Multiple ovulations are uncommon in humans, cattle and many breeds of sheep. Pituitary gonadotrophins and as yet unidentified ovarian factors precisely regulate follicular development so that, normally, only one follicle is selected to ovulate. The Inverdale (FecXI) sheep, however, carries a naturally occurring X-linked mutation that causes increased ovulation rate and twin and triplet births in heterozygotes (FecXI/FecX+; ref. 1), but primary ovarian failure in homozygotes (FecXI/FecXI; ref. 2). Germ-cell development, formation of the follicle and the earliest stages of follicular growth are normal in FecXI/FecXI sheep, but follicular development beyond the primary stage is impaired. A second family unrelated to the Inverdale sheep also has the same X-linked phenotype (Hanna, FecXH). Crossing FecXI with FecXH animals produces FecXI/FecXH infertile females phenotypically indistinguishable from FecXI/FecXI females. We report here that the FecXI locus maps to an orthologous chromosomal region syntenic to human Xp11.2-11.4, which contains BMP15, encoding bone morphogenetic protein 15 (also known as growth differentiation factor 9B (GDF9B)). Whereas BMP15 is a member of the transforming growth factor beta (TGFbeta) superfamily and is specifically expressed in oocytes, its function is unknown. We show that independent germline point mutations exist in FecXI and FecXH carriers. These findings establish that BMP15 is essential for female fertility and that natural mutations in an ovary-derived factor can cause both increased ovulation rate and infertility phenotypes in a dosage-sensitive manner.

uRNA and Cxbladder showed improved sensitivity for the detection of urothelial carcinoma compared to the NMP22 assays. Stratification with Cxbladder provides a potential method to prioritize patients for the management of waiting lists.

Prostaglandin (PG) E 2 release is induced in pulmonary A549 cells by the NF-U UB-activating stimuli interleukin-1L L (IL-1L L) and phorbol 12-myristate 13-acetate (PMA). Adenoviral over-expression of IU UBKv KvN, a dominant NF-U UB inhibitor, prevents NF-U UB-dependent transcription and was used to qualify the role of NF-U UB in the release of PGE 2 . IU UBKv KvN repressed IL-1L L-induced, but not PMA-induced, cycloxygenase-2 (COX-2) and microsomal prostaglandin E synthase (mPGES) expression. These data conclusively demonstrate a substantial role for NF-U UB in the co-ordinate induction of COX-2, mPGES and in the corresponding release of PGE 2 by IL-1L L. However, other pathways are primarily responsible for PGE 2 release induced by PMA.

cAMP-elevating drugs are thought to mediate their biological effects by activating the cAMP/cAMP-dependent protein kinase (PKA) cascade. However, this hypothesis is difficult to confirm due to a lack of selective inhibitors. Here, we have probed the role of PKA in mediating inhibitory effects of several cAMP-elevating drugs in BEAS-2B epithelial cells using an adenovirus vector encoding a PKA inhibitor protein (PKI␣) and have compared it to H-89, a commonly used small molecule PKA inhibitor. Initial studies established efficient gene transfer and confirmed functionality of PKI␣ 48 h after virus infection. All cAMP-elevating drugs tested promoted the phosphorylation of cAMP response element-binding protein (CREB), activated a cAMP response element (CRE)-driven luciferase reporter gene, and suppressed both granulocyte/ macrophage colony-stimulating factor (GM-CSF) generation and [ 3 H]arachidonic acid (AA) release in response to interleukin-1␤ and monocyte chemotactic protein (MCP)-1, respectively. These effects were abolished by PKI␣. In contrast, H-89 behaved unpredictably under the same conditions. Thus, although CREB phosphorylation evoked by a range of cAMP-elevating drugs was abolished by H-89, neither activation of the CRE-dependent luciferase reporter gene construct nor the inhibition of GM-CSF generation was inhibited. Paradoxically, H-89 antagonized MCP-1-induced [ 3 H]AA release and enhanced the inhibitory effect of submaximal concentrations of rolipram and 8-bromo-cAMP. We suggest that expression of PKI␣ in susceptible cells provides a simple and unambiguous way to assess the role of PKA in cAMP signaling and to probe the mechanism of action of other drugs and cAMP-dependent responses where the participation of PKA is equivocal. Furthermore, these data suggest that H-89 is not a selective inhibitor of PKA and should be avoided.Through highly coordinated changes in the rate of synthesis and degradation, cAMP mediates the effect of a large number of hormones, autacoids, and neurotransmitters. Current dogma holds that agonism of Gs-coupled receptors augments the basal activity of one or more isoforms of adenylyl cyclase. The cAMP signal then is propagated and amplified through the activation of PKA, ultimately to effect a change in cell function (see Beavo and Brunton, 2002). In the inactive state, PKA is a tetramer composed of two catalytic and two regulatory subunits (Francis and Corbin, 1999). cAMP, when elevated, binds to the regulatory subunits, resulting in the dissociation of the inactive holoenzyme and the release of

1 In common with human bronchial epithelial cells, pulmonary A549 cells release prostaglandin (PG) E 2 in response to pro-in¯ammatory cytokines. We have therefore used these cells to examine the e ect of the selective mitogen activated protein (MAP) kinase inhibitors; PD098059, a mitogen activated and extracellular regulated kinase kinase (MEK) 1 inhibitor, UO126, a dual MEK1 & MEK2 inhibitor, and SB203580, a p38 MAP kinase inhibitor in the IL-1b-dependent release of PGE 2 . 2 Following IL-1b treatment the extracellular regulated kinases (ERKs) and the p38 MAP kinases were rapidly phosphorylated. 3 PD09059, UO126 and SB203580 prevented IL-1b-induced PGE 2 release at doses that correlated closely with published IC 50 values. Small or partial e ects at the relevant doses were observed on induction of cyclo-oxygenase (COX) activity or COX-2 protein suggesting that the primary e ects were at the level of arachidonate availability. 4 Neither PD098059 nor SB203580 showed any e ect on IL-1b-induced arachidonate release. We therefore speculate that the MEK1/ERK and p38 kinase cascades play a role in the functional coupling of arachidonate release to COX-2. 5 In contrast, UO126 was highly e ective at inhibiting IL-1b-dependent arachidonate release, implicating MEK2 in the activation of the PLA 2 that is involved in IL-1b-dependent PGE 2 release. 6 We conclude that the MEK1, MEK2 and p38 MAP kinase inhibitors, PD098059, UO126 and SB203580, are highly potent in respect of in¯ammatory PG release. Finally, we conclude that these inhibitors act via mechanistically distinct processes, which may have anti-in¯ammatory bene®ts.

Purpose: New markers that enable the percentage of transitional cell carcinomas (TCC) of the bladder that are diagnosed before invasion of the bladder muscle layers to be increased would reduce the morbidity and mortality associated with this disease. The purpose of this study was to develop a simple, accurate urine test based on mRNA markers and simple gene signatures that (a) could detect TCC before muscle invasion while maintaining high specificity in patients with hematuria or urinary tract infections and (b) identify patients most likely to have grade 3 or stage zT1disease. Experimental Design: RNA markers with high overexpression in stage Ta tumors and/or T1 to T4 tumors but low expression in blood or inflammatory cells were characterized by quantitative reverse transcription-PCR using 2 mL of voided urine from 75 TCC patients and 77 control patients with other urological diseases. Results: A combination of the RNAs CDC2, MDK, IGFBP5, and HOXA13 detected 48%, 90%, and 100% of stage Ta, T1, and >T1 TCCs, respectively, at a specificity of 85%. Detection of Ta tumors increased to 60% for primary (non-recurrent) Ta tumors and 76% forTa tumors z1cm in diameter. Test specificity was 80% for the 20 control patients with urinary tract infections. The combination of CDC2 and HOXA13 distinguished between grade 1 to 2 TCCs and grade 3 or stage zT1TCCs with f80% specificity and sensitivity. Conclusions: Simple gene expression signatures can be used as urine markers for the accurate detection and characterization of bladder cancer.There are f360,000 new cases of bladder cancer worldwide annually and f145,000 deaths from the disease (1). About 30% of tumors have invaded into or beyond the muscularis propria at the time of diagnosis. Patients with these muscleinvasive tumors (stages T2-T4) have 10-year recurrence-free survival rates ranging from >80% for organ-confined tumors to f30% for tumors with regional lymph node metastases (2). Muscle-invasive tumors are generally treated by radical cystectomy with bilateral pelvic lymph node dissection followed by urinary diversion. In contrast, tumors that have not invaded the muscularis propria (stage Ta, T1, and in situ carcinomas) are usually resected by organ-preserving transurethral resection and present 10-year survival rates of 70% to 85% (2). Screening high-risk groups, such as the elderly, cigarette smokers, and certain occupational groups including truck drivers, painters, and workers in the textile dye and rubber tire industries (3, 4), would be predicted to lead to a higher proportion of tumors being identified at the pre -muscle-invasive stage and an overall decrease in the bladder cancer burden.Recent studies using high-throughput microarray analysis have shown that mRNA expression profiles are a powerful method for discriminating between bladder tumors and normal urothelium and defining subgroups of bladder tumors (5-8). The informativeness of these expression profiles and the high frequency of tumor cell exfoliation into urine have raised the prospect of...

In A549 pulmonary cells, the dexamethasone-and budesonide-dependent repression of interleukin-1b-induced prostaglandin E 2 release was mimicked by the steroid antagonist, RU486. Conversely, whereas dexamethasone and budesonide were highly effective inhibitors of interleukin-1b-induced cyclooxygenase (COX)/prostaglandin E synthase (PGES) activity and COX-2 expression, RU486 (< 1 lM) was a poor inhibitor, but was able to efficiently antagonize the effects of dexamethasone and budesonide. In addition, both dexamethasone and RU486 repressed [ 3 H]arachidonate release, which is consistent with an effect at the level of phospholipase A 2 activity. By contrast, glucocorticoid response element-dependent transcription was unaffected by RU486 but induced by dexamethasone and budesonide, whilst dexamethasone-and budesonide-dependent repression of nuclear factor-jB-dependent transcription was maximally 30-40% and RU486 (< 1 lM) was without significant effect. Thus, two pharmacologically distinct mechanisms of glucocorticoid-dependent repression of prostaglandin E 2 release are revealed. First, glucocorticoiddependent repression of arachidonic acid is mimicked by RU486 and, second, repression of COX/PGES is antagonized by RU486. Finally, whilst all compounds induced glucocorticoid receptor translocation, no role for glucocorticoid response element-dependent transcription is supported in these inhibitory processes and only a limited role for glucocorticoid-dependent inhibition of nuclear factor-jB in the repression of COX-2 is indicated.

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