Differences in polyadenylation site choice between somatic and male germ cells

Background  

We have previously noted that there were differences in somatic and male germ cell polyadenylation site choices. First, male germ cells showed a lower incidence of the sequence AAUAAA (an important element for somatic polyadenylation site choice) near the polyadenylation site choice. Second, the polyadenylation sites chosen in male germ cells tended to be nearer the 5' end of the mRNA than those chosen in somatic cells. Finally, a number of mRNAs used a different polyadenylation site in male germ cells than in somatic cells. These differences suggested that male germ cell-specific polyadenylation sites may be poor substrates for polyadenylation in somatic cells. We therefore hypothesized that male germ cell-specific polyadenylation sites would be inefficiently used in somatic cells.     

Selective activation of Fyn/PI3K and p38 MAPK regulates IL-4 production in BMMC under nontoxic stress condition

Mast cells have the ability to react to multiple stimuli, implicating these cells in many immune responses. Specific signals from the microenvironment in which mast cells reside can activate different molecular events that govern distinct mast cells responses. We previously demonstrated that hydrogen peroxide (H(2)O(2)) promotes IL-4 and IL-6 mRNA production and potentates FcepsilonRI-induced cytokine release in rat basophilic leukemia RBL-2H3 cells. To further evaluate the effect of an oxidative microenvironment (which is physiologically present in an inflammatory site) on mast cell function and the molecular events responsible for mast cell cytokine production in this environment, we analyzed the effect of H(2)O(2) treatment on IL-4 production in bone marrow-derived, cultured mast cells. Our findings show that nanomolar concentrations of H(2)O(2) induce cytokine secretion and enhance IL-4 production upon FcepsilonRI triggering. Oxidative stimulation activates a distinct signal transduction pathway that induces Fyn/PI3K/Akt activation and the selective phosphorylation of p38 MAP kinase. Moreover, H(2)O(2) induces AP-1 and NFAT complexes that recognize the IL-4 promoter. The absence of Fyn and PI3K or the inhibition of p38 MAPK activity demonstrated that they are essential for H(2)O(2)-driven IL-4 production. These findings show that mast cells can respond to an oxidative microenvironment by initiating specific signals capable of eliciting a selective response. The findings also demonstrate the dominance of the Fyn/p38 MAPK pathway in driving IL-4 production.     

Cdc42 regulates E-cadherin ubiquitination and degradation through an epidermal growth factor receptor to Src-mediated pathway

E-cadherins play an essential role in maintaining epithelial polarity by forming Ca2+-dependent adherens junctions between epithelial cells. Here, we report that Ca2+ depletion induces E-cadherin ubiquitination and lysosomal degradation and that Cdc42 plays an important role in regulating this process. We demonstrate that Ca2+ depletion induces activation of Cdc42. This in turn up-regulates epidermal growth factor receptor (EGFR) signaling to mediate Src activation, leading to E-cadherin ubiquitination and lysosomal degradation. Silencing Cdc42 blocks activation of EGFR and Src induced by Ca2+ depletion, resulting in a reduction in E-cadherin degradation. The role of Cdc42 in regulating E-cadherin ubiquitination and degradation is underscored by the fact that constitutively active Cdc42(F28L) increases the activity of EGFR and Src and significantly enhances E-cadherin ubiquitination and lysosomal degradation. Furthermore, we found that GTP-dependent binding of Cdc42 to E-cadherin is critical for Cdc42 to induce the dissolution of adherens junctions. Our data support a model that activation of Cdc42 contributes to mesenchyme-like phenotype by targeting of E-cadherin for lysosomal degradation.     

Embryonic stem cells contribute to mouse chimeras in the absence of detectable cell fusion

Embryonic stem (ES) cells are capable of differentiating into all embryonic and adult cell types following mouse chimera production. Although injection of diploid ES cells into tetraploid blastocysts suggests that tetraploid cells have a selective disadvantage in the developing embryo, tetraploid hybrid cells, formed by cell fusion between ES cells and somatic cells, have been reported to contribute to mouse chimeras. In addition, other examples of apparent stem cell plasticity have recently been shown to be the result of cell fusion. Here we investigate whether ES cells contribute to mouse chimeras through a cell fusion mechanism. Fluorescence in situ hybridization (FISH) analysis for X and Y chromosomes was performed on dissociated tissues from embryonic, neonatal, and adult wild-type, and chimeric mice to follow the ploidy distributions of cells from various tissues. FISH analysis showed that the ploidy distributions in dissociated tissues, notably the tetraploid cell number, did not differ between chimeric and wild-type tissues. To address the possibility that early cell fusion events are hidden by subsequent reductive divisions or other changes in cell ploidy, we injected Z/EG (lacZ/EGFP) ES cells into ACTB-cre blastocysts. Recombination can only occur as the result of cell fusion, and the recombined allele should persist through any subsequent changes in cell ploidy. We did not detect evidence of fusion in embryonic chimeras either by direct fluorescence microscopy for GFP or by PCR amplification of the recombined Z/EG locus on genomic DNA from ACTB-cre::Z/EG chimeric embryos. Our results argue strongly against cell fusion as a mechanism by which ES cells contribute to chimeras.     

AUTOPHAGIC VACUOLES PRODUCED IN VITRO : I. Studies on Cultured Macrophages Exposed to Chloroquine

Mouse macrophages exposed to 30 µg/ml of chloroquine in vitro develop autophagic vacuoles containing various cytoplasmic components and acid phosphatase. The early toxic vacuoles appear in the perinuclear region within 15 min; on electron microscopy, they show irregular shape, amorphous moderately dense content, apparent double membranes, and in some instances curved thin tubular extensions with a central, dark linear element. Cytoplasmic structures are probably transported into the vacuoles by invagination of the vacuolar membrane. After exposure to chloroquine for 1–4 hr, macrophages display large vacuoles containing degraded cytoplasmic structures, membranous whorls, and amorphous material. When chloroquine is removed by changing the culture medium after 4 hr, the cells survive and 24 hr later they exhibit no abnormality except for large cytoplasmic dense bodies packed with membrane lamellae. During recovery chloroquine disappears from the cells. 24 hr after exposure to chloroquine the macrophages have accumulated less hydrolases than control cells.     

Assessment of cell death studies by monitoring hydrogen peroxide in cell culture

Hydrogen peroxide (H₂O₂) has been widely used to study the oxidative stress response. However, H₂O₂ is unstable and easily decomposes into H₂O and O₂. Consequently, a wide range of exposure times and treatment concentrations has been described in the literature. In the present study, we established a ferrous oxidation-xylenol orange (FOX) assay, which was originally described for food and body liquids, as a method for the precise quantification of H₂O₂ concentrations in cell culture media. We observed that the presence of FCS and high cell densities significantly accelerate the decomposition of H₂O₂, therefore acting as a protection against cell death by accidental necrosis.     

Comparative efficacy of recombinant modified vaccinia virus Ankara expressing simian immunodeficiency virus (SIV) Gag-Pol and/or Env in macaques challenged with pathogenic SIV

Prior studies demonstrated that immunization of macaques with simian immunodeficiency virus (SIV) Gag-Pol and Env recombinants of the attenuated poxvirus modified vaccinia virus Ankara (MVA) provided protection from high levels of viremia and AIDS following challenge with a pathogenic strain of SIV (V. M. Hirsch et al., J. Virol. 70:3741-3752, 1996). This MVA-SIV recombinant expressed relatively low levels of the Gag-Pol portion of the vaccine. To optimize protection, second-generation recombinant MVAs that expressed high levels of either Gag-Pol (MVA-gag-pol) or Env (MVA-env), alone or in combination (MVA-gag-pol-env), were generated. A cohort of 24 macaques was immunized with recombinant or nonrecombinant MVA (four groups of six animals) and was challenged with 50 times the dose at which 50% of macaques are infected with uncloned pathogenic SIVsmE660. Although all animals became infected postchallenge, plasma viremia was significantly reduced in animals that received the MVA-SIV recombinant vaccines as compared with animals that received nonrecombinant MVA (P = 0.0011 by repeated-measures analysis of variance). The differences in the degree of virus suppression achieved by the three MVA-SIV vaccines were not significant. Most importantly, the reduction in levels of viremia resulted in a significant increase in median (P < 0.05 by Student's t test) and cumulative (P = 0.010 by log rank test) survival. These results suggest that recombinant MVA has considerable potential as a vaccine vector for human AIDS.     

Localization and signaling of G(beta) subunit Ste4p are controlled by a-factor receptor and the a-specific protein Asg7p

Haploid yeast cells initiate pheromone signaling upon the binding of pheromone to its receptor and activation of the coupled G protein. A regulatory process termed receptor inhibition blocks pheromone signaling when the a-factor receptor is inappropriately expressed in MATa cells. Receptor inhibition blocks signaling by inhibiting the activity of the G protein beta subunit, Ste4p. To investigate how Ste4p activity is inhibited, its subcellular location was examined. In wild-type cells, alpha-factor treatment resulted in localization of Ste4p to the plasma membrane of mating projections. In cells expressing the a-factor receptor, alpha-factor treatment resulted in localization of Ste4p away from the plasma membrane to an internal compartment. An altered version of Ste4p that is largely insensitive to receptor inhibition retained its association with the membrane in cells expressing the a-factor receptor. The inhibitory function of the a-factor receptor required ASG7, an a-specific gene of previously unknown function. ASG7 RNA was induced by pheromone, consistent with increased inhibition as the pheromone response progresses. The a-factor receptor inhibited signaling in its liganded state, demonstrating that the receptor can block the signal that it initiates. ASG7 was required for the altered localization of Ste4p that occurs during receptor inhibition, and the subcellular location of Asg7p was consistent with its having a direct effect on Ste4p localization. These results demonstrate that Asg7p mediates a regulatory process that blocks signaling from a G protein beta subunit and causes its relocalization within the cell.     

The C2B Domain Is the Primary Ca Sensor in DOC2B: A Structural and Functional Analysis

DOC2B (double-C₂ domain) protein is thought to be a high-affinity Ca^2 + sensor for spontaneous and asynchronous neurotransmitter release. To elucidate the molecular features underlying its physiological role, we determined the crystal structures of its isolated C2A and C2B domains and examined their Ca^2 +-binding properties. We further characterized the solution structure of the tandem domains (C2AB) using small-angle X-ray scattering. In parallel, we tested structure–function correlates with live cell imaging tools. We found that, despite striking structural similarity, C2B binds Ca^2 + with considerably higher affinity than C2A. The C2AB solution structure is best modeled as two domains with a highly flexible orientation and no difference in the presence or absence of Ca^2 +. In addition, kinetic studies of C2AB demonstrate that, in the presence of unilamellar vesicles, Ca^2 + binding is stabilized, as reflected by the ~ 10-fold slower rate of Ca^2 + dissociation than in the absence of vesicles. In cells, isolated C2B translocates to the plasma membrane (PM) with an EC₅₀ of 400 nM while the C2A does not translocate at submicromolar Ca^2 + concentrations, supporting the biochemical observations. Nevertheless, C2AB translocates to the PM with an ~ 2-fold lower EC₅₀ and to a greater extent than C2B. Our results, together with previous studies, reveal that the C2B is the primary Ca^2 + sensing unit in DOC2B, whereas C2A enhances the interaction of C2AB with the PM.     

Microbial colonization of aquifer sediment exposed in a groundwater well in northern Germany.

Microbial growth within the water-saturated subsurface environment was investigated by exposing sandy sediments to groundwater for 12 weeks at a depth of 10 or 20 m in a stainless-steel groundwater well. Washing and heating the sediment to 600 degrees C (removal of organic carbon) prior to the exposure did not prevent the natural microbial community from colonizing the sterilized sediment samples. Total cell counts of more than 10(7) or 10(8) per g of dried sediment were obtained. Viable cell counts of 10(5) cells per g on oligotrophic media indicated the presence, within the exposed sediment, of a highly active and multiplying biota. Microscopic analysis of enrichments inoculated with exposed sediment samples revealed a total of 45 different morphotypes, approximately 42% of the microbial community observed in previous studies of this site. The interstitial water running off of the retrieved sediment contained only 17 morphotypes and had up to 6 x 10(5) viable cells per ml.