HIV-1 tat, a strong transactivator, is essential for the HIV-1 replication and AIDS progression. The Tat function is markedly inhibited by human anti-oncogene p53. This work was initiated to identify the p53-associated inhibitory mechanism on tat-mediated transactivation. Inhibitory function of P53 was confirmed by co-transfection of tat-expressing Jurkat cells with LTR-CAT plasmid, or H3Tl cells (LTR-CAT integrated HeLa cells) with different ratio of pSV-tat/pCDNA-p53 plasmids. Results from the direct protein-protein .interaction between soluble p53 and tat, and yeast two-hybrid experiments showed that the co-suppression mechanism is unlikely to be due to the direct interaction. CAT activity was not affected by tat in Jurkat cells which were transfected with p53-promoter-CAT or p53-enhancer-CAT, suggesting that the tat-mediated p53 suppression is not directly associated with p53-promoter. Finally, we have tested protein kinase activity in p53-tranfected Jurkat cells, which might phosphorylate HIV-1 tat, resulting in inhibition of tat function. Some of our data lead us to assume that the p53-mediated tat inhibition is likely to be associated with p53-associated, signaling-mediated phosphorylation of tat, resulting in the dysfunction of tat. This study is now under investigation.
Animals ; Cats ; Genes, Tumor Suppressor ; HIV-1* ; Humans ; Jurkat Cells ; Phosphorylation ; Plasmids ; Protein Kinases ; RNA Interference ; Trans-Activators ; Transcriptional Activation ; Yeasts

BACKGROUND: Uterine leiomyoma is the most common pelvic tumor, occurring in 20-25% of women in reproductive age. Gonadotropin releasing hormone agonist (GnRHa) has been reognized as a temporary medical management for this disorder. The etiology of these tumors is unknown but it has been shown that the insulin-like growth factors (IGF-I, IGF-II) are promoters of growth in nongynecologic tumors. Several recent studies have suggested the possible role of IGFs in human leiomyoma growth. The IGF binding proteins (IGFBPs) are believed to modulate actions of IGF and to have IGF-independent actions. The purpose of this study was to evaluate the type of IGF and IGFBP which may be involved in leiomyoma growth and to investigate a possible IGF related mechanism of action of GnRHa. METHOD: The IGFs and IGFBPs were measured by double antibody radioimmunoassay, western ligand blot and immunoprecipitation in the tissue cytosols of normal uterine myometria (n=15), nontumorous myometria adjacent to a leiomyoma and leiomyoma from patients nontreated (n=15) and treated (n=10) with GnRHa. RESULTS: The mean IGF-I and IGF-II level were significantly higher in leiomyoma from untreated patients than in the adjacent myometrium and normal myometrium but no significant differences in these IGF levels between normal myometrium and adjacent myometrium were noted. The IGFBP-2, IGFBP-3 and 26kDa IGFBP were detected variably but IGFBP-4 was consistently present in all tissues. There were no significant differences in the relative intensity for IGFBP-4 and the frequency of IGFBPs between leiomyoma, adjacent myometrium and normal myometrium from untreated patients. The IGF-I, IGF-II levels and the relative intensity of IGFBP-4 in leiomyoma from GnRHa-treated patients were significantly lower than those in untreated patients, but these levels in the adjacent myometrium were comparable. The frequency of each IGFBP in leiomyoma and the adjacent myornetrium from GnRHa-treated patients did not significantly differ from untreated patients. CONCLUSION: Both IGF-I and IGF-II are involved in the growth of leiomyoma and GnRHa may in part act to decrease size of leiomyoma by regulating the local levels of IGF-I, IGF-II and IGFBP-4.
Animals ; Cytosol ; Female ; Gonadotropin-Releasing Hormone ; Humans ; Immunoprecipitation ; Insulin-Like Growth Factor Binding Protein 2 ; Insulin-Like Growth Factor Binding Protein 3 ; Insulin-Like Growth Factor Binding Protein 4 ; Insulin-Like Growth Factor Binding Proteins ; Insulin-Like Growth Factor I ; Insulin-Like Growth Factor II ; Leiomyoma* ; Mice ; Myometrium ; Radioimmunoassay ; Somatomedins*

PURPOSE: This study was designed to identify the possible mechanism of insensitivity of DU145 prostate cancer cells to the transforming growth factor (TGF)-beta1-mediated growth inhibition. MATERIALS AND METHODS: DU145 cells were treated with 4, 40, 100 pM TGF-beta1 for 3, 6, 9 days. Initially we performed the growth assay. After that, we analysed the change of cell cycle using fluorescence flow cytometry. At each time point, Western blot analysis with cell pellets was performed to investigate the change of expressions of epidermal growth factor(EGF), TGF-alpha, EGF receptor(EGFR) and TGF receptorII(TbetaR-II) proteins. RESULTS: The growth rate of TGF-beta1-treated cells was initially suppressed, but over time returned to control level. Flow cytometric analysis revealed that TGF-beta1-treated cells showed an increase in apoptotic/G1 phases, and concurrent decrease in S, G2/M phases until 6days. On day 9, however, TGF-beta1-treated cells showed a persistent increase of apoptotic unclei in spite of recovery of apoptotic/G1, S and G2/M phases. Western blot analysis showed that the intensity of EGF or TGF-alpha band decreased in dose-sependent manner on day 6. However, the intensity of each band increased up to the control level on day 9. the expression of EGFR or TbetaR-II protein did not change after treatment of TGF-beta1. CONCLUSIONS: these results suggest that EGF and TGF-alpha sould mediate in part the escape fron the inhibitory effect of TGF-beta1 in DU145 cells.
Blotting, Western ; Cell Cycle ; Epidermal Growth Factor* ; Flow Cytometry ; Fluorescence ; Prostate* ; Prostatic Neoplasms* ; Transforming Growth Factor alpha ; Transforming Growth Factor beta1 ; Transforming Growth Factors ; United Nations

No abstract available.
Amino Acid Sequence* ; HIV* ; Phenotype*

AIMS OF STUDY: Present study designed to observe inhibitory effects of propiverine HC1 and tiropramide against the smooth muscle contraction of female rat bladder. Propiverine has both direct smooth muscle relaxation and anticholinergic effect and has relatively fewer side effect than conventionally used drugs such as oxybutinin. Tiropramide has been known as modulatory agents of gastrointestinal motility but also has inhibitory effects against the bladder contraction. METHODS: 30 adult female Sprague-Dawley rats were used. Bladder body above ureteral orifice was resected under pentobarbital anesthesia. 1 x 0.5 cm sized smooth muscle strip was made, and incubated in Tyrode`s solution aerated with 95% oxygen. After reaching equilibrium state, each strip was stimulated by field stimulation (FS, 1-32 Hz) and bethanechol administration (0.0000001-0.0001M). From each strip, degree of muscle contraction was recorded by physiograph (Gilson IC-MP). After the control stimulations, each strip was treated by atropine, tiropramide, oxybutinin and propiverine HC1. After 30 minutes, same stimulation were repeated and degree of muscle contraction was compared to pre incubation data. RESULTS: Frequency and dose dependent muscle contractions were noted for both FS and bethanechol stimulation. Greater degree of contractions were noted for FS than for bethanechol stimulation. Inhibitory effects of tiropramide, propiverine HC1 and oxybutinin were greater than those of atropine at FS (1-32 Hz). At high concentration (0.0001M), all of the drugs but atropine inhibited field stimulated smooth muscle contraction more than 90%. At lower concentration (0. 0000001-0.000001M), inhibitory actions of oxybutinin and propiverine HC1 were greater than that of tiropramide (p>0.05). Propiverine HC1 and oxybutinin had similar inhibitory effect for all con-centration. At higher concentration (0.0001M), inhibitory effects of tiro-pramide were more than 98% whereas those of oxybutinin and propiverine HC1 were 88%. At low concentration (0.0000001-0.000001M), oxybutinin exhibited greater inhibition against the bethanechol induced contraction than did tiropramide and propiverine HC1. With these results, it was suggested that in low concentration, oxybutinin and propiverine HCI had greater inhibitory effect than did tiropramide against smooth muscle contraction of the bladder. In high concentration though, tiropramide had superior inhibitory effect than did oxybutinin and propiverine HC1. Since, no difference was noted between oxybutinin and propiverine HC1 for the inhibitory action of bladder contraction, propiverine HC1 seems reasonable substitute for the treatment of detrusor hyperreflexia with less side effects. Also these results indicate that tiropramide can be used for the management of unstable bladder.
Adult ; Anesthesia ; Animals ; Atropine ; Bethanechol ; Female ; Gastrointestinal Motility ; Humans ; Muscle Contraction ; Muscle, Smooth* ; Oxygen ; Pentobarbital ; Rats* ; Rats, Sprague-Dawley ; Reflex, Abnormal ; Relaxation ; Ureter ; Urinary Bladder*

OBJECTIVES: To test the hypothesis that the endometrial stromal cells from patients with endometriosis responds differently to the insulin-like growth factor (IGF) compared with those from patients without endometriosis. METHODS: IGFs in peritoneal fluid (PF) from patients with endometriosis(n=18) and without endometriosis(n=12;control patients) were measured by radioimmunoassay. Endometrial stromal cells from patients with endometriosis and control patients were cultured in serum free media(SFM) in the presence or absence of PF or IGF-I(0.25-25 ng/ml) or IGF-II(5-50 ng/ml) and the proliferation of endometrial stromal cells were evaluated by [3H] thymidine incorporation test. All statistics were performed by ANOVA test and student's t-test. RESULTS: When added to SFM, IGF-I(1-25 ng/ml) increased thymidine incorporation in both endometrial stromal cells from patients with endometriosis and control patients in dose dependent manner and IGF-II(5-25 ng/ml) gave similar response in latter cells but not in former cells. Within low IGF-I(less than 100 ng/ml) PF group or high IGF-I(more than 100ng/ml) PF group, the type of endometrial stromal cells did not result in any difference in thymidine incorporation. However, regardless of the source of stromal cells, high IGF-I PF group produced a greater extent of thymidine incorporation than low IGF-I PF group in patients with endometriosis but not in control patients. Also, thymidine incorporation was higher in high IGF-I PF group of former patients than in the same group of latter patients. PF induced higher thymidine incorporation in endometrial stromal cells than the same levels(0.25-2.5 ng/ml) of IGF-I directly added to SFM. CONCLUSIONS: The effects of IGF-I in PF on endometrial stromal cells are similar regardless of their source and IGF-I is one of several growth factors that may participate in the growth of endometrial stromal cells in pelvic endometriosis.
Ascitic Fluid ; DNA ; Endometriosis* ; Female ; Humans ; Insulin-Like Growth Factor I ; Intercellular Signaling Peptides and Proteins ; Radioimmunoassay ; Somatomedins* ; Stromal Cells* ; Thymidine

No abstract available.
Agranulocytosis* ; Granulocytes* ; Humans

No abstract available.
Antibodies* ; Endometriosis* ; Female ; Humans

No abstract available.
Chorionic Gonadotropin* ; Gonadotropin-Releasing Hormone* ; Gonadotropins* ; Humans* ; Progesterone* ; Superovulation*

No abstract available.
Fertilization in Vitro* ; Superovulation*