The ability of the bovine C-type leukemia virus to induce syncytia formation in monolayer cell cultures has been used to develop a specific and simple infectivity assay for the virus. Using bovine ...embryonic spleen cells or human diploid embryonic lung cells as indicator cells, the results of the assay can be evaluated in 4 to 6 or 6 to 8 days, respectively. Pretreatment of the indicator cells with DEAE-dextran greatly increases the sensitivity of the assay. The assay is quantitative and can be applied as a direct method for the identification of bovine C-type leukemia virus-infected animals; it also provides a simple, and sensitive procedure for the detection and titration of virus-neutralizing antibodies.
Hyperoxia has been reported to be protective against gut-derived sepsis. Although secretory immunoglobulin A (IgA) is primarily responsible for humoral defense of mucosal surfaces, a potential ...synergistic effect with hyperoxia is unknown. An asanguineous cell monolayer system was used to study these aspects in vitro.
MDCK cells were grown as polarized monolayers in a two-chamber culture system. Apical chambers were inoculated with 10(8) Escherichia coli M14 with or without polyclonal IgA and incubated in a 21 or 95% O2 environment. Basal medium was sampled at 90 and 180 minutes for bacterial translocation. In a second experiment, MDCK cells were lysed at 90 minutes and intracellular bacteria were quantitated.
Bacterial translocation was decreased versus normoxia by the treatment groups IgA without hyperoxia or IgA with hyperoxia at 90 minutes. Bacterial internalization at 90 minutes was reduced to the greatest extent by the combined effects of hyperoxia and IgA. Translocation data at 180 minutes confirmed the additional protective effect of hyperoxia with IgA.
Hyperoxia exerts a significant protective effect on barrier function independent of enhanced leukocyte function. Hyperoxia has an added effect to the mucosal defense provided by IgA.
A microvascular endothelial cell line (CD clone 4) isolated from murine lung adheres to and spreads well on fibronectin, vitronectin, and fibrinogen, but poorly on collagen type IV and laminin. ...Ligating cell surface av, b3, a4, a5, or b1 integrin receptors with monospecific antibodies promoted a dramatic cell spreading and motility on vitronectin or collagen IV. Antibodies directed to other adhesion molecules, including aIIb, PECAM-1, and P-selectin were ineffective. Ligation with monoclonal anti-av or -b3, but not -a4, -a5, or -b1 antibodies, induced a rapid, and dose-dependent tyrosine phosphorylation of a ~30 kD protein, which preceded CD clone 4 endothelial cell spreading and motility and was partially inhibited by genistein and completely inhibited by BAPTA. All other antibodies tested did not induce the tyrosine phosphorylation of the 30 kD protein as well as cell spreading and motility. The present results suggest that b1 and b3 integrins employ different biochemical mechanisms in signaling endothelial cell spreading and motility and that the tyrosine phosphorylation of the 30 kD protein (and probably other proteins) may play an important role in signaling b3 integrin-mediated endothelial cell interaction with other cells (e.g., tumor cells) and extracellular matrix.
Results of a search for new phenomena in events with an energetic photon and large missing transverse momentum with the ATLAS experiment at the LHC are reported. Data were collected in proton--proton ...collisions at a center-of-mass energy of 8 TeV and correspond to an integrated luminosity of 20.3 fb$^{-1}$. The observed data are well described by the expected Standard Model backgrounds. The expected (observed) upper limit on the fiducial cross section for the production of such events is 6.1 (5.3) fb at 95% confidence level. Exclusion limits are presented on models of new phenomena with large extra spatial dimensions, supersymmetric quarks, and direct pair production of dark-matter candidates.
Results of a search for decays of massive particles to fully hadronic final states are presented. This search uses 20.3 fb$^{-1}$ of data collected by the ATLAS detector in $\sqrt{s} = 8$ TeV ...proton--proton collisions at the LHC. Signatures based on high jet multiplicities without requirements on the missing transverse momentum are used to search for $R$-parity-violating supersymmetric gluino pair production with subsequent decays to quarks. The analysis is performed using a requirement on the number of jets, in combination with separate requirements on the number of $b$-tagged jets, as well as a topological observable formed from the scalar sum of the mass values of large-radius jets in the event. Results are interpreted in the context of all possible branching ratios of direct gluino decays to various quark flavors. No significant deviation is observed from the expected Standard Model backgrounds estimated using jet-counting as well as data-driven templates of the total-jet-mass spectra. Gluino pair decays to ten or more quarks via intermediate neutralinos are excluded for a gluino with mass $m_{\tilde{g}} < 1$ TeV for a neutralino mass $m_{\tilde{\chi}^0_1} = 500$ GeV. Direct gluino decays to six quarks are excluded for $m_{\tilde{g}} < 917$ GeV for light-flavor final states, and results for various flavor hypotheses are presented.
A search has been performed for pair production of heavy vector-like down-type ($B$) quarks. The analysis explores the lepton-plus-jets final state, characterized by events with one isolated charged ...lepton (electron or muon), significant missing transverse momentum and multiple jets. One or more jets are required to be tagged as arising from $b$-quarks, and at least one pair of jets must be tagged as arising from the hadronic decay of an electroweak boson. The analysis uses the full data sample of $pp$ collisions recorded in 2012 by the ATLAS detector at the LHC, operating at a center-of-mass energy of 8 TeV, corresponding to an integrated luminosity of 20.3 fb$^{-1}$. No significant excess of events is observed above the expected background. Limits are set on vector-like $B$ production, as a function of the $B$ branching ratios, assuming the allowable decay modes are $B \rightarrow Wt/Zb/Hb$. In the chiral limit with a branching ratio of 100% for the decay $B \rightarrow Wt$, the observed (expected) 95% CL lower limit on the vector-like $B$ mass is 810 GeV (760 GeV). In the case where the vector-like $B$ quark has branching ratio values corresponding to those of an $SU(2)$ singlet state, the observed (expected) 95% CL lower limit on the vector-like $B$ mass is 640 GeV (505 GeV). The same analysis, when used to investigate pair production of a colored, charge 5/3 exotic fermion $T_{5/3}$, with subsequent decay $T_{5/3} \rightarrow Wt$, sets an observed (expected) 95% CL lower limit on the $T_{5/3}$ mass of 840 GeV (780 GeV).
Regulation of the plasma membrane Ca super(2+) pump in the cell is of critical importance in maintaining calcium homeostasis. Since protein kinase C is known to regulate functions of cellular ...proteins by direct phosphorylation or by inducing their gene expression, we investigated the possible involvement of protein kinase C in the regulation of the plasma membrane Ca super(2+) pump. Our results suggest that protein kinase C is involved in the regulation of the Ca super(2+) pump in endothelial cells.
Clinical data indicate that gut perfusion deficits must be rectified within 24 hours after traumatic injury to decrease organ failure and death. Ischemia/reperfusion injury to the gut causes ...enterocyte apoptosis (Apo), which may contribute to intestinal barrier failure. The temporal response of enterocyte Apo to acidosis and hypoxia/reoxygenation (H/R) in vitro is unknown. The purpose of this study was to examine the effect of various time points of acidosis or H/R on enterocyte apoptosis and monolayer integrity in an in vitro model.
Caco-2 cell monolayers were made acidic (Dulbecco's modified Eagle's medium, pH 6.9) by hydrochloric acid or exposed to 95% nitrogen/5% carbon dioxide (hypoxia) and then 21% oxygen (reoxygenation). Escherichia coli C-25 were added to the apical media in subsets. Apo and necrosis were quantified by flow cytometry. Permeability was determined by fluorescein isothiocyanate-dextran. Transepithelial electrical resistance (TEER) indexed monolayer.
Extracellular acidosis and C-25 significantly increased apoptosis of Caco-2 cells at 18 hours (extracellular acidosis EC + C-25, 14.5 +/- 3.0; control, 3.8 +/- 0.8; p < 0.001 by analysis of variance). Similarly, the H/R + C-25 group showed a significant increase in apoptosis at 12 hours (H/R + C-25 vs. control, 22.86 +/- 2.12 vs. 3.74 +/- 0.7; p < 0.001 by analysis of variance). The permeability difference was not significant for EC + C-25 versus control at 18 hours (0.68 +/- 0.25 vs. 0.43 +/- 0.0.0.36, respectively; p > 0.05). The H/R + C-25 group had a profound increase in permeability over control at 12 hours (10.8 +/- 0.5 vs. 2.1 +/- 0.3, respectively; p < 0.001). The TEER was significantly lowered for EC versus control at 18 hours (458 +/- 1.5 vs. 468 +/- 8.2) and at 0, 6, and 18 hours for EC + C-25 (409 +/- 28.1, 443 +/- 16.8, and 438 +/- 8.9 vs. 455 +/- 6.5, 467 +/- 6.5, and 469 +/- 8.2, respectively). There was no significant change in the H/R and H/R + C-25 groups.
Synergism of H/R or tissue acidosis and bacteria caused increased Apo, TEER, and permeability in vitro.
Adult calcium tolerant rat ventricular myocytes were maintained under serum-free culture conditions for five days. beta-adrenergic and muscarinic cholinergic receptor expression was assessed by ...radioligand binding determinations using 125I-iodocyanopindolol (ICYP) and 3H-quinuclidinyl benzilate (QNB), respectively. The binding data were correlated with myocyte structural integrity and contractile responsiveness to norepinephrine (NE). During the 5 days in primary culture, beta-adrenergic and muscarinic cholinergic receptor binding capacity diminished Bmax = 17.1 to 9.2 fmol/mg protein and Bmax = 169.0 to 26.6 fmol/mg protein, respectively. The affinity of both autonomic receptors was unaltered during the period of observation. The majority of isolated myocytes were viable (65 to 85%) and remained rod-shaped for 5 days as assessed by phase contrast microscopy. Up to 2 days in vitro the rod-shaped myocytes appeared ultrastructurally similar to their in vivo counterparts and displayed intact nuclei and the usual complement of cellular organelles. From day 3, phase contrast as well as transmission electron microscopy revealed a progressive increase in autophagic vacuoles consisting primarily of disrupted mitochondria. The number of myocytes that contracted in response to norepinephrine (NE) decreased from 57.2 to 2.3% by day 5. These data indicate that adult rat cardiac myocytes maintained in serum free culture for 5 days, express beta-adrenergic and muscarinic cholinergic receptors. There is a rapid decline (50%) in muscarinic cholinergic receptor number and contractile response to NE by day 2. However, the decrease in beta-receptor Bmax by day two is insufficient to explain the severe loss of cell responsiveness to NE. This functional loss may be related, at least in part, to the ultrastructural abnormalities that are first evident at day 2 in culture. Thus, short-term myocyte cultures that retain phenotypic and physiologic characteristics of in vivo cardiac myocytes could provide a useful in vitro system for exploring pharmacologic-functional interactions in the myocardium.
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