Indian hedgehog is a major mediator of progesterone signaling in the mouse uterus.

The hedgehog family of morphogens are regulators of cell proliferation, differentiation and cell-cell communication. These morphogens have been shown to have important roles in organogenesis, spermatogenesis, stem cell maintenance and oncogenesis. Indian hedgehog (encoded by Ihh) has been shown to be expressed in the uterine epithelium under the control of the steroid hormone, progesterone. Although in vivo and in vitro studies have shown that progesterone achieves its effects on uterine function through epithelial-stromal cross-talk, molecular mediator(s) for this cellular communication pathway have not been elucidated. Using new experimental approaches that ablate Ihh specifically in Pgr-positive uterine cells of the mouse, we demonstrate that Ihh is an essential mediator of Pgr action in the uterus, and expression of this factor is critical in mediating the communication between the uterine epithelium and stroma required for embryo implantation.

Identification of antigenic Edwardsiella tarda surface proteins and their role in pathogenesis.

Edwardsiella tarda causes an infectious fish disease called edwardsiellosis. Several outer membrane proteins (OMPs) are associated with virulence factors and are attractive as vaccine candidates. In this study, 4 immuno-reactive OMPs of E. tarda were detected using anti-sera from flounder infected with E. tarda. Using matrix-assisted laser desorption/ionization mass spectrometry analyses, 2 of the 4 OMPs were identified as OmpA and murein lipoprotein (Lpp), which are highly conserved surface proteins in gram-negative bacteria. For further characterization of these surface proteins, we generated ompA- and lpp-inactivated mutants by insertion of a kanamycin cassette in the corresponding genes, and named these mutants E. tarda CK99 and CK164, respectively. As expected, immuno-reactive OmpA and Lpp proteins were absent in E. tarda CK99 and CK164, respectively, confirming that OmpA and Lpp are antigenic surface proteins. Interestingly, the LD(50) value of E. tarda CK164 in fish (2.0 × 10(8) colony-forming unit [CFU]/fish) was greater than that of the parental strain (3.0 × 10(7) CFU/fish). The LD(50) of E. tarda CK99 did not differ from that of its parental strain. After administering attenuated E. tarda CK164 to fish, we monitored the E. tarda-specific immune response profile. We observed that the E. tarda-specific serum IgM titer increased in a time-dependent manner, and was much higher than the value observed after the administration of a heat-killed E. tarda control. Moreover, fish vaccinated with E. tarda CK164 were 100% protected when challenged by CK41, a pathogenic strain. Our results suggest that E. tarda CK164 can potentially be used for developing an effective live attenuated vaccine for edwardsiellosis that can be applied in the aquaculture industry.

The antibacterial activity of various saturated and unsaturated fatty acids against several oral pathogens.

The antibacterial activity of various saturated fatty acids (SFA) and unsaturated fatty acids (USFA) against different oral pathogens which are implicated in the cause of dental caries, stomatitis, gingivitis, and periodontitis was examined. The saturated fatty acids Pa, StA and ArA, and the unsaturated omega-7 fatty acids PLA and omega-9 fatty acids OA showed either none to low antimicrobial activity against all of the 12 oral pathogenic strains used in this study. In contrast, the omega-3 PUFAs, ALA, SDA, EPA and DHA, and the omega-6 PUFAs, LA, GLA, and AA showed considerable antimicrobial activity against 8, 7, 6 and 5 strains, and 6, 10 and 5 strains, respectively. In particular, the omega-3 and omega-6 PUFAs showed strong antimicrobial activity against Porphyromonas gingivalis KCTC 381, the cause of periodontitis, and against Aggregatibacter segnis KCTC 5968, Fusobacterium nucleatum subsp. Polymorphum KCTC 5172 and Prevotella intermedia KCTC 25611, all organisms implicated in the cause of gingivitis. To date, no bacterial resistance to free fatty acids has been encountered and no resistance phenotype has emerged. Therefore, these results suggest that PUFAs may be useful in the development of therapeutic agents for oral diseases, and in particular, in the development of agents that have minimal side effects and against which there is no bacterial resistance.

Foxa2 is essential for mouse endometrial gland development and fertility.

During embryonic development, Foxa2 is required for the formation of the node and notochord, and ablation of this gene results in defects in gastrulation, neural tube patterning, and gut morphogenesis. Foxa2 has been shown to be expressed specifically in the glandular epithelium of the murine uterus. To study the uterine function of Foxa2, this gene was conditionally ablated in the mouse uterus by crossing mice with floxed Foxa2 alleles, Foxa2(loxP/loxP), with the Pgr(cre) mouse model. Pgr(cre/+) Foxa2(loxP/loxP) mice showed significantly reduced fertility. Analysis of the uterus on Day 5.5 of pregnancy showed disrupted blastocyst implantation. Pgr(cre/+) Foxa2(loxP/loxP) mice also showed a severe impairment of the uterus to respond to the artificial induction of the decidual response. Morphological examination of the uteri of these mice showed a severe reduction in the number of endometrial glands. The loss of endometrial glands resulted in the reduction of leukemia inhibitory factor (Lif) expression. The lack of a decidual response could be partially rescued by an intrauterine injection of LIF before the initiation of the decidual response. This analysis demonstrates that Foxa2 regulates endometrial gland development and that mice with a loss of endometrial glands cannot support implantation in part due to the loss of LIF, which is a requisite for fertility in the mouse.

Role of the steroid receptor coactivator SRC-3 in cell growth.

Steroid receptor coactivator 3 (SRC-3/p/CIP/AIB1/ACTR/RAC3/TRAM-1) is a member of the p160 family of nuclear receptor coactivators, which includes SRC-1 (NCoA-1) and SRC-2 (TIF2/GRIP1/NCoA2). Previous studies indicate that SRC-3 is required for normal animal growth and is often amplified or overexpressed in many cancers, including breast and prostate cancers. However, the mechanisms of SRC-3-mediated growth regulation remain unclear. In this study, we show that overexpression of SRC-3 stimulates cell growth to increase cell size in prostate cancer cell lines. Furthermore, our results indicate that overexpression of SRC-3 can modulate the AKT signaling pathway in a steroid-independent manner, which results in the activation of AKT/mTOR signaling concomitant with an increase in cell size. In contrast, down-regulation of SRC-3 expression in cells by small interfering RNA decreases cell growth, leading to a smaller cell size. Similarly, in SRC-3 null mutant mice, AKT signaling is down-regulated in normally SRC-3-expressing tissues. Taken together, these results suggest that SRC-3 is an important modulator for mammalian cell growth.

The genomic analysis of the impact of steroid receptor coactivators ablation on hepatic metabolism.

Members of the steroid receptor coactivator (SRC) family, which include SRC-1 (NcoA-1/p160), SRC-2(TIF2/GRIP1/NcoA-2) and SRC-3(pCIP/RAC3/ACTR/pCIP/ AIB1/TRAM1), are critical mediators of steroid receptor action. Gene ablation studies previously identified SRC-1 and SRC-2 as being involved in the control of energy homeostasis. A more precise identification of the molecular pathways regulated by these coactivators is crucial for understanding the role of steroid receptor coactivators in the control of energy homeostasis and obesity. A genomic approach using microarray analysis was employed to identify the subsets of genes that are altered in the livers of SRC-1-/-, SRC-2-/-, and SRC-3-/- mice. Microarray analysis demonstrates that gene expression changes are specific and nonoverlapping for each SRC member in the liver. The overall pattern of altered gene expressions in the SRC-1-/- mice was up-regulation, whereas SRC-2-/- mice showed an overall down-regulation. Several key regulatory enzymes of energy metabolism were significantly altered in the liver of SRC-2-/- mice, which are consistent with the prior observation that SRC-2-/- mice have increased energy expenditure. This study demonstrates that the molecular targets of SRC-2 regulation in the murine liver stimulate fatty acid degradation and glycolytic pathway, whereas fatty acid, cholesterol, and steroid biosynthetic pathways are down-regulated.

Enhancer- and silencer-like sequences that mediate insulin-like growth factor-binding protein-2 gene expression in uterine cells of pregnancy.

The regulation of insulin-like growth factor-binding protein-2 (IGFBP-2) gene transcription in specific cell and developmental contexts is not well understood. Here, we identified DNA regions that mediate IGFBP-2 gene transcription in two of the three major cell types of the uterine endometrium of the early pregnant pig. Two clusters of transcriptional start sites at nucleotides -109/-105 and -96/-87 (+1, translational initiation site) in the porcine IGFBP-2 gene were localized in uterine endometrium and in primary cultures of endometrial glandular epithelial (GE) and stromal (ST) fibroblastic cells. Upstream regions of this gene (spanning -1,397/+73) were fused to a luciferase reporter gene, and the constructs were transiently transfected into endometrial GE and ST cells representative of pregnancy days 12 and 18 (day 115 = parturition). A short (110 bp) upstream region (-874/-765) stimulated the IGFBP-2 and heterologous SV40 promoters in the two cell types at both pregnancy days. Two noncontiguous copies of the novel sequence motif TCAGGG within the 110-bp fragment were implicated in transcriptional activity, since block mutation of these sequences led to a repression of SV40 basal promoter activity in endometrial cells. Southwestern blotting identified an endometrial nuclear protein of 34-kDa molecular weight that bound an oligonucleotide containing this motif, and EMSA suggested robust expression of this protein in early pregnancy endometrium and in ovary but at much reduced levels in endometrium at later pregnancy. A pair of E-box elements (CANNTG) within the 110 bp region was stimulatory to IGFBP-2 promoter activity; block mutation of these converted the 110-bp region into a potent transcriptional silencer in all but day 18 ST cells. Results identify novel DNA motifs that regulate the IGFBP-2 gene promoter in uterine endometrium in pregnancy-associated fashion.

Identification of murine uterine genes regulated in a ligand-dependent manner by the progesterone receptor.

Progesterone (P4) acting through its cognate receptor, the progesterone receptor (PR), plays an important role in uterine physiology. The PR knockout (PRKO) mouse has demonstrated the importance of the P4-PR axis in the regulation of uterine function. To define the molecular pathways regulated by P4-PR in the mouse uterus, Affymetrix MG U74Av2 oligonucleotide arrays were used to identify alterations in gene expression after acute and chronic P4 treatments. PRKO and wild-type mice were ovariectomized and then treated with vehicle or 1 mg P4 every 12 h. Mice were killed either 4 h after the first injection (acute P4 treatment) or after the fourth injection of P4 (chronic P4 treatment). At the genomic level, the major change in gene expression after acute P4 treatment was an increase in the expression of 55 genes. Conversely, the major change in gene expression after chronic P4 treatment was an overall reduction in the expression of 102 genes. In the analysis, retinoic acid metabolic genes, cytochrome P 450 26a1 (Cyp26a1), alcohol dehydrogenase 5, and aldehyde dehydrogenase 1a1 (Aldh1a1); kallikrein genes, Klk5 and Klk6; and specific transcription factors, GATA-2 and Cited2 [cAMP-corticosterone-binding protein/p300-interacting transactivator with glutamic acid (E) and aspartic acid (D)-rich tail], were validated as regulated by the P4-PR axis. Identification and analysis of these responsive genes will help define the role of PR in regulating uterine biology.

Exploration of receptor binding of Bacillus thuringiensis toxins

Wild type and mutant toxins of Bacillus thuringiensis delta-endotoxins were examined for their binding to midgut brush border membrane vesicles (BBMV). CryIAa, CryIAb, and CryIAc were examined for their binding to Gypsy moth (Lymantria dispar) BBMV. The binding of CryIAa and CryIAc was directly correlated with their toxicity, while CryIAb was observed to have lower binding than expected from its toxicity. The latter observation confirms the observation of Wolfersberger (1990). The "rule" of reciprocity of binding and toxicity is apparently obeyed by CryIAa and CryIAc, but broken by CryIAb on L. dispar. Alanine substitutions were made in several positions of the putative loops of CryIAa to test the hypothesis that the loops are intimately involved in binding to the receptor. The mutant toxins showed minor shifts in heterologous binding to Bombyx mori BBMV, but not enough to conclude that the residues chosen play critical roles in receptor binding.