Progesterone has been implicated to play a critical role in mediating LH induction of ovulation and possibly luteinization. The present study was undertaken to determine the effects of various agonists (LH, FSH, forskolin, and GnRH) known to stimulate ovulation on their abilities to induce progesterone receptor (PR) mRNA and protein in rat preovulatory follicles and in cultured rat granulosa cells exhibiting a preovulatory phenotype. In cultured granulosa cells, PR mRNA was induced by LH in a dose- and time-dependent manner. Transcripts of approximately 11.0, 7.2, 6.8, 6.2, 3.4, and 3.1 kilobases in size were induced by ovulatory (500 ng/ml), but not low (50 ng/ml), concentrations of LH and FSH as well as by forskolin (10 microM), GnRH (1 microM), and phorbol 12-myristate 13-acetate (200 nM). Two forms (A and B) of PR protein were also induced in an agonist- and time-dependent manner, with the shorter form A (mol wt, 83,000-85,000) appearing in greater abundance than the longer form B (mol wt, 115,000). Indirect immunofluorescent analyses verified nuclear localization of the induced receptor. Forskolin and progesterone, but not progesterone alone, were able to activate a glucocorticoid (progesterone) response element-E1b-chloramphenicol acetyltransferase reporter construct (12-fold) after transfection into cultured granulosa cells. The antiprogestins RU486 and ZK98299 did not inhibit induction of PR mRNA and protein, but effectively blocked agonist activation of glucocorticoid response element2-E1b-chloramphenicol acetyltransferase, as well as LH stimulation of luteinization in vitro. These results provide direct evidence that agonists stimulating diverse intracellular pathways can induce PR in granulosa cells, that progesterone plays a functional role in the luteinization process triggered by the LH surge, and that the effects are mediated at least in part by induction of PR.

Animal growth and development is dependent on reiterative use of key signaling pathways such as Hedgehog (Hh) pathway. It is widely believed that Cubitus-interruptus (Ci) mediates all functions of Hh pathway. Here we report that CG32062, the Drosophila homologue of Ataxin-2 Binding Protein 1 (dA2BP1), functions as a cofactor of Ci to specify intervein region between L3 and L4 veins of the adult wing. Specifically, Ci-mediated transactivation of knot/collier (kn) in this region of the developing wing imaginal disc is dependent on dA2BP1 function. Protein interaction studies and chromatin-immunoprecipiation experiments suggest that Ci helps dA2BP1 to bind kn promoter, which in turn may help Ci to activate kn expression. These results suggest a mechanism by which Ci may activate targets such as kn, which do not have classical Ci/Gli-binding sites.

The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the βH-βI loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies–D6F10 (targeting abrin), anti-α-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies.

The mammalian estrogen induced oviductal glycoprotein (OGP) has been known to associate with capacitated sperm, oocytes and developing embryos. This study aimed to identify the putative binding partner of OGP on gametes using N-terminal peptide of bonnet monkey (Macaca radiata) OGP, Nmon, as bait. A protein(s) of molecular size approximately 54 kDa was detected by far-western blot analysis of detergent solubilized human sperm proteins. MALDI-TOF mass spectra analysis of approximately 54 kDa tryptic peptides gave a significant hit to non-muscle myosin heavy chain. Biochemical characterization of approximately 54 kDa was done with antibodies specific to non-muscle myosin IIA, MYH9. The approximately 54 kDa protein, possible breakdown product of MYH9, immunoreacted with MYH9 antibody in western blot analysis. OGP binding to approximately 54 kDa could also be demonstrated in far-western blot analysis of detergent solubilized human sperm proteins and nuclear matrix intermediate filament (NM-IF) preparations from human sperm and mouse oocytes. Far-western blot analysis of MYH9 enriched by immunoprecipitation identified the native approximately 220 kDa protein as OGP-binding partner. The identical and characteristic immunogold localization pattern of Nmon and MYH9 on sperm NM-IF preparation substantiated these findings. The results suggest that OGP binds to both gametes through its interaction with MYH9 through the non-glycosylated N-terminal conserved region of OGP, spanning the residues 11-137.

The capsid protein (CP) of Sesbania mosaic virus (SeMV, a T=3 plant virus) consists of a disordered N-terminal R-domain and an ordered S-domain. Removal of the R-domain results in the formation of T=1 particles. In the current study, the R-domain was replaced with unrelated polypeptides of similar lengths: the B-domain of Staphylococcus aureus SpA, and SeMV encoded polypeptides P8 and P10. The chimeric proteins contained T=3 or larger virus-like particles (VLPs) and could not be crystallized. The presence of metal ions during purification resulted in a large number of heterogeneous nucleoprotein complexes. N∆65-B (R domain replaced with B domain) could also be purified in a dimeric form. Its crystal structure revealed T=1 particles devoid of metal ions and the B-domain was disordered. However, the B-domain was functional in N∆65-B VLPs, suggesting possible biotechnological applications. These studies illustrate the importance of N-terminal residues, metal ions and robustness of the assembly process.

The importance of riboflavin-carrier protein (RCP) in the maintenance of pregnancy in mice has been studied. Selective passive immunoneutralization of the maternal RCP resulted in fetal death and resorption. Six hours after chicken RCP antiserum treatment, the following observations were made: there was profuse vaginal bleeding in all the animals, a 60% reduction in embryonic ornithine decarboxylase (ODC) activity, a 70% reduction in the maternal progesterone levels, and a 50% reduction in the 14C-riboflavin uptake by the embryo. The above observations are indicative of fetal distress and resorption. By 24 h after treatment, there was 100% resorption of fetuses and the mouse progesterone levels dropped to 20% of untreated or normal rabbit serum (NRS)-treated values. Cytological studies of the fetal liver revealed the classical signs of cellular degeneration in hepatocytes as well as hematopoietic cells. The effect was apparent as early as 1 h after antiserum administration. The erythroid aplasia supports the biochemical evidence that fetal demise is due to preferential riboflavin deficiency of the fetus.

The secretory cells lining the lumen of the mammalian oviduct synthesize and secrete high molecular weight glycoprotein (OGP). Molecular cDNA cloning of most of the mammalian OGP has been accomplished. The nucleotide and deduced amino acid sequences show a remarkable homology across species and also to chitinase protein. Even though OGP has been shown to interact with gametes and the early embryo, the protein's direct function has not yet been established. A prerequisite for such studies is the availability of well-characterized protein in bulk. We used recombinant DNA technology to obtain OGP (rOGP). An authentic partial cDNA clone encoding bonnet monkey (Macaca radiata) OGP (accession number AF132 215) was recloned into expression vector pET20b. Overexpression of the protein could be demonstrated after induction with isopropylthio-beta-galactopyranoside. Recombinant protein was purified by gel filtration of Escherichia coli lysate through Sephadex G75. The protein migrated with a molecular weight of approximately 14 kDa on SDS-PAGE. The molecular weight as assessed by matrix-assisted laser adsorption time-of-flight was 14 439 daltons. With Western blot procedures the protein could be immunostained with antibodies to human OGP, baboon OGP, and antipeptide antibodies generated against a well-conserved region of mammalian OGP. The monospecificity of rabbit antibodies generated against rOGP was established by its ability to immunostain human OGP (100-110 kDa) isolated from hydrosalpinx by Western blot analysis, and the antibody immunostained epithelial cells that secrete OGP in human fallopian tubes. OGP binding sites on the head and tail region of monkey sperm could be demonstrated by using antibody against rOGP.

Progesterone has been implicated to play a critical role in mediating LH induction of ovulation and possibly luteinization. The present study was undertaken to determine the effects of various agonists (LH, FSH, forskolin, and GnRH) known to stimulate ovulation on their abilities to induce progesterone receptor (PR) mRNA and protein in rat preovulatory follicles and in cultured rat granulosa cells exhibiting a preovulatory phenotype. In cultured granulosa cells, PR mRNA was induced by LH in a dose- and time-dependent manner. Transcripts of approximately 11.0, 7.2, 6.8, 6.2, 3.4, and 3.1 kilobases in size were induced by ovulatory (500 ng/ml), but not low (50 ng/ml), concentrations of LH and FSH as well as by forskolin (10 microM), GnRH (1 microM), and phorbol 12-myristate 13-acetate (200 nM). Two forms (A and B) of PR protein were also induced in an agonist- and time-dependent manner, with the shorter form A (mol wt, 83,000-85,000) appearing in greater abundance than the longer form B (mol wt, 115,000). Indirect immunofluorescent analyses verified nuclear localization of the induced receptor. Forskolin and progesterone, but not progesterone alone, were able to activate a glucocorticoid (progesterone) response element-E1b-chloramphenicol acetyltransferase reporter construct (12-fold) after transfection into cultured granulosa cells. The antiprogestins RU486 and ZK98299 did not inhibit induction of PR mRNA and protein, but effectively blocked agonist activation of glucocorticoid response element2-E1b-chloramphenicol acetyltransferase, as well as LH stimulation of luteinization in vitro. These results provide direct evidence that agonists stimulating diverse intracellular pathways can induce PR in granulosa cells, that progesterone plays a functional role in the luteinization process triggered by the LH surge, and that the effects are mediated at least in part by induction of PR.