RNAi in mouse oocytes and preimplantation embryos: effectiveness of hairpin dsRNA

Wolbachia are intracellular symbionts mainly found in arthropods, causing various sexual alterations on their hosts by unknown mechanisms. Here we report the results that strongly suggest that Wolbachia have virus-like particles of phage WO, which was previously identified as a prophage-like element in the Wolbachia genome. Wolbachia (strain wTai) infection in an insect was detected with the antibody against Wsp, an outer surface protein of Wolbachia, by fluorescence microscopy and immunoelectron-microscopy for the first time. Virus-like particles in Wolbachia were observed by electron-microscopy. The 0.22-microm filtrate of insect ovary contained DAPI-positive particles, and PCR analysis demonstrated that a phage WO DNA passed through the filter while Wolbachia DNA were eliminated, suggesting that the DAPI-positive particles were phage WO.     

YfiD of Escherichia coli and Y06I of bacteriophage T4 as autonomous glycyl radical cofactors reconstituting the catalytic center of oxygen-fragmented pyruvate formate-lyase.

Reaction of oxygen with the glycyl radical in pyruvate formate-lyase (PFL) leads to cleavage of the polypeptide backbone between N-Calpha of Gly734. A recombinant protein comprising the core of PFL (Ser1-Ser733) is shown here to associate with the YfiD protein (14 kDa) of Escherichia coli and likewise with the homologous T4 encoded Y06I protein, yielding upon reaction with PFL activase a heterooligomeric PFL enzyme that has full catalytic activity (35 U/nmol). Treatment of the activated complexes with oxygen led to cleavage of the 14 kDa proteins into 11 and 3 kDa polypeptides as expected for the localization of the putative glycyl radical at Gly102 (YfiD) or Gly95 (Y06I). For the isolated fragments from Y06I, mass spectrometric analysis (nanoESI-MS) determined a C-terminal serine carboxamide in the 11 kDa fragment, and a N-terminal oxalyl modification in the 3 kDa fragment. We speculate that YfiD in E. coli and other facultative anaerobic bacteria has evolved as a "spare part" for PFL's glycyl radical domain, utilized for rapid recovery of PFL activity (and thus ATP generation) in cells that have experienced oxidative stress.     

Adenylyl cyclase type II domains involved in Gbetagamma stimulation

Mammalian particulate adenylyl cyclases contain two transmembrane regions (M(1) and M(2)) and two cytosolic domains (C(1) and C(2)) forming the catalytic core. The cytosolic domains are subdivided into a highly conserved region (part a) and a region with lower similarity (part b). Hypothetical models exist that account for the mechanism by which Galpha(s) and forskolin stimulate mammalian adenylyl cyclase. In contrast, little is known about how Gbetagamma dimers regulate catalysis. The so-called QEHA region located in the C(2a) domain of type II adenylyl cyclase has been proposed to represent a site of interaction. Here we show (i) that the QEHA region directly interacts with Gbetagamma but (ii) that it is of minor importance for the stimulation of type II adenylyl cyclase because it can be replaced by corresponding, nonidentical regions of other adenylyl cyclase isoforms without altering the stimulatory effect of Gbetagamma and (iii) that the C(1b) region is necessary for Gbetagamma to exert a stimulatory effect on adenylyl cyclase type II as in a C(1b) deletion mutant the Gbetagamma regulation was specifically impeded whereas the Galpha(s)- and forskolin-mediated stimulation was maintained.     

Pleiotropic effects of Pasteurella multocida toxin are mediated by Gq-dependent and -independent mechanisms. involvement of Gq but not G11

Pasteurella multocida toxin (PMT) is a highly potent mitogen for a variety of cell types. PMT has been shown to induce various cellular signaling processes, and it has been suggested to function through the heterotrimeric G-proteins G(q)/G(11). To analyze the role of G(q)/G(11) in the action of PMT, we have studied the effect of the toxin in Galpha(q)/Galpha(11) double-deficient fibroblasts as well as in fibroblasts lacking only Galpha(q) or Galpha(11). Interestingly, formation of inositol phosphates in response to PMT was exclusively dependent on Galpha(q) but not on the closely related Galpha(11). Although Galpha(q)/Galpha(11) double-deficient and Galpha(q)-deficient cells did not respond with any production of inositol phosphates to PMT, PMT was still able to induce various other cellular effects in these cells, including the activation of Rho, the Rho-dependent formation of actin stress fibers and focal adhesions, as well as the stimulation of c-Jun N-terminal kinase and extracellular signal-regulated kinase. These data show that PMT leads to a variety of cellular effects that are mediated only in part by the heterotrimeric G-protein G(q).     

Distribution of mitochondrial DNA lineages among Native American tribes of Northeastern North America

The mtDNA haplogroups of 185 individuals from Native American tribes in Northeast North America were determined. A subset of these individuals was analyzed by sequencing hypervariable segments I and II of the control region. The haplogroup frequency distributions of populations in the Northeast exhibit regional continuity that predates European contact. A large amount of gene flow has occurred between Siouan-and Algonquian-speaking groups, probably due to an Algonquian intrusion into the Northeast. The data also support both the Macro-Siouan hypothesis and a relatively recent intrusion of Northern Iroquoians into the Northeast. These conclusions are consistent with archaeological and linguistic evidence.     

Regulation of cytosolic phospholipase A2 in rat endometrial stromal cells: the role of epidermal growth factor

The effect of epidermal growth factor on the levels of cytosolic phospholipase A2 mRNA and protein in cultured rat endometrial stromal cells isolated from uteri sensitized for the decidual cell reaction was examined. Treatment with epidermal growth factor increased the steady-state cytosolic phospholipase A2 mRNA and protein levels as demonstrated by Northern and Western blot analyses, respectively. Immunocytochemical analysis demonstrated an increase of cytosolic phospholipase A2 protein in most cells, as opposed to a small subpopulation of cells in culture. These results show that epidermal growth factor causes an increase in steady-state cytosolic phospholipase A2 mRNA and protein levels in rat endometrial stromal cells from uteri sensitized for the decidual cell reaction. Epidermal growth factor receptor ligands may regulate cytosolic phospholipase A2 and thus prostaglandin production in the endometrial stromal cells during implantation.