Visna virus synthesized in absence of host-cell division and DNA synthesis

Visna virus is similar to the avian and the murine oncornaviruses. Oncornavirus replication is dependent upon the provirus being integrated into the host cell's DNA but integration and subsequent oncornavirus synthesis is blocked when the host cells are prevented from synthesizing cellular DNA or dividing. The synthesis of visna virus is restricted in vivo and may be dependent upon the host cell's ability to synthesize cellular DNA or divide. Treatment of sheep choroid plexus (SCP) cells with ultraviolet light or with mitomycin C prior to infection irreversibly inhibited plexus (ScP) cells with ultraviolet light or with mitomycin C prior to infection irreversibly inhibited both cell division and cellular nucleic acid synthesis but did not inhibit visna virus synthesis. Similarly, the synthesis of visna virus in cultures of SCP cells which had been prevented from dividing by being deprived of serum and in cultures of SCP cells which were incapable of synthesizing host cell nucleic acids by being treated with miracil D or sodium hexachloroiridate was equivalent to the synthesis of visna virus in cultures of SCP cells which were allowed to both synthesize cellular nucleic acids and divide. The synthesis of visna virus in the presence of ethidium bromide further demonstrated that integration of the visna provirus into the host cell's DNA is not required for visna virus synthesis to occur.     

Ovine cells: their long-term cultivation and susceptibility to visna virus.

Sheep choroid plexus (SCP) cells have been subcultured more than 120 times and have undergone over 300 cell generations. These fibroblastic-appearing SCP II-B cells contain ovine-specific antigens, have an absolute plating efficiency of 23 to 28% and are as susceptible to visna virus infection and virus-induced cytopathology as their low passage level counterparts. Cultures of low, relatively high and high passage level SCP cells produced equivalent amounts of visna virus at similar rates when infected with equal amounts of visna virus. The passage level of the SCP II-B cells, their elapsed number of cell generations, their possession of ovine-specific antigens and their full susceptibility to visna virus allow these cells to be considered an established line of sheep cells.     

Equilibrium unfolding of the PDZ domain of β2-syntrophin

β2-syntrophin, a dystrophin-associated protein, plays a pivotal role in insulin secretion by pancreatic β-cells. It contains a PDZ domain (β2S-PDZ) that, in complex with protein-tyrosine phosphatase ICA512, anchors the dense insulin granules to actin filaments. The phosphorylation state of β2-syntrophin allosterically regulates the affinity of β2S-PDZ for ICA512, and the disruption of the complex triggers the mobilization of the insulin granule stores. Here, we investigate the thermal unfolding of β2S-PDZ at different pH and urea concentrations. Our results indicate that, unlike other PDZ domains, β2S-PDZ is marginally stable. Thermal denaturation experiments show broad transitions and cold denaturation, and a two-state model fit reveals a significant unfolded fraction under physiological conditions. Furthermore, T(m) and T(max) denaturant-dependent shifts and noncoincidence of melting curves monitored at different wavelengths suggest that two-state and three-state models fail to explain the equilibrium data properly and are in better agreement with a downhill scenario. Its higher stability at pH >9 and the results of molecular dynamics simulations indicate that this behavior of β2S-PDZ might be related to its charge distribution. All together, our results suggest a link between the conformational plasticity of the native ensemble of this PDZ domain and the regulation of insulin secretion.     

Optical Absorption Modeling of Plasmonic Organic Solar Cells Embedding Silica-Coated Silver Nanospheres

We numerically study plasmonic solar cells in which a square periodic array of core–shell Ag@SiO₂ nanospheres (NSs) are placed on top of the indium tin oxide (ITO) layer using a 3D finite-difference time-domain (FDTD) method. We investigate the influence of various parameters such as the periodicity of the array, the Ag core diameter, the active layer thickness, the shell thickness, and the refractive index of the shell materials on the optical performance of the organic solar cells (OSC). Our results show that the optimal periodicity of the array of NSs is dependent on the size of Ag core NSs in order to maximize optical absorption in the active layer. A very thin active layer (<70 nm) and an ultrathin (<5 nm) SiO₂ shell are needed in order to obtain the highest optical absorption enhancement. Strong electric field localization is observed around the plasmonic core–shell nanoparticles as a result of localized surface plasmon resonance (LSPR) excited by Ag NSs with and without silica shell. Embedding 50 nm Ag NSs with 1-nm-thick SiO₂ shell thickness on top of ITO leads to an enhanced intrinsic optical absorption in a 40-nm-thick poly(3-hexylthiophene):phenyl-C₆₁-butyric acid methyl ester (P3HT:PCBM) active layer by 24.7% relative to that without the NSs. The use of 1-nm-thick ZnO shell instead of SiO₂ leads to an enhanced intrinsic absorption in a 40-nm-thick P3HT:PCBM active layer by 27%.

     

Tuning of Light Trapping and Surface Plasmon Resonance in Silver Nanoparticles/c-Si Structures for Solar Cells

In this work, we investigate silver (Ag) nanoparticle-related plasmonic effect on light absorption in Si substrate. Ag nanoparticles (Ag-NPs) deposited on top of Si were used to capture and couple incident light into these structures by forward scattering. We demonstrate that we can control nanoparticle size and shape while varying deposition time and annealing parameters. By the increase of the total time of the reaction process, morphology of Ag-NPs evolutes affecting the number and the width of surface plasmon resonance peaks, whereas for changed annealing parameters (temperature and time), the effect is more pronounced on the broadening and the position of peaks. Specific morphology of Ag-NPs can exhibit an interesting enhancement of optical properties which enables plasmon-related application in photovoltaic solar cells.     

Role of Alveolar β2-Adrenergic Receptors on Lung Fluid Clearance and Exercise Ventilation in Healthy Humans

Background

In experimental conditions alveolar fluid clearance is controlled by alveolar β₂-adrenergic receptors. We hypothesized that if this occurs in humans, then non-selective β-blockers should reduce the membrane diffusing capacity (D_M), an index of lung interstitial fluid homeostasis. Moreover, we wondered whether this effect is potentiated by saline solution infusion, an intervention expected to cause interstitial lung edema. Since fluid retention within the lungs might trigger excessive ventilation during exercise, we also hypothesized that after the β₂-blockade ventilation increased in excess to CO₂ output and this was further enhanced by interstitial edema.

Methods and Results

22 healthy males took part in the study. On day 1, spirometry, lung diffusion for carbon monoxide (DLCO) including its subcomponents D_M and capillary volume (V_Cap), and cardiopulmonary exercise test were performed. On day 2, these tests were repeated after rapid 25 ml/kg saline infusion. Then, in random order 11 subjects were assigned to oral treatment with Carvedilol (CARV) and 11 to Bisoprolol (BISOPR). When heart rate fell at least by 10 beats·min⁻¹, the tests were repeated before (day 3) and after saline infusion (day 4). CARV but not BISOPR, decreased D_M (−13±7%, p = 0.001) and increased V_Cap (+20±22%, p = 0.016) and VE/VCO₂ slope (+12±8%, p<0.01). These changes further increased after saline: −18±13% for D_M (p<0.01), +44±28% for V_Cap (p<0.001), and +20±10% for VE/VCO₂ slope (p<0.001).

Conclusions

These findings support the hypothesis that in humans in vivo the β₂-alveolar receptors contribute to control alveolar fluid clearance and that interstitial lung fluid may trigger exercise hyperventilation.     

Candida zemplinina can reduce acetic acid produced by Saccharomyces cerevisiae in sweet wine fermentations

In this study we investigated the possibility of using Candida zemplinina, as a partner of Saccharomyces cerevisiae, in mixed fermentations of must with a high sugar content, in order to reduce its acetic acid production. Thirty-five C. zemplinina strains, which were isolated from different geographic regions, were molecularly characterized, and their fermentation performances were determined. Five genetically different strains were selected for mixed fermentations with S. cerevisiae. Two types of inoculation were carried out: coinoculation and sequential inoculation. A balance between the two species was generally observed for the first 6 days, after which the levels of C. zemplinina started to decrease. Relevant differences were observed concerning the consumption of sugars, the ethanol and glycerol content, and acetic acid production, depending on which strain was used and which type of inoculation was performed. Sequential inoculation led to the reduction of about half of the acetic acid content compared to the pure S. cerevisiae fermentation, but the ethanol and glycerol amounts were also low. A coinoculation with selected combinations of S. cerevisiae and C. zemplinina resulted in a decrease of ~0.3 g of acetic acid/liter, while maintaining high ethanol and glycerol levels. This study demonstrates that mixed S. cerevisiae and C. zemplinina fermentation could be applied in sweet wine fermentation to reduce the production of acetic acid, connected to the S. cerevisiae osmotic stress response.     

Effects of chest wall strapping on mechanical response to methacholine in humans.

We examined the effects of chest wall strapping (CWS) on the response to inhaled methacholine (MCh) and the effects of deep inspiration (DI). Eight subjects were studied on 1 day with MCh inhaled without CWS (CTRL), 1 day with MCh inhaled during CWS (CWSon/on), and 1 day with MCh inhaled during temporary removal of CWS (CWSoff/on). On the CWSon/on day, MCh caused greater increases in pulmonary resistance, upstream resistance, dynamic elastance, residual volume, and greater decreases in maximal expiratory flow than on the CTRL day. On the CWSoff/on day, the changes in these parameters with MCh were not different from the CTRL day. Six of the subjects were again studied using the same protocol on CTRL and CWSon/on days, except that, on a third day, MCh was given after applying the CWS, but the measurements before and after the inhalation were made without CWS (CWSon/off). The latter sequence was associated with more severe airflow obstruction than during CTRL, but less than with CWSon/on. The bronchodilator effects of a DI were blunted when CWS was applied during measurements (CWSon/on and CWSoff/on) but not after it was removed (CWSon/off). We conclude that CWS is capable of increasing airway responsiveness only when it is applied during the inhalation of the constrictor agent. We speculate that breathing at low lung volumes induced by CWS enhances airway narrowing because the airway smooth muscle is adapted at a length at which the contractile apparatus is able to generate a force greater than normal.     

Feasibility studies of oncornavirus production in microcarrier cultures

Summary Studies conducted with virus-infected monolayer cell cultures have demonstrated the feasibility of producing several tumor-associated viruses in microcarrier (mc) cultures (Sephadex G50 beads treated with DEAE-chloride). The efficiency of cell adherence to mc varied with the cell type, the pH of the growth medium, and the stirring force applied to keep the mc in suspension. Most cells attached firmly to the mc and could not be removed easily with routine trypsinization procedures. Techniques using Enzar-T and Pronase were effective in detaching cells from mc in 10 to 15 min while retaining 95% cell viability. After detachment, Ficoll gradients were used for rapid and complete separation of viable cell suspensions from the mc. Retrovirus production in large volumes of mc cultures was investigated with periodic harvesting of growth fluids. Physical, biochemical, and biological properties of the Mason-Pfizer monkey virus and the RD114 virus recovered from the mc cultures were identical to those produced in conventional cultures. The utilization of mc has several applications in research and short-term cultures, but the as-yet-unsolved technical problems met were found to be serious limitations when attempting mass cell culturing on a long-term basis. For reprint requests address: Dr. Keith Jensen, Pfizer, Inc., Groton, CT 06340. This work was supported in part under contract N01-CP-33234 within the Virus Cancer Program of the National Cancer Institute.