Cyclic AMP induces rapid increases in gap junction permeability and changes in the cellular distribution of connexin43

The rapid effects of cAMP on gap junction-mediated intercellular communication were examined in several cell types which express different levels of the gap junction protein, connexin43 (Cx43), including immortalized rat hepatocyte and granulosa cells, bovine coronary venular endothelial cells, primary rat myometrial and equine uterine epithelial cells. Functional analysis of changes in junctional communication induced by 8-bromo-cAMP was monitored by a fluorescence recovery after photobleaching assay in subconfluent cultures in the presence or absence of 1.0 mm 1-octanol (an agent which uncouples cells by closing gap junction channels). Communicating cells treated with 1.0 mm 8-bromo-cAMP alone exhibited significant increases in the percent of fluorescence recovery which were detected within 1–3 min depending on cell type, and junctional communication remained significantly elevated for up to 24 hr. Addition of 1.0 mm 8-bromo-cAMP to cultured cells, which were uncoupled with 1.0 mm octanol for 1 min, exhibited partial restoration of gap junctional permeability beginning within 3–5 min. Identical treatments were performed on cultures that were subsequently processed for indirect immunofluorescence to monitor Cx43 distribution. The changes in junctional permeability of cells correlated with changes in the distribution of immunoreactive Cx43. Cells treated for 2 hr with 10 m monensin exhibited a reduced communication rate which was accompanied by increased vesicular cytoplasmic Cx43 staining and reduced punctate surface staining of junctional plaques. Addition of 1.0 mm 8-bromo-cAMP to these cultures had no effect on the rate of communication or the distribution of Cx43 compared to cultures treated with monensin alone. These data suggest that an effect of cyclic AMP on Cx43 gap junctions is to promote increases in gap junctional permeability by increasing trafficking and/or assembly of Cx43 to plasma membrane gap junctional plaques.We acknowledge the technical assistance of Richard Lewis and Meghan Abella. We thank Dr. Hugh Dookwah for contributions to the myometrial cell isolation protocol and Drs. Stephen H. Safe, Timothy D. Phillips, and Evelyn Tiffany-Castiglioni for helpful discussions. This work was funded by NIH (HD-26182, P42-ES04917, ES05871-01A1), the March of Dimes Birth Defects Foundation Basic Research grant #1-0796, and USDA 92-37203-7952.     

Physiological and ultrastructural responses of Catharanthus roseus cell suspension to salt stress

Catharanthus roseus (L.) cell response to salinity was investigated. Seven days after cell treatment with 100 mM NaCl, they showed a decrease in dry weight and an increase in sodium and chloride contents (about 12.4- and 1.5-fold, respectively, in comparison to the control). At the ultrastructural level, NaCl treatment reduced cell size and increased plastid density. In addition, it reduced the starch grain size and their number per plastid; however, starch content was 1.5-fold increased, which was due to the increase in the plastid density. At the ultrastructural level, the applied salinity had no obvious effects, such as swelling or disorganization of plastids except a slight decrease in the stroma electron density. Equally, no deleterious effect was observed on mitochondria except a small increase of their crista volume and matrix electron density. It was shown that, although the relative sensitivity of C. roseus cells to salt stress pointed by the reduction in the dry weight, a decrease in the cell size, and the high accumulation of toxic ions, they preserved the integrity of their plastids and mitochondria.     

PTPN22 gene polymorphism in Egyptian alopecia areata patients and its impact on response to diphencyprone immunotherapy

PTPN22 1858C>T gene polymorphism has been associated with several autoimmune disorders including alopecia areata. The aim of the current study was to investigate the effect of the inherited genetic polymorphism 1858C>T of PTPN22 gene on the predisposition to severe forms of alopecia areata and its effect on the response to DPC treatment. To achieve our aim, PTPN22 1858C>T genotyping was performed by PCR-based restricted fragment length polymorphism (PCR-RFLP) analysis. The study included 103 Egyptian patients with extensive alopecia areata treated by DPC. Hundred healthy age and sex matched blood donors were included in the current study as a control group. Results of genotyping showed that PTPN22 CT and TT mutant genotypes were significantly higher in AA patients compared to controls and conferred increase risk of AA (OR = 2.601, 95% CI = 1.081–6.255). Statistical comparison between AA patients with wild and mutant genotypes revealed that the duration of the illness was significantly longer in those harboring the mutant genotypes. Moreover, the association of other autoimmune diseases as atopy and diabetes mellitus was higher in patients with mutant genotypes. Furthermore, PTPN22 1858C>T genetic polymorphism did not affect the patients' response to DPC immunotherapy.     

Two dimensional electrophoresis of the exo-proteome produced from community acquired methicillin resistant Staphylococcus aureus belonging to clonal complex 80

Two-dimensional electrophoresis (2DE) combined with mass spectrometry was used to characterize the exo-proteome secreted by two strains (ER13 and ER21) representing community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) belonging to clonal complex 80 (CC80). Common spots were detected between the 2 gels using the Progenesis SameSpots software. Two hundred and fifty-one and 312 spots from the exo-proteome of ER13 and ER21 were resolved, respectively. 2DE overlap comparison showed that 59 spots were shared. LC–MS/MS analysis identified 57 proteins from these spots comprising about 21% extracellular, 48% cytoplasmic, 2% cytoplasmic membrane, 2% cell wall, and 26% with unknown localization. The identified proteins were classified with respect to their Gene Ontology (GO) annotation as ～24% virulence determinants and toxins, ～17% involved in carbohydrate metabolism, ～14% involved in environmental stress, and ～12% associated with cell division. The identification of the enterotoxin B from the exo-products of both strains used in our study, as belonging to CC80 was interesting.     

Effect of salt stress on photosynthesis and chlorophyll fluorescence in Medicago truncatula

In the present study, photosynthetic parameters including gas exchanges, pigment contents, and chlorophyll fluorescence, were compared in two contrasting local Medicago truncatula lines TN6.18 and TN8.20, in response to salt added to the nutrient solution. Plants were cultivated under symbiotic nitrogen fixation (SNF) after inoculation with a reference strain Sinorhizobium meliloti 2011, a very tolerant strain to salinity (700 mM NaCl), and grown in a controlled glasshouse. On one month old plants (with active SNF), salt treatment (75 mM NaCl) was gradually applied. Photosynthesis, assimilating pigments and chlorophyll fluorescence were monitored throughout the experiment during both short and long terms, compared to control (non-saline) conditions. A genotypic variation in salt tolerance was found; TN6.18 was the more sensitive to salinity. The relative tolerance of TN8.20 was concomitant with the highest photochemical quenching coefficient (q_P) affecting the maximum quantum yield of PSII (Y); the real quantum yield (?_exc) was the most affected in the sensitive line. Moreover, stomatal and PSII reaction centers activities differed clearly between the studied lines. We found that the effect of salinity on photosynthesis of M. truncatula was related to PSII activity reduction rather than to stomatal conductance limitation. Photosynthesis was reduced by the inhibition of CO₂ assimilation caused by PSII damage. This was clearly estimated by the Y, ?_exc and especially by the quantum yield of electron transport of PSII (Φ_PSII). Thus, on the basis of our results on the two local M. truncatula lines, we recommend the use of chlorophyll fluorescence as non-destructive screening method to discriminate susceptible and resistant legumes to salt stress.     

Antagonism of CD95 signaling blocks butyrate induction of apoptosis in young adult mouse colonic cells

There is greatinterest in utilizing butyrate as a chemopreventive agent for colontumorigenesis because of its ability to promote apoptosis in colontumor cell lines. Because CD95 (APO-1/Fas) transduces signals resultingin apoptosis, we tested the hypothesis that butyrate-dependentcolonocyte apoptosis is mediated by this death receptor. Butyrate (1 mM) exposure for 24 h upregulated expression of Fas andits ligand in young adult mouse colon (YAMC) cells. To delineate theproapoptotic effect of butyrate and to avoid the confounding effects ofdetachment from the extracellular matrix, adherent cell apoptosis wasmonitored as loss of plasma membrane asymmetry and dissipation ofmitochondrial membrane potential (_mt) by lasercytometry. Soluble Fas receptor protein (Fas:Fc chimera) and caspaseinhibitors (z-VAD-fmk and z-IETD-fmk) blocked butyrate induction ofapoptosis. Treatment with Fas agonistic antibody (clone Jo-2)significantly induced cell death, indicating that Fas in colonocytes isfunctional. In addition, butyrate promoted apoptosis by inducing lossof _mt and phospholipidasymmetry of the plasma membrane after 12 and 24 h of exposure,respectively, before cell detachment. Therefore, Fas receptor-dependentsignal transduction is involved in butyrate induction of apoptosis in colonocytes.

     

Lebecetin, a potent antiplatelet C-type lectin from Macrovipera lebetina venom

A novel C-type lectin protein (CLP), lebecetin, was purified to homogeneity from the venom of Macrovipera lebetina by gel filtration on a Sephadex G75 column and ion exchange chromatography on Mono S column. Lebecetin is a basic protein with a pHi=9.9 and migrates in SDS-PAGE as a single band or two distinct bands under nonreducing and reducing conditions, respectively. These results are further confirmed by MALDI-TOF mass spectrometry that indicates a molecular mass of 29779 Da for native lebecetin and molecular masses of 15015 and 16296 Da for alpha and beta subunits, respectively. The N-terminal amino acid sequences of lebecetin subunits show a high degree of similarity with those of C-type lectin-like proteins. In addition, functional studies showed that lebecetin has a potent inhibitory effect on platelet aggregation induced by thrombin in a concentration-dependent manner. In contrast, no inhibitory effect is observed when platelets are exposed to thromboxane A2 (TxA2) mimetic (U46619) or arachidonic acid. Moreover, there was no effect either on blood coagulation or A, B and O washed human erythrocytes agglutination. Furthermore, flow cytometric analysis revealed that fluoro-isothiocyanate (FITC)-labelled lebecetin bound to human formalin fixed platelets in a saturable and concentration manner and this binding was specifically prevented by anti-glycoprotein Ib (GPIb) mAb. These observations suggest that lebecetin is a C-type lectin-like protein that selectively binds to platelet GPIb.     

Inhibitory effects of cytomegalovirus proteins US2 and US11 point to contributions from direct priming and cross-priming in induction of vaccinia virus-specific CD8(+) T cells

The extent to which naive CD8(+) CTLs (T(CD8)(+)) are primed by APCs presenting endogenous Ags (direct priming) or Ags acquired from other infected cells (cross-priming) is a critical topic in basic and applied immunology. To examine the contribution of direct priming in the induction of VV-specific T(CD8)(+), we generated recombinant vaccinia viruses that express human CMV proteins (US2 and US11) that induce the destruction of newly synthesized MHC class I molecules. Expression of US2 or US11 was associated with a 24-63% decrease in numbers of primary or secondary VV-specific T(CD8)(+) responding to i.p. infection. Using HPLC-isolated peptides from VV-infected cells, we show that US2 and US11 selectively inhibit T(CD8)(+) responses to a subset of immunogenic VV determinants. Moreover, VV-US2 and lysates from VV-infected histoincompatible cells elicit T(CD8)(+) specific for a similar subset of VV determinants. These findings indicate that US2 and US11 can function in vivo to interfere with the activation of virus-specific T(CD8)(+). Furthermore, they suggest that 1) both cross-priming and direct priming contribute significantly to the generation of VV-specific T(CD8)(+), 2) the sets of immunogenic vaccinia virus determinants generated by cross-priming and direct priming are not completely overlapping, and 3) cross-priming overrides the effects of cis-acting viral interference with the class I Ag presentation pathway.     

Unrestricted modification search reveals lysine methylation as major modification induced by tissue formalin fixation and paraffin embedding

Formalin‐fixed paraffin‐embedded (FFPE) tissue is considered as an appropriate alternative to frozen/fresh tissue for proteomic analysis. Here we study formalin‐induced alternations on a proteome‐wide level. We compared LC‐MS/MS data of FFPE and frozen human kidney tissues by two methods. First, clustering analysis revealed that the biological variation is higher than the variation introduced by the two sample processing techniques and clusters formed in accordance with the biological tissue origin and not with the sample preservation method. Second, we combined open modification search and spectral counting to find modifications that are more abundant in FFPE samples compared to frozen samples. This analysis revealed lysine methylation (+14 Da) as the most frequent modification induced by FFPE preservation. We also detected a slight increase in methylene (+12 Da) and methylol (+30 Da) adducts as well as a putative modification of +58 Da, but they contribute less to the overall modification count. Subsequent SEQUEST analysis and X!Tandem searches of different datasets confirmed these trends. However, the modifications due to FFPE sample processing are a minor disturbance affecting 2–6% of all peptide‐spectrum matches and the peptides lists identified in FFPE and frozen tissues are still highly similar.