Negative regulation of c-kit-mediated cell proliferation by Fc gamma RIIB

Fc gamma RIIB are single-chain low-affinity receptors for IgG that bear an immunoreceptor tyrosine-based inhibition motif in their intracytoplasmic domain and that negatively regulate immunoreceptor tyrosine-based activation motif-dependent cell activation. They are widely expressed by cells of hematopoietic origin. We investigated here whether Fc gamma RIIB could also negatively regulate protein tyrosine kinase receptor (RTK)-dependent cell proliferation. As an experimental model, we used growth factor-dependent mast cells that constitutively express Fc gamma RIIB and c-kit, an RTK prototype. We found that anti-c-kit Abs mimicked the effect of stem cell factor and induced thymidine incorporation in Fc gamma RIIB-/-, but not in wild-type (wt) mast cells unless Fc gamma RIIB were blocked or anti-c-kit F(ab')2 were used. When coaggregated with c-kit by intact Abs in wt mast cells, Fc gamma RIIB inhibited thymidine incorporation, as well as cell proliferation, and inhibition was correlated with an arrest of cells in G1 during the cell cycle. The coaggregation of c-kit with Fc gamma RIIB did not affect ligand-induced c-kit phosphorylation and induced the tyrosyl-phosphorylation of Fc gamma RIIB, which selectively recruited the Src homology 2 domain-bearing inositol 5-phosphatase SHIP. Our results indicate that IgG Abs to growth factors or growth factor receptors may control RTK-dependent proliferation of a variety of cells that express Fc gamma RIIB.     

Cofilin 1 is revealed as an inhibitor of glucocorticoid receptor by analysis of hormone-resistant cells

Significant knowledge about glucocorticoid signaling has accumulated, yet many aspects remain unknown. We aimed to discover novel factors involved in glucocorticoid receptor regulation that do not necessarily require direct receptor interaction. We achieved this by using a functional genetic screen: a stable cell line which cannot survive hormone treatment was engineered, randomly mutated, and selected in the presence of glucocorticoid. A hormone-resistant clone was analyzed by two-dimensional gel electrophoresis. Differentially expressed proteins were identified and tested as candidates for regulation of the glucocorticoid receptor. An unexpected candidate, cofilin 1, inhibited receptor activity. Cofilin is known to promote actin depolymerization and filament severing. Several experiments suggest that this feature of cofilin is involved in its inhibitory action. Both its actin depolymerization activity and its inhibitory action on the receptor are dependent on its phosphorylation state. Treatment of cells with a cytoskeleton-disrupting agent decreased receptor activity, as did overexpression of actin, particularly a mutant actin that does not polymerize. In addition, overexpression of cofilin and actin as well as chemical cytoskeleton disruption changed the subcellular receptor distribution and upregulated c-Jun, which could constitute the inhibitory mechanism of cofilin. In summary, cofilin represents a novel factor that can cause glucocorticoid resistance.     

Disruption of the mouse mTOR gene leads to early postimplantation lethality and prohibits embryonic stem cell development

The mammalian target of rapamycin (mTOR) is a key component of a signaling pathway which integrates inputs from nutrients and growth factors to regulate cell growth. Recent studies demonstrated that mice harboring an ethylnitrosourea-induced mutation in the gene encoding mTOR die at embryonic day 12.5 (E12.5). However, others have shown that the treatment of E4.5 blastocysts with rapamycin blocks trophoblast outgrowth, suggesting that the absence of mTOR should lead to embryonic lethality at an earlier stage. To resolve this discrepancy, we set out to disrupt the mTOR gene and analyze the outcome in both heterozygous and homozygous settings. Heterozygous mTOR (mTOR(+/-)) mice do not display any overt phenotype, although mouse embryonic fibroblasts derived from these mice show a 50% reduction in mTOR protein levels and phosphorylation of S6 kinase 1 T389, a site whose phosphorylation is directly mediated by mTOR. However, S6 phosphorylation, raptor levels, cell size, and cell cycle transit times are not diminished in these cells. In contrast to the situation in mTOR(+/-) mice, embryonic development of homozygous mTOR(-/-) mice appears to be arrested at E5.5; such embryos are severely runted and display an aberrant developmental phenotype. The ability of these embryos to implant corresponds to a limited level of trophoblast outgrowth in vitro, reflecting a maternal mRNA contribution, which has been shown to persist during preimplantation development. Moreover, mTOR(-/-) embryos display a lesion in inner cell mass proliferation, consistent with the inability to establish embryonic stem cells from mTOR(-/-) embryos.     

Pharmacodynamic equivalence of a decapeptyl 3-month SR formulation with the 28-day SR formulation in patients with advanced prostate cancer

AIMS: The objective of the study was to assess the pharmacodynamic equivalence of LHRH analogue triptorelin 3-month and 28-day SR formulations. METHODS: Patients with documented locally advanced or metastatic prostate cancer were randomized to receive one injection of the 3-month formulation (n = 63) or three injections at 28-day intervals of the 28-day formulation (n = 68). Group-chemical castration rates defined as the percentage of patients reaching a testosterone plasma level 相似文献   

Sequential dimerization of human zipcode-binding protein IMP1 on RNA: a cooperative mechanism providing RNP stability

Active cytoplasmic RNA localization depends on the attachment of RNA-binding proteins that dictate the destination of the RNA molecule. In this study, we used an electrophoretic mobility-shift assay in combination with equilibrium and kinetic analyses to characterize the assembly of the human zipcode-binding protein IMP1 on targets in the 3′-UTR from Igf-II mRNA and in H19 RNA. In both cases, two molecules of IMP1 bound to RNA by a sequential, cooperative mechanism, characterized by an initial fast step, followed by a slow second step. The first step created an obligatory assembly intermediate of low stability, whereas the second step was the discriminatory event that converted a putative RNA target into a ‘locked’ stable RNP. The ability to dimerize was also observed between members of the IMP family of zipcode-binding proteins, providing a multitude of further interaction possibilities within RNP granules and with the localization apparatus.     

Phase I/II trial of immunogenicity of a human papillomavirus (HPV) type 16 E7 protein–based vaccine in women with oncogenic HPV-positive cervical intraepithelial neoplasia

Purpose: Infection with oncogenic human papillomavirus (HPV) and HPV-16 in particular is a leading cause of anogenital neoplasia. High-grade intraepithelial lesions require treatment because of their potential to progress to invasive cancer. Numerous preclinical studies have demonstrated the therapeutic potential of E7-directed vaccination strategies in mice tumour models. In the present study, we tested the immunogenicity of a fusion protein (PD-E7) comprising a mutated HPV-16 E7 linked to the first 108 amino acids of Haemophilus influenzae protein D, formulated in the GlaxoSmithKline Biologicals adjuvant AS02B, in patients bearing oncogenic HPV-positive cervical intraepithelial neoplasia (CIN). Methods: Seven patients, five with a CIN3 and two with a CIN1, received three intramuscular injections of adjuvanted PD-E7 at 2-week intervals. Three additional CIN1 patients received a placebo. CIN3 patients underwent conization 8 weeks postvaccination. Cytokine flow cytometry and ELISA were used to monitor antigen-specific cellular and antibody responses from blood taken before and after vaccine or placebo injection. Results: Some patients had preexisting systemic IFN- CD4⁺ (1/10) and CD8⁺ (5/10) responses to PD-E7. Vaccination, not placebo injection, elicited systemic specific immune responses in the majority of the patients. Five vaccinated patients (71%) showed significantly increased IFN- CD8⁺ cell responses upon PD-E7 stimulation. Two responding patients generated long-term T-cell immunity toward the vaccine antigen and E7 as well as a weak H. influenzae protein D (PD)–directed CD4⁺ response. All the vaccinated patients, but not the placebo, made significant E7- and PD-specific IgG. Conclusions: The encouraging results obtained from this study performed on a limited number of subjects justify further analysis of the efficacy of the PD-E7/AS02B vaccine in CIN patients.     

Cytological, genetic, and molecular analysis to characterize compatible and incompatible interactions between Medicago truncatula and Colletotrichum trifolii

In this study, a new pathosystem was established using the model plant Medicago truncatula and Colletotrichum trifolii, the causal agent of anthracnose on Medicago sativa. Screening of a few M. truncatula lines identified Jemalong and F83005.5 as resistant and susceptible to Colletotrichum trifolii race 1, respectively. Symptom analysis and cytological studies indicated that resistance of Jemalong was associated with a hypersensitive response of the plant. The two selected lines were crossed, and inoculations with C. trifolii were performed on the resulting F1 and F2 progenies. Examination of the disease phenotypes indicated that resistance was dominant and was probably due to a major resistance gene. Molecular components of the resistance were analyzed through macroarray experiments. Expression profiling of 126 expressed sequence tags corresponding to 92 genes, which were selected for their putative functions in plant defense or signal transduction, were compared in Jemalong and F83005.5 lines. A strong correlation was observed between the number of up-regulated genes and the resistance phenotype. Large differences appeared at 48 h postinoculation; more than 40% of the tested genes were up-regulated in the Jemalong line compared with only 10% in the susceptible line. Interestingly, some nodulin genes were also induced in the resistant line upon inoculation with C. trifolii.     

Incorporation of oxyphytosterols in tissues of hamster

Oxyphytosterols (OPS) were fed to hamsters, at different concentrations, in order to observe their eventual incorporation into plasma, aorta, liver, kidneys and heart. The animals receiving the very high level (2500 ppm) presented 7beta-hydroxycampesterol, beta-epoxycampesterol, campestanetriol, 7-ketocampesterol, 7beta-hydroxysitosterol, beta-epoxysitosterol, sitostanetriol and 7-ketositosterol in all tissues. The same compounds were observed in the tissues of animals receiving 500 ppm of OPS in their diet, but with much lower levels. In hamsters fed 100 ppm of OPS, as well as in control animals, in most cases, the only observed OPS was sitostanetriol, which seems to be difficult to eliminate from the animal.