構築主義は規範をどこまで語ることができるのか？ : 政治的構築主義・節合・民主主義

2014-03-28departmental bulletin pape

HIF-1α protein during murine RSV pneumonia in vivo.

<p>Female, 6- to 8-week-old BALB/c mice were inoculated intranasally with purified RSV at 1×10⁷ plaque-forming units (PFUs), diluted in sterile 0.9 % sodium chloride for a total inoculation volume of 50 µl. As mock treatment, control mice were inoculated in the same way with an equivalent volume of sucrose diluted in 0.9 % sodium chloride. Lungs were removed at indicated time-points, and HIF-1α protein levels were determined by Western blot analysis (A) or quantified by densitometry, relative to beta-actin (B; *p<0.01).</p

Transcript levels of HIF-1-dependent genes following RSV infection.

<p>Total RNA was isolated from RSV-infected (multiplicity of infection, MOI 1, 3 and 5) or non-infected A549 cells (control) and (A) CD73, (B) VEGG, (C) FN1, (D) COX2 mRNA levels were determined by real-time RT-PCR. Data were calculated relative to internal housekeeping gene (β-actin) and are expressed as fold increase over uninfected control-cells ±SEM at each infection dose (*P<0.05, different from uninfected control-cells).</p

HIF-1α during RSV infected of pulmonary epithelia.

<p>(A) ∼1,5×10⁵ A549-cells were seeded on glass slides and infected with RSV (multiplicity of infection, MOI 3). After 24 h they were fixed, permeabilized and incubated with anti-HIF1α and anti-RSV IgG as primary antibodies. Alexa Fluor 488 and Alexa Fluor 594 were used for staining. In addition, slides were counter-stained with Dapi. The cells were visualized with confocal laser scanning microscopy. Uninfected cells were used as controls (B).</p

Protein levels of HIF-1-dependent genes following RSV infection.

<p>Total protein was isolated from RSV-infected (multiplicity of infection, MOI 1, 3 and 5) or non-infected A549 cells. Protein levels were determined by Western blot. The same blots were probed for β-actin expression as a control for protein loading. In addition, densitometric analysis of protein levels relative to β-actin were performed. Data are expressed as fold increase over uninfected control-cells ±SEM at each infection dose. (A) COX2; (B) FN1 (*<i>P</i><0.01, different from control, n  =  3).</p

Figure 7

<p>(a) <i>Measurements of oxygen partial pressures (pO₂) in the supernatants of RSV infected pulmonary epithelia.</i> (A) A549 cells were cultured and infected at a multiplicity of infection (MOI) of 1 or 5 in gas-tight sealed flasks. Oxygen partial pressure was measured in the supernatants at indicated time points following infection. (B) A samples was assessed for HIF-1α protein levels by Western blot 24 h after RSV infection or control conditions. Blots were probed for β-actin expression as a control for protein loading</p

HIF-1α protein measurements during RSV infection in vitro.

<p>(A, B) Cultured pulmonary epithelia (A549) were grown to 80% confluency, infected with intact (multiplicity of infection, MOI 1, 3 or 5) or UV-inactivated RSV (MOI 3). In other studies A549 cells were exposed over 24 h to ambient hypoxia (2% oxygen). Cells were lysed and nuclear proteins were isolated, and Western immunoblotting for RSV G-protein (A) or HIF1α was performed. Uninfected cells were used as control (Co). The same blots were probed for β-actin expression as a control for protein loading. A representative blot of 3 is shown, in addition to densitometric analysis of HIF-1α protein levels relative to β-actin (C;*<i>P</i><.01, different from control, n  =  3).</p

Transcript levels of HIF-dependent genes following HIF-1α siRNA repression during RSV infection.

<p>(A) HIF-1α protein levels in A549 cells following hairpin siRNA repression of HIF-1α (HIF-/-; A549 cells transfected with control siRNA:SCR). Cells were grown to 80% confluency and exposed to normoxia or hypoxia (2% oxygen) over indicated time period. Nuclear proteins were isolated and Western Blot analysis was performed for HIF-1α. The same blots were probed for β-actin expression as a control for protein loading. A representative blot of 3 is shown, in addition to densitometric analysis of HIF-1α protein levels relative to β-actin (B;*<i>P</i><0.01, different from control, n  =  3).(C, D, E) Total RNA was isolated from RSV-infected (multiplicity of infection, MOI 3) or non-infected A549 following HIF-1α repression (A549 HIF-/-) or transfection with control siRNA (A549 scr). (C) VEGF, (D) CD73, (E) FN1 transcript levels were determined by RT-PCR. Data were calculated relative to internal housekeeping gene (β-actin) and are expressed as fold increase over uninfected control-cells ±SEM (*P<0.05).</p

Transcript levels of HIF-dependent genes following infection with inactivated RSV.

<p>Total RNA was isolated from uninfected, RSV-infected or UV-inactivated RSV infected A549 cells. (A) VEGF, (B) CD73, (C) FN1, (D) COX2 transcript levels were determined by real-time RT-PCR. Data were calculated relative to internal housekeeping gene (β-actin) and are expressed as fold increase over uninfected control-cells ±SEM at each infection dose (*P<0.05, different from uninfected control-cells).</p

(A) Structures of BadA, Hia and YadA heads with the three domains colored according to the domain annotation from the alignment

The superimpositions of the individual domains from all three proteins are shown in the left panel. Note the different order of domains between Hia and BadA. In the BadA Trp-ring domain, 43 of 45 residues could be superimposed to the equivalent Hia domain with an r.m.s.d. of 2.02 Å, and in the GIN domain, 26 of 30 residues could be superimposed with an r.m.s.d. of 1.58 Å. In the BadA neck region, 19 residues could be superimposed to the YadA neck with an r.m.s.d. of 0.28 Å and to the Hia neck with an r.m.s.d. of 1.32 Å. All r.m.s.d. values refer to the C atoms. (B) Sequence alignment of the BadA head with other TAAs. The sequences of Hia and YadA are taken from the published structures; alignments based on these structures were used for homology modeling of the BadA head. The conserved residues that were used to name the domains are marked in bold. Abbreviations used: BhBadA – BadA g