Utilizing yeast strains containing promoter mutations, we demonstrate that transcription of the HSP82 gene causes nucleosomes toward the 3′‐end to become DNase I sensitive and ‘split’ into structures that exhibit a ‘half‐nucleosomal’ cleavage periodicity. Splitting occurs even when only a few RNA polymerase II molecules are engaged in basal level transcription or during the first round of induced transcription. The split nucleosomal structure survives nuclear isolation suggesting that it may be stabilized by post‐translational modifications or non‐histone proteins, and may require DNA replication for reversal to a whole nucleosomal structure. Split nucleosomes represent a structure for DNase I sensitive chromatin and are probably of common occurrence but difficult to detect experimentally. We suggest that transient positive supercoils downstream of traversing RNA polymerase lead to nucleosome splitting.

Serum deprivation-triggered increases in reactive oxygen species (ROS) are known to induce apoptotic cell death. However, the mechanism by which serum deprivation causes ROS production is not known. Since mitochondria are the main source of ROS and since mitochondrial ROS modulator 1 (Romo1) is involved in ROS production, we sought to determine if serum deprivation triggered ROS production through Romo1. To examine the relationship between Romo1 and the serum deprivation-triggered increase in ROS, we transfected Romo1 siRNA into various cell lines and looked for inhibition of mitochondrial ROS generation. Romo1 knockdown by Romo1 siRNA blocked the mitochondrial ROS production caused by serum deprivation, which originates in the mitochondrial electron transport chain. We also found that Romo1 knockdown inhibited serum deprivation-induced apoptosis. These findings suggest that Romo1-derived ROS play an important role in apoptotic cell death triggered by withdrawal of cell survival factors.

3 ؊ transport. Stimulation of CFTR with forskolin markedly inhibited NBC3 activity. This inhibition was prevented by the inhibition of protein kinase A. NBC3 and CFTR could be reciprocally coimmunoprecipitated from transfected HEK cells and from the native pancreas and submandibular and parotid glands. Precipitation of NBC3 or CFTR from transfected HEK293 cells and from the pancreas and submandibular gland also coimmunoprecipitated EBP50. Glutathione S-transferase-EBP50 pulled down CFTR and hNBC3 from cell lysates when expressed individually and as a complex when expressed together. Notably, the deletion of the C-terminal PDZ binding motifs of CFTR or hNBC3 prevented coimmunoprecipitation of the proteins and inhibition of hNBC3 activity by CFTR. We conclude that CFTR and NBC3 reside in the same HCO 3 ؊ -transporting complex with the aid of PDZ domain-containing scaffolds, and this interaction is essential for regulation of NBC3 activity by CFTR. Furthermore, these findings add additional evidence for the suggestion that CFTR regulates the overall trans-cellular HCO 3 ؊ transport by regulating the activity of all luminal HCO 3 ؊ secretion and salvage mechanisms of secretory epithelial cells. HCO 3Ϫ concentration is tightly controlled in all biological fluids including fluids secreted by exocrine glands. The ductal systems or their equivalents are the sites of active regulation of HCO 3 Ϫ content of the secreted fluids. This is also the site of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) 1 (1-5). The transporters participating in ductal HCO 3 Ϫ homeostasis and their regulation are only partially known. Probably, the best results are available in the salivary glands and pancreatic ducts. Active regulation of luminal HCO 3 Ϫ concentration and pH i requires the regulation of both HCO 3 Ϫ secretory and absorptive mechanisms. HCO 3 Ϫ secretion is believed to occur by HCO 3 Ϫ influx across the basolateral membrane mediated by a Na ϩ -HCO 3 Ϫ cotransport mechanism (6, 7). The transporter mediating this activity is probably pNBC1, the pancreatic isoform of the electrogenic Na ϩ -HCO 3 Ϫ cotransporter family (8, 9). HCO 3 Ϫ efflux across the luminal membrane (LM) requires the activity of a Cl Ϫ /HCO 3 Ϫ exchange mechanism (6, 10, 11) and is dependent on the expression of CFTR both in human and in animal models (11,12).In the resting state, secretory glands have to absorb HCO 3 Ϫ . The transporters involved in HCO 3 Ϫ absorption are only beginning to emerge. HCO 3 Ϫ influx across the LM is in part the result of Na ϩ /H ϩ exchange mediated by NHE3 (13,14). However, in recent studies with the pancreatic (13) and the submandibular gland (SMG) ducts (9), we showed that Ͼ50% HCO 3 Ϫ absorption (H ϩ secretion) is mediated by more than one Na ϩ -dependent mechanism that is different from any known NHE isoform. Furthermore, we found that the SMG duct and acinar cells express several splice variants of NBC3 (rat orthologues NBCn1B-D) and used anti-NBC3 antibodies to localize the proteins to the LM (...

Low serum levels of adiponectin are a high risk factor for various types of cancer. Although adiponectin inhibits proliferation and metastasis of breast cancer cells, the underlying molecular mechanisms remain obscure. In this study, we show that adiponectin-activated AMPK reduces the invasiveness of MDA-MB-231 cells by stimulating dephosphorylation of AKT by increasing protein phosphatase 2A (PP2A) activity. Among the various regulatory B56 subunits, B56; was directly phosphorylated by AMPK at Ser 298 and Ser 336 , leading to an increase of PP2A activity through dephosphorylation of PP2Ac at Tyr 307 . We also show that both the blood levels of adiponectin and the tissue levels of PP2A activity were decreased in breast cancer patients and that the direct administration of adiponectin into tumor tissues stimulates PP2A activity. Taken together, these findings show that adiponectin, derived from adipocytes, negatively regulates the invasiveness of breast cancer cells by activating the tumor suppressor PP2A. [Cancer Res 2009;69(9):4018-26]

NK cells are a key component of innate immune systems, and their activity is regulated by cytokines and hormones. Adiponectin, which is secreted from white adipose tissues, plays important roles in various diseases, including hypertension, cardiovascular diseases, inflammatory disorders, and cancer. In this study the effect of adiponectin on NK cell activity was investigated. Adiponectin was found to suppress the IL-2-enhanced cytotoxic activity of NK cells without affecting basal NK cell cytotoxicity and to inhibit IL-2-induced NF-κB activation via activation of the AMP-activated protein kinase, indicating that it suppresses IL-2-enhanced NK cell cytotoxicity through the AMP-activated protein kinase-mediated inhibition of NF-κB activation. IFN-γ enhances NK cell cytotoxicity by causing an increase in the levels of expression of TRAIL and Fas ligand. The production of IFN-γ, one of the NF-κB target genes in NK cells, was also found to be suppressed by adiponectin, accompanied by the subsequent down-regulation of IFN-γ-inducible TRAIL and Fas ligand expression. These results clearly demonstrate that adiponectin is a potent negative regulator of IL-2-induced NK cell activation and thus may act as an in vivo regulator of anti-inflammatory functions.