2–14 mineralized vertebrae were observed in wild-type animals. Decreases in the number of calcified vertebrae were also observed in bcs mutants when examined at 9 and 11 dpf, respectively. Interestingly, by 13 dpf the defects in bcs mutants were no longer evident. There were no significant differences in the number of calcified vertebrae between wild-type and mutant animals. We examined the expression of bone development marker genes (e.g., Sox9b, Bmp2b, and Cyp26b1, which play important roles in bone formation and calcification). In mutant fish, we observed slight increases in Sox9b expression, no alterations in Bmp2b expression, but significant increases in Cyp26b1 expression. Together, the data suggest that bcs delays axial skeletal calcification, but does not affect bone formation and maturation.     

Antiemetic Role of Thalidomide in a Rat Model of Cisplatin-Induced Emesis

The efficacy of thalidomide to attenuate cisplatin-induced emesis was evaluated in a rat model. Four groups were utilized: control group (peritoneal injection and gastric lavage with normal saline), cisplatin group (peritoneal injection of cisplatin at 10 mg/kg and gastric lavage with normal saline), thalidomide group (cisplatin as above and gastric lavage with thalidomide at 10 mg/kg), and granisetron group (positive control for antiemetic effects; cisplatin given as above and gastric lavage done with granisetron at 0.5 mg/kg). The cisplatin-induced kaolin consumption (pica behavior) was used as a model of emesis in patients. The animals’ kaolin and food intakes were measured. Further, medulla and gastric tissues were obtained 5 and 33 h after peritoneal injections to quantify the levels of Substance P and Neurokinin-1 receptor (NK-1R). The cisplatin-induced kaolin consumption was significantly (p < 0.05 vs. cisplatin group) attenuated by thalidomide 72 h after the injection. The levels of Substance P in the medulla and gastric tissue were increased 5 h after the injection in both cisplatin and thalidomide groups, however, returned faster to normal levels in the thalidomide group (p < 0.05 vs. cisplatin group). Further, levels of NK-1R in the cisplatin, thalidomide, and granisetron group were significantly increased at both 5 and 33 h (p < 0.05 vs. control group), with no obvious difference among these three groups. In conclusion, thalidomide attenuates animal equivalent of cisplatin-induced emesis, and this beneficial effect is associated with decreased levels of Substance P levels in the medulla and gastric tissue.     

Cloning and expression analysis of a water stress-induced gene from Brassica oleracea.

Plant growth and productivity are greatly affected by water stress, such as drought and salinity. Here we report on the cloning and expression analysis of a water stress-induced gene from Brassica oleracea (designated as BoWS, GenBank accession number AY571333) by rapid amplification of cDNA ends (RACE). The full-length cDNA of BoWS consisted of 594 bp and contained a 285 bp open reading frame (ORF) encoding a 95-amino-acid protein. The deduced protein had a calculated molecular mass of 10.53 kDa and an isoelectric point of 6.93. The sequence similarity and comparative analysis showed that BoWS was 84% identical to Arabidopsis thaliana putative water stress-induced protein (GenBank accession number AAM67282). Southern blot analysis indicated that BoWS was a low-copy gene. Northern blot analysis revealed that the expression of BoWS was upregulated by abscisic acid (ABA), mannitol, NaCl, drought, salicylic acid (SA) and hydrogen peroxide (H(2)O(2)). Our results indicate that BoWS is extremely related to the water-deficit stress in B. oleracea.     

Transient Ischemia Differentially Increases Tyrosine Phosphorylation of NMDA Receptor Subunits 2A and 2B

Abstract: Activation of the N -methyl- d -aspartate (NMDA) receptor has been implicated in the events leading to ischemia-induced neuronal cell death. Recent studies have indicated that the properties of the NMDA receptor channel may be regulated by tyrosine phosphorylation. We have therefore examined the effects of transient cerebral ischemia on the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in different regions of the rat brain. Transient (15 min) global ischemia was produced by the four-vessel occlusion procedure. The tyrosine phosphorylation of NR2A and NR2B subunits was examined by immunoprecipitation with anti-tyrosine phosphate antibodies followed by immunoblotting with antibodies specific for NR2A or NR2B, and by immunoprecipitation with subunit-specific antibodies followed by immunoblotting with anti-phosphotyrosine antibodies. Transient ischemia followed by reperfusion induced large (23–29-fold relative to sham-operated controls), rapid (within 15 min of reperfusion), and sustained (for at least 24 h) increases in the tyrosine phosphorylation of NR2A and smaller increases in that of NR2B in the hippocampus. Ischemia-induced tyrosine phosphorylation of NR2 subunits in the hippocampus was higher than that of cortical and striatal NR2 subunits. The enhanced tyrosine phosphorylation of NR2A or NR2B may contribute to alterations in NMDA receptor function or in signaling pathways in the postischemic brain and may be related to pathogenic events leading to neuronal death.