[Interferon α sensitizes human osteosarcoma cells to doxorubicin-induced apoptosis through p53-dependent pathway].

Objective: To investigate the effect of IFNα on doxorubicin-induced apoptosis in human osteosarcoma cells with its molecular mechanisms to provide evidences for improving the treatment of osteosarcoma. Methods: Osteosacoma U2OS and MG63 cells were treated with IFNα and Doxorubicin, alone or in combination, for 72 h . Cytotoxicity was determined with MTT. Apoptosis was evaluated through fluorescence-activated cell sorting, Hoechst33258 staining and DNA ladder assay. The expression of p53, Bax, Bcl-2, Mdm2, p21, caspase-3 and PARP was determined with Western blot. siRNA interference was used to silence p53. Results: IFNα treatment for 72 h did not induce cytotoxicity but greatly enhanced doxorubicin-induced cytotoxicity and apoptosis in p53-wild U2OS cells but not p53-mutant MG63 cells. Compared with other groups, the combination of IFNα and doxorubicin induced more obvious apoptotic morphological changes and DNA ladder. IFNα did not alter the expression of the indicate genes. The expression of p53, Bax, Mdm2 and p21 was up-regulated by doxorubicin and further increased in response to combination. The expression of Bcl-2 was down-regulated by doxorubicin and further decreased in response to combination.There were no differences among groups in MG63 cells. The expression of p53 was effectively blocked by p53-siRNA in U2OS cells. The p53 silencing greatly reduced the cytotoxicity mediated by combination for 72 h, compared with non- and control-siRNA groups. The activation of caspase-3 and PARP mediated by combination was largely suppressed by p53 silence. Conclusion: IFNα sensitizes human osteosarcoma cells to doxorubicin-induced apoptosis through p53-dependent pathway. The combination of IFNα and traditional chemotherapy can be used in osteosarcoma treatment.

Temporal contrast enhancement of picosecond pulses based on phase-conjugate wave generation.

A practical technique based on the phase-conjugate wave (PCW) generation is proposed to improve the temporal contrast of the picosecond pulses. Our theory predicts the temporal contrast of the picosecond pulses can be enhanced to about the cube of the temporal contrast of the input pulse via the PCW generation, in which the conversion efficiency from the incident pulse to the PCW is about 25%. In a proof-of-principle experiment, the temporal contrast of picosecond pulses was enhanced from 1.7×10(3) to 8.6×10(8) with the conversion efficiency of 10%. This technique is promising to effectively eliminate the background of the ultrashort and ultraintense laser pulses in the future.

Effect of high-dose progestogen on hemostatic properties of blood in patients with endometrial cancer.

Fifteen cases of endometrial cancer were administered daily doses of 600 mg of MPA after surgery to prevent the recurrence of cancer. The initiation times of coagulation (time necessary for fibrin network formation) were measured with a highly sensitive damped oscillation rheometer and compared with those of 15 control patients who were not administered MPA. Biochemical studies of blood coagulation and fibrinolysis were also done. The initiation times of coagulation were 19.0+/-1.8 minutes (min mean +/- standard deviation) after 3-6 months and 16.0+/-2.0 min after 9-12 months of MPA administration, both times being significantly shorter compared with the controls (24.0+/-2.5 min). Hematocrit values, platelet counts and fibrinogen levels were similar between the two groups. Activated partial thromboplastin time (APTT) was significantly decreased and antithrombin III activity (AT III), thrombin-antithrombin complex (TAT), plasminogen level, plasmin-alpha(2) plasmin inhibitor complex level (PIC) and the fibrin degradation product level (FDP) were significantly increased in the MPA group compared with the control group. Accelerated coagulation of blood was definitely induced by high-dose MPA but antithrombin and fibrinolytic activities were also induced, and, thus, thromboembolic complications were prevented.

Induction of experimental autoimmune Graves' disease in BALB/c mice.

We immunized BALB/c mice with M12 cells (H-2d) expressing either mouse (mM12 cells) or human thyrotropin receptor (TSHR) (hM12 cells). Immunized mice developed autoantibodies to native TSHR by day 90 and, by day 180, showed considerable stimulatory Ab activity as measured by their ability to enhance cAMP production (ranging from 6. 52 to 20.83 pmol/ml in different treatment groups relative to 1.83 pmol/ml for controls) by TSHR-expressing Chinese hamster ovary cells. These mice developed severe hyperthyroidism with significant elevations in both tetraiodothyronine and triiodothyronine hormones. Tetraiodothyronine levels in different experimental groups ranged from a mean of 8.66-12.4 microg/dl, relative to 4.8 microg/dl in controls. Similarly, mean triiodothyronine values ranged from 156.18 to 195.13 ng/dl, relative to 34.99 ng/dl for controls. Next, we immunized BALB/c mice with a soluble extracellular domain of human TSHR (TBP), or TBP expressed on human embryonic kidney cells (293 cells) (293-TBP cells). These mice showed severe hyperthyroidism in a manner very similar to that described above for mice immunized with the mouse TSHR or human TSHR, and exhibited significant weight loss, with average weight for treatment groups ranging from 20.6 to 21.67 g, while controls weighed 24.2 g. Early after onset of the disease, histopathological examination of thyroids showed enlargement of colloids and thinning of epithelial cells without inflammation. However, later during disease, focal necrosis and lymphocytic infiltration were apparent. Our results showed that conformationally intact ectodomain of TSHR is sufficient for disease induction. Availability of a reproducible model in which 100% of the animals develop disease should facilitate studies aimed at understanding the molecular pathogenesis of Graves' disease.

Characterization of two LGR genes homologous to gonadotropin and thyrotropin receptors with extracellular leucine-rich repeats and a G protein-coupled, seven-transmembrane region.

The receptors for LH, FSH, and TSH belong to the large G protein-coupled, seven-transmembrane (TM) protein family and are unique in having a large N-terminal extracellular (ecto-) domain containing leucine-rich repeats important for interaction with the glycoprotein ligands. We have identified two new leucine-rich repeat-containing, G protein-coupled receptors and named them as LGR4 and LGR5, respectively. The ectodomains of both receptors contain 17 leucine-rich repeats together with N- and C-terminal flanking cysteine-rich sequences, compared with 9 repeats found in known glycoprotein hormone receptors. The leucine-rich repeats in LGR4 and LGR5 are arrays of 24 amino acids showing similarity to repeats found in the acid labile subunit of the insulin-like growth factor (IGF)/IGF binding protein complexes as well as slit, decorin, and Toll proteins. The TM region and the junction between ectodomain and TM 1 are highly conserved in LGR4, LGR5, and seven other LGRs from sea anemone, fly, nematode, mollusk, and mammal, suggesting their common evolutionary origin. In contrast to the restricted tissue expression of gonadotropin and TSH receptors in gonads and thyroid, respectively, LGR4 is expressed in diverse tissues including ovary, testis, adrenal, placenta, thymus, spinal cord, and thyroid, whereas LGR5 is found in muscle, placenta, spinal cord, and brain. Hybridization analysis of genomic DNA indicated that LGR4 and LGR5 genes are conserved in mammals. Comparison of overall amino acid sequences indicated that LGR4 and LGR5 are closely related to each other but diverge, during evolution, from the homologous receptor found in snail and the mammalian glycoprotein hormone receptors. The identification and characterization of new members of the LGR subfamily of receptor genes not only allow future isolation of their ligands and understanding of their physiological roles but also reveal the evolutionary relationship of G protein-coupled receptors with leucine-rich repeats.

Pathway of complex formation between DNA and three subunits of CBF/NF-Y. Photocross-linking analysis of DNA-protein interaction and characterization of equilibrium steps of subunit interaction and dna binding.

In this study, we used a photocross-linking method to identify specific contact of CCAAT-binding factor (CBF) subunits in a CBF-DNA complex. The analysis showed that all three subunits in the CBF-DNA complex were cross-linked to DNA and that CBF-B and CBF-C were cross-linked more strongly than CBF-A. None of the CBF-A and CBF-C subunits, which together formed a CBF-A/CBF-C heterodimer, were cross-linked without CBF-B; in contrast, CBF-B was cross-linked in the absence of CBF-A/CBF-C. No subunit of heterotrimeric CBF containing DNA-binding domain mutant of either CBF-B or CBF-C was cross-linked to DNA, and interestingly, cross-linking of CBF-B that occurred without CBF-A/CBF-C was inhibited in presence of mutant CBF-C/CBF-A heterodimer. Altogether, these results indicated that the specific DNA contact surface of each CBF subunit is generated as a result of interaction between CBF-B and CBF-A/CBF-C heterodimer and that the three CBF subunits interact interdependently with DNA to form a CBF-DNA complex. Equilibrium interactions among the three CBF subunits and between CBF subunits and DNA were studied by electrophoretic mobility shift assay. This showed that at equilibrium DNA-binding conditions, the CBF-A/CBF-C heterodimer is very stable, but association between CBF-B and CBF-A/CBF-C is very weak. The nature of the association of CBF-B with CBF-A/CBF-C was also revealed by studying the inhibition of CBF-DNA complex formation by the mutant CBF-B. This study indicated that the association between CBF-B and CBF-A/CBF-C is stabilized upon interaction with DNA, a process likely to favor formation of a high-affinity CBF-DNA complex.

Soluble ecto-domain mutant of thyrotropin (TSH) receptor incapable of binding TSH neutralizes the action of thyroid-stimulating antibodies from Graves' patients.

A soluble form of the amino-terminal extracellular (ecto-) domain of the human TSH receptor was generated. This protein was capable of binding TSH and autoimmune antibodies found in Graves' patients. A deletion mutant of the ectodomain lacking nine amino acids in the C-terminal region lost its ability to interact with TSH but retained binding to Graves' IgGs. In cells expressing recombinant TSH receptors, cotreatment with the mutant protein blocked the cAMP production induced by stimulating antibodies from all Graves' patients tested but was without effect on TSH action. The ability to dissociate the actions of TSH and Graves' IgGs provides a tool with which to study the mechanisms underlying Graves' disease and the possibility of neutralizing the undesirable effects of thyroid-stimulating antibodies without altering the normal responses to TSH.

Acute myocarditis with eosinophilia presenting as asymmetric septal hypertrophy during pregnancy.

A pregnant female at 24 weeks' gestation developed acute myocarditis with eosinophilia presenting as asymmetric septal hypertrophy. Her clinical course improved with ordinary treatment within a few weeks.

Modulatory role of epidermal growth factor in follicle-stimulating hormone-induced DNA synthesis in cultured rat granulosa cells.

Epidermal growth factor (EGF) modestly increased DNA synthesis by cultured rat granulosa cells. FSH also stimulated DNA synthesis dose-relatedly with the maximal effect occurring at FSH 100 ng/ml. The stimulatory effect of FSH was still greater than that of EGF. However, in the presence of EGF, the stimulatory effect of FSH at any concentration was regulated to the level as high as when EGF alone stimulates. In addition, EGF inhibited DNA synthesis induced by forskolin, but enhanced the action of (Bu)2cAMP additively, which indicates that EGF attenuates DNA synthesis of granulosa cells by suppressing the activity of adenylate cyclase or cAMP production. As it is suggested that cAMP is the most likely intracellular second messenger for FSH, the regulatory effect of EGF on FSH-induced DNA synthesis could be, in part, due to suppression of cAMP production. These results suggest that EGF is involved in granulosa cell proliferation, irrespective of the presence of FSH, in a different pathway from FSH and additionally modulates the FSH growth-promoting effect as a local regulator. The interaction between EGF and FSH may be important in the control of follicular development.

Is personality involved in the expression of dysmenorrhea in patients with endometriosis?

OBJECTIVE: To investigate whether a relationship exists between personality and expression of pain in women with endometriosis. STUDY DESIGN: With use of the Rosenzweig picture frustration study, the personality of women with endometriosis was assessed. These results were then correlated with the manifestation of pain. RESULTS: Women without dysmenorrhea tended to be less assertive compared with women who complained of dysmenorrhea and women without endometriosis. Those women without dysmenorrhea showed a destructive attitude in face of a problem. CONCLUSIONS: Personality affected the expression of pain. Pain caused by endometriosis was influenced by each personality type.

Estradiol stimulates the binding of epidermal growth factor in cultured rat granulosa cells.

The effect of estradiol on the binding of epidermal growth factor (EGF) was investigated in a rat granulosa cell culture system. Cultured granulosa cells without estradiol possess an EGF receptor with a Kd value of 7.4 x 10(-10) M and binding sites of 1.45 x 10(3) molecules per cell. The addition of estradiol to the culture media resulted in an increase in the binding of EGF in a dose-dependent manner at concentration between 10(-9) M and 10(-7) M. Estradiol at 10(-7) M produced a 2.5-fold increase in the number of binding sites without affecting the binding affinity. An increase in the binding was observed as early as after 6 h incubation. The addition of testosterone alone had no effect on the EGF binding. However, in the presence of follicle stimulating hormone, it increased the binding dose-dependently. The stimulatory effect of testosterone was completely abolished by an aromatase inhibitor, thus suggesting that estradiol converted from testosterone could be a contributing factor in the mechanism of testosterone action. These observations imply that estradiol may play a role in regulating growth and the differentiation of granulosa cells by modulating the EGF binding.

[Regulation of aromatase activity by gonadotropin-releasing hormone and its agonists in cultured rat granulosa cells].

The effects of Gn-RH and its agonists on the activity of aromatase were examined by means of a rat granulosa cell culture system. A potent Gn-RH agonist, buserelin ([D-Ser (but)6 des-Gly-NH2(10)] Gn-RH ethylamide), alone stimulated aromatase activity in a dose-dependent manner at concentrations between 10(-13) and 10(-6) M. Both Gn-RH and another Gn-RH agonist, TAP144 ([D-Leu6, des-Gly-NH2(10)] Gn-RH ethylamide), also exhibited a stimulatory effect. Buserelin enhanced the activity of aromatase stimulated by FSH at concentrations of 1 and 10 ng/ml. However, it decreased the activity stimulated by a higher level of FSH (100 ng/ml). These results indicate that Gn-RH agonist increases the activity of aromatase in the absence of FSH. However, in the presence of FSH it exerts different effects on the aromatase activity depending on the concentrations of FSH.