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The clone MO15 which codes for a 40 kd protein (p40MO15) with 40% amino acid identity to the human cdc2 protein kinase has been isolated from a Xenopus cDNA library using a synthetic oligonucleotide probe. MO15 mRNA is accumulated during oogenesis, becomes de‐adenylated during meiotic maturation, and is degraded after the mid‐blastula‐transition stage of embryogenesis. Translation of p40MO15 is restricted to non‐mature oocytes. Specific inhibition of p40MO15 synthesis in stage VI oocytes by antisense oligonucleotide depletion of MO15 mRNA increases the rate of progesterone induced H1 kinase activation and oocyte maturation. This effect can be reversed by subsequent injection of synthetic MO15 mRNA. These results suggest that p40MO15 is involved in negatively regulating meiosis.

We have constructed a high density, 13 270-clone cDNA array for the sentinel fish species European flounder (Platichthys flesus), combining clones from suppressive subtractive hybridization and a liver cDNA library; DNA sequences of 5211 clones were determined. Fish were treated by single intraperitoneal injection with 50 micrograms cadmium chloride per kilogram body weight, a dose relevant to environmental exposures, and hepatic gene expression changes were determined at 1, 2, 4, 8, and 16 days postinjection in comparison to saline-treated controls. Gene expression responses were confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR). Blast2GO gene ontology analysis highlighted a general induction of the unfolded protein response, response to oxidative stress, protein synthesis, transport, and degradation pathways, while apoptosis, cell cycle, cytoskeleton, and cytokine genes were also affected. Transcript levels of cytochrome P450 1A (CYP1A) were repressed and vitellogenin altered, real-time PCR showed induction of metallothionein. We thus describe the establishment of a useful resource for ecotoxicogenomics and the determination of the temporal molecular responses to cadmium, a prototypical heavy metal pollutant.

2-Aminoethoxydiphenyl Borate (2-APB) has been extensively used recently as a membrane permeable modulator of inositol-1,4,5-trisphosphate-sensitive Ca 2+ channels and store-operated Ca 2+ entry. Here, we report that 2-APB is also an inhibitor of sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) Ca 2+ pumps, and additionally increases ion leakage across the phospholipid bilayer. Therefore, we advise caution in the interpretation of results when used in Ca 2+ signalling experiments. The inhibition of 2-APB on the SERCA Ca 2+ pumps is isoform-dependent, with SERCA 2B being more sensitive than SERCA 1A (IC 50 values for inhibition being 325 and 725 lM, respectively, measured at pH 7.2). The Ca 2+ -ATPase is also more potently inhibited at lower pH (IC 50 ¼ 70 lM for SERCA 1A at pH 6). 2-APB decreases the affinity for Ca 2+ binding to the ATPase by more than 20-fold, and also inhibits phosphoryl transfer from ATP (by 35%), without inhibiting nucleotide binding. Activity studies performed using mutant Ca 2+ -ATPases show that Tyr837 is critical for the inhibition of activity by 2-APB. Molecular modeling studies of 2-APB binding to the Ca 2+ ATPase identified two potential binding sites close to this residue, near or between transmembrane helices M3, M4, M5 and M7. The binding of 2-APB to these sites could influence the movement of the loop between M6 and M7 (L6-7), and reduce access of Ca 2+ to their binding sites.Keywords: 2-APB; Ca 2+ -ATPase; Inhibition; SERCA.Ca 2+ plays a very important role in a number of signalling pathways, both within and between cells. The modulation of its levels in the cytosol is crucial to the viability and survival of the cell. Prolonged exposure to Ca 2+ can result in apoptosis, whereas a lack of rise in cytosolic [Ca 2+ ] may lead to the failure of signal transduction [1]. Specific pharmacological agents have been of great use as probes to aid our understanding of Ca 2+ signalling processes [2][3][4]. One such agent, 2-aminoethoxydiphenylborate (2-APB), has been reported to be a membrane permeable inhibitor of the inositol-1,4,5-trisphosphate (InsP 3 )-sensitive Ca 2+ channel with an IC 50 value of 42 lM (in the presence of 100 nM InsP 3 ) [5]. However, the effectiveness of 2-APB as a modulator of the InsP 3 receptor (InsP 3 R) has recently been questioned. We have recently shown that 2-APB is a lower affinity inhibitor of the type 1 InsP 3 R than was originally reported [6]. Our results show that the potency of 2-APB to inhibit InsP 3 -induced Ca 2+ release is dependent upon InsP 3 concentration used. At 0.25 lM InsP 3 , an IC 50 value of 220 lM was observed, while at 10 lM InsP 3 , the concentration of 2-APB required to half maximally inhibit Ca 2+ release is 1 mM.2-APB and xestospongin C (another cell permeant InsP 3 receptor inhibitor) have been used to characterize the mechanism of store-operated Ca 2+ entry, whereby Ca 2+ influx from the extracellular matrix is triggered by the emptying of Ca 2+ stores [7][8][9][10]. The concentrations of 2-APB used in these studies were in the ...

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SummaryEnteroaggregative Escherichia coli (EAEC) is a major cause of diarrhoea in developing countries. EAEC 042 is the prototypical strain. EAEC 042 secretes the functionally well-characterized Pet autotransporter toxin that contributes to virulence through its cytotoxic effects on intestinal epithelial cells. Following a global transposon mutagenesis screen of EAEC 042, the transcription factors, CRP and Fis, were identified as essential for transcription of the pet gene. Using both in vivo and in vitro techniques, we show that the pet promoter is co-dependent on CRP and Fis. We present a novel co-activation mechanism whereby CRP is placed at a non-optimal position for transcription initiation, creating dependence on Fis for full activation of pet. This study complements previous findings that establish Fis as a key virulence regulator in EAEC 042.

The Escherichia coli lac operon promoter is widely used as a tool to control recombinant protein production in bacteria. Here, we give a brief review of how it functions, how it is regulated, and how, based on this knowledge, a suite of lac promoter derivatives has been developed to give a controlled expression that is suitable for diverse biotechnology applications.

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