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We have previously demonstrated that the tetraspanin CD9 is necessary for membrane fusion between sperm and oocyte during fertilization. While knockout mice for CD9 are viable, CD9(-/-) females are sterile due to the inability of their oocytes to fuse with sperm. While CD9 is not essential for subsequent development, a role in embryonic stem (ES) cell self-renewal was hypothesised on the basis of two observations: CD9 is highly expressed in murine and human ES cells and the CD9-blocking antibody inhibits mouse ES cell colony formation and survival. To investigate whether CD9 has a direct effect on ES cells, we generated and characterised several CD9 knockout murine ES cell lines. These CD9(-/-) ES cell lines exhibited equivalent morphology and growth properties to wild-type ES cells. Furthermore, the CD9(-/-) ES cell lines also displayed similar expression of pluripotency factors Oct3/4, Sox2 and Nanog. CD9(-/-) ES cells were found to be pluripotent in vivo, as their cells injected into immunocompromised mice gave rise to teratomas consisting of tissues representative of all three germ layers. Additionally several high contribution mouse chimeras were generated by blastocyst injection with several CD9(-/-) ES cell lines. Taken together, our results reveal that CD9 is dispensable for mouse ES cell self-renewal and pluripotency. The generation of CD9(-/-) ES cells should prove to be a useful tool with which to study the function of this protein and a range of other associated cellular processes.

Unique ATP-inhibitable K+ channels (KATP) in the kidney determine the rate of urinary K+ excretion and play an essential role in extracellular K+ balance. Here, we demonstrate that functionally similar low sulfonylurea affinity KATP channels are formed by two heterologous molecules, products of Kir1.1a and cystic fibrosis transmembrane conductance regulator (CFTR) genes. Co-injection of CFTR and Kir1.1a cRNA into Xenopus oocytes lead to the expression of K+ selective channels that retained the high open probability behavior of Kir1.1a but acquired sulfonylurea sensitivity and ATP-dependent gating properties. Similar to the KATP channels in the kidney but different from KATP channels in excitable tissues, the Kir1.1a/CFTR channel was inhibited by glibenclamide with micromolar affinity. Since the expression of Kir1.1a and CFTR overlap at sites in the kidney where the low sulfonylurea affinity KATP are expressed, our study offers evidence that these native KATP channels are comprised of Kir1.1a and CFTR. The implication that Kir subunits can interact with ABC proteins beyond the subfamily of sulfonylurea receptors provides an intriguing explanation for functional diversity in KATP channels.

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a high affinity cellular substrate for protein kinase C. The MARCKS gene is under multiple modes of transcriptional control, including cytokine- and transformation-dependent, cell-specific, and developmental regulation. This study evaluated the transcriptional control of MARCKS gene expression during early development of Xenopus laevis. Xenopus MARCKS was highly conserved with its mammalian and avian homologues; its mRNA and protein were abundant in the maternal pool and increased after the mid-blastula transition (MBT). The Xenopus MARCKS gene was similar to those of other species, except that a second intron interrupted the 5'- untranslated region. By transiently transfecting XTC-2 cells and microinjecting Xenopus embryos with reporter gene constructs containing serial deletions of 5'-flanking MARCKS sequences, we identified a 124-base pair minimal promoter that was critical for promoter activity. Developmental gel shift assays revealed that a CBF/NF-Y/CP-1-like factor and an Sp1-like factor bound to this region in a manner correlating with the onset of Xenopus MARCKS transcription at MBT. Mutations in the promoter that abolished binding of these two factors also completely inhibited transcriptional activation of the MARCKS gene at MBT. The binding sites for these two factors are highly conserved in the human and mouse MARCKS promoters, suggesting that these elements might also regulate MARCKS transcription in other species. These studies not only increase our knowledge of the transcriptional regulation of the MARCKS genes but also have implications for the mechanisms responsible for zygotic activation of the Xenopus genome at MBT.

Background: The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is activated by cAMP-dependent phosphorylation. CFTR channel activity is also stimulated by cGMP-dependent protein kinase and protein kinase C. Results Here, we show that CFTR channel activation by cGMP may also occur directly. In oocytes from one-third of Xenopus donors, the activation of CFTR by cGMP averaged 87 % of the level achieved by cAMP. The currents activated by either cyclic nucleotide displayed similar current–voltage relationships, kinetics, pharmacology and halide selectivity. Sequential stimulation by cAMP and cGMP was not additive, suggesting that both cyclic nucleotides activate the same channel; cGMP was one order of magnitude more potent than cAMP, and its action was insensitive to protein kinase inhibitors. Analysis of the amino-acid sequence of CFTR revealed a domain in the amino-terminal portion of the third cytoplasmic loop that resembles a class of cyclic-nucleotide-binding domains related to that of the catabolite-gene activator protein, CAP. Two CFTR residues in this domain — Val397 and Lys420 — were identified which, when changed to alanine, altered the response to cGMP independently of the response to cAMP. Conclusion We conclude that direct cyclic nucleotide binding may play a role in channel gating of CFTR. The cGMP-binding domain may provide a useful target for pharmacologic intervention in cystic fibrosis.

Background: Ileal HCO 3− secretion is not well understood. The aim of this study was to examine its Na + and Cl − dependencies, electrogenicity, and responses to amiloride, 4-acetamido-4′-isothiocyano-stilbene-2,2′-disulfonate (SITS), and cyclic nucleotides. Methods: The serosa to mucosa HCO 3 − flux (J sm ) across rabbit ileal mucosa mounted between HCO 3 − -free mucosal solution and HCO 3 − -containing serosal solutions was determined by titration. Results: In SO 4 2− -containing Ringer's solution, J sm varied with [Na + ] in two phases, one with a high and one with a low affinity for Na + ; amiloride inhibited the high- and SITS inhibited the low-affinity phase. Switching from SO 4 2− -to Cl − -containing Ringer's solution caused a SITS-inhibitable 42% increase in J sm . Changes in J sm were coupled 3:2 with changes in short-circuit current. Cyclic nucleotide effects on J sm were as follows. In SO 4 2− -containing Ringer's solution at 141 (but not 80) mmol/L Na + , theophylline caused equal increases in J sm and short-circuit current that equaled the combined effects of 8-Br-5′-cyclic guanosine monophosphate (cGMP) and 8-Br-5′-cyclic adenosine monophosphate (cAMP). Serosal SITS blocked these effects, but amiloride did not. In Cl − -containing Ringer's solution, theophylline and bumetanide together (but not separately) increased J sm . Conclusions: (1) Basolateral HCO 3 − entry occurs via Na + /H exchange and a SITS-inhibitable process (Na + -HCO 3 − cotransport?). (2) Most HCO 3 − exit across the brush border occurs by a Cl − -independent process and some by Cl − /HCO 3 − exchange. (3) At low cellular [Cl − ], HCO 3 − can be secreted via anion channels activated by cAMP and cGMP. (4) Ileal HCO 3 − secretion is electrogenic.