Effect of carbon monoxide treatment on neuronal apoptosis.

<p>(<b>A</b>) Representative micrographs of neurons treated or not with 20 µM of glutamate and 10 µM of CO. Apoptotic hallmarks were analyzed by fluorescent microscopy. <i>Upper panel</i>, for the photos taken with the filter for phase contrast, <i>middle panel</i>, for Hoechst (white arrows for nuclei with condensed chromatin) and <i>lower panel</i>, for propidium iodide (white arrows for cells which membrane integrity was lost). (<b>B</b>) Primary cultures of neuronal cells were pre-treated with 10 µM CO, followed by 24 h of glutamate (10–30 µM) treatment. Cell viability was assessed by counting cells containing normal nuclei and plasmatic membrane integrity. For each coverslip, at least 1500 cells were counted. All values are mean ± SD (error bars), n = 5; *<i>p</i><0.05 compared to control. (<b>C</b>) The effect of 10 µM CO treatment on Bcl-2 expression was assessed by its mRNA quantification.</p

BEVS-driven FL-gB expression screening.

<p>(<b>A</b>) bBst2x was used to infect Sf9 (left panel) or High Five (right panel) cells at 0.3 (circles), 1 (diamonds), 2.5 (triangles) or 4 (crosses) ×10⁶ cell/mL (CCI)) with mutliplicities of infection (m.o.i.) of 0.1, 1, 3, 5 or 10 pfu/cell. Cells were harvested at 72 hours after infection and detergent-soluble protein extracts were analysed by densitometric analysis of anti-gB TM immunoblots. Overall relative quantification was made by comparing samples from different experimental series in the same immunoblots (hereafter n = 3 with error bars representing 95% confidence interval, C.I.) and setting as 100% the mean of most intense signals. (<b>B</b>) Comparison of FL-gB expression pattern and kinetics in Sf9 and High Five cell lines, bBst2x-infected with m.o.i. 5 at CCI 0.3 (Sf9) or 2.5 (High Five, Hi5). Cells were sampled at the indicated hours <i>post</i>-infection (h.p.i.) and detergent-soluble cell extracts probed in immunoblot (IB) with anti-gB TM. Positions of the molecular weight markers are indicated and expressed in kDa. (<b>C</b>) Theroretical FL-gB relative volumetric yields computed from (A) for m.o.i. 5 series, according to CCI and cell viability at 72 h.p.i.</p

BEVS-driven FL-gB expression screening.

<p>(<b>A</b>) bBst2x was used to infect Sf9 (left panel) or High Five (right panel) cells at 0.3 (circles), 1 (diamonds), 2.5 (triangles) or 4 (crosses) ×10⁶ cell/mL (CCI)) with mutliplicities of infection (m.o.i.) of 0.1, 1, 3, 5 or 10 pfu/cell. Cells were harvested at 72 hours after infection and detergent-soluble protein extracts were analysed by densitometric analysis of anti-gB TM immunoblots. Overall relative quantification was made by comparing samples from different experimental series in the same immunoblots (hereafter n = 3 with error bars representing 95% confidence interval, C.I.) and setting as 100% the mean of most intense signals. (<b>B</b>) Comparison of FL-gB expression pattern and kinetics in Sf9 and High Five cell lines, bBst2x-infected with m.o.i. 5 at CCI 0.3 (Sf9) or 2.5 (High Five, Hi5). Cells were sampled at the indicated hours <i>post</i>-infection (h.p.i.) and detergent-soluble cell extracts probed in immunoblot (IB) with anti-gB TM. Positions of the molecular weight markers are indicated and expressed in kDa. (<b>C</b>) Theroretical FL-gB relative volumetric yields computed from (A) for m.o.i. 5 series, according to CCI and cell viability at 72 h.p.i.</p

Analysis of purified FL-gB.

<p>(<b>A</b>) Purified reduced (left panel) or non-reduced (right panel) FL-gB was loaded on SDS-PAGE; amounts of FL-gB loaded in the reducing SDS-PAGE are indicated; for non-reducing electrophoresis, 2.5 µg of FL-gB were heated or not in the absence of reducing agents. The positions of the uncleaved FL-gB, TM and SU chains are indicated as migrating before or after the reduction of the gB protomer disulfide bridges. (<b>B</b>) 5 µg of purified FL-gB were analysed by reducing SDS-PAGE after incubating the protein for 4 hours at 30°C with or without the indicated units (U) of recombinant furin and loaded on reducing SDS-PAGE. (<b>C</b>) FL-gB glycosylation was analysed by incubating 2.5 µg of either denatured or native FL-gB with PNGase F for 3 hours or endoglycosidase F isoforms for 1 hour, respectively; the control sample was left undigested. Denaturation was by heating FL-gB at 99°C for 20 min in 0.2% SDS and 1% 2-mercaptoethanol.</p

Experimental groups and time-points schematic representation.

<p><b>Control group</b>, <i>n = 22</i>, untreated animals that did not suffer any treatment; <b>Carbon Monoxide (CO) group</b>, <i>n = 16</i>, subjected to 3 exposures of 250 ppm, for 1 h at P4, P5 and P6; <b>Hypoxia-Ischemia (HI) group</b>, <i>n = 17</i>, animals that underwent surgery and hypoxia (8% of O₂ in nitrogen) exposure for 75 minutes; <b>CO+HI group</b>, <i>n = 19</i>, CO treatment plus hypoxia-ischemia. Animals were euthanized at 6 and 24 h <i>post</i>-HI. Brains were collected and analyzed for lesion volume and cell death markers, as described in the methods section. <i>Histo</i>, for brains analyzed by histological methods; <i>WB</i>, for brains collected and processed for western blot analysis.</p

The concentration of assembly intermediates for three initial protein concentration scenarios:

<p>1) <b>[vp2]₀</b> = 1.2 M, <b>[vp6]₀</b> = 7.8 M and <b>[vp7]₀</b> = 7.8 M (stoichiometric ratios – white bars); 2) <b>[vp2]₀</b> = 1.2 M, <b>[vp6]₀</b> = 7.8 M and <b>[vp7]₀</b> = 0.78 M (limitation of vp7 – black bars); 3) <b>[vp2]₀</b> = 12 M, <b>[vp6]₀</b> = 7.8 M and <b>[vp7]₀</b> = 7.8 M (excess of vp2 – grey bars). The Gibbs free energy of vp2, vp6 and vp7 subunit association was assumed to be equal (<b>ΔG⁰_vp2,3-mer</b> = <b>ΔG⁰_vp6,13-mer</b> = <b>ΔG⁰_vp7,13-mer</b> = −4.08 kcal.mol⁻¹).</p

Carbon monoxide effect in hippocampus after perinatal hypoxia-ischemia – apoptotic profiles.

<p>Whereas contralateral hippocampus displayed a preserved morphology (<b>A</b>) following HI, diffuse tissue disruption was detected in the hippocampus ipsilateral to the occlusion (<b>B</b>). C–E are representative pictures of ischemic hippocampus, where diffuse apoptosis was documented; with peculiar morphological features including pyknotic nuclei (<b>C</b>), indicating early stage of apoptosis, progressive nuclear fragmentation (<b>D</b>) and karyorrhexis as confirmed by detectable apoptotic bodies (<b>E</b>). Compared to HI group, the number of apoptotic profiles was significantly lower when animals were exposed to CO prior to HI (<b>F</b>). All values are mean ± SD (error bars); *<i>p</i><0.05 compared to Control group for the corresponding side and **<i>p</i><0.05 compared to HI group ischemic hippocampus. (<b>G</b>) For each group there is no significant difference in cytotoxic edema volume (mm³) between the ipsi- and the contralateral hippocampus.</p

The effect of ΔG⁰_vp7,13-mer on RLP assembly efficiency when the initial protein concentration of vp2, vp6 and vp7 is 1.2 M, 7.8 M and 7.8 M, respectively, and the Gibbs free energy of vp2 and vp6 subunit association (ΔG⁰_vp2,3-mer and ΔG⁰_vp6,13-mer, respectively) is equal to −4.08 kcal.mol⁻¹.

<p><b>ΔG⁰_vp7,13-mer</b> varies between −4.08×[0.05 to 1.4] kcal.mol⁻¹.</p

Half maximal inhibitory concentrations (IC50) of MDCK-E1 and MDCK-E1-Cre cell clones in response to increasing concentrations of <i>tert</i>-butyl hydroperoxide (<i>t</i>-BHP) (causing oxidative stress injury) in the culture medium.

<p>MDCK-E1#121 and MDCK-E1#106 represent high and low E1B expression, while MDCK-E1#106-Cre#10 and MDCK-E1#106-Cre#19 represent high and low Cre-activity, respectively. Error bars correspond to standard-deviation of quadruplicate assays. *<i>p</i><4×10⁻⁵, **<i>p</i><0.05, ***p<0.04, indicating the significance of a single factor Anova analysis.</p

gB peptides identified by MS/MS.

1<p>start and end amino acids as by conceptual translation of HCMV UL55 ORF.</p>2<p>Confidence Interval.</p