PMC

Molecular properties of purified Drosophila Rh7. (A–D) Absorption spectra of Rh7-Cap purified after it was regenerated with 11-cis-retinal (A,B) or 11-cis-3-hydroxyretinal (C,D). (A,C) Spectra were measured in the dark (black curve), after UV light irradiation (red curve), after subsequent yellow light (>500 nm) irradiation (green curve), after UV light re-irradiation (blue curve), and after yellow light re-irradiation (violet curve). (B,D) Difference spectra calculated based on the spectra in (A) or (C). The red curve was calculated by subtracting the spectrum before from that after UV light irradiation. The green curve was calculated by subtracting the spectrum after UV light irradiation from that after subsequent yellow light irradiation. The blue curve was calculated by subtracting the spectrum after yellow light irradiation from that after UV light re-irradiation. The violet curve was calculated by subtracting the spectrum after UV light re-irradiation from that after yellow light re-irradiation. The inset of (D) shows a comparison of the spectral properties of Rh7-Cap reconstituted with 11-cis-retinal (black curve) and 11-cis-3-hydroxyretinal (red curve). Difference spectra caused by UV light irradiation shown by red curve in (B) and (D) were normalized to be ~1.0 at the positive maxima. (E) Light-dependent retinal configuration changes of 11-cis-retinal bound Rh7-Cap. Left-hand panel, the retinal configurations were analyzed with HPLC after extraction of the chromophore as retinal oximes (syn and anti forms of 11-cis-, 13-cis-, and all-trans-retinal oximes). Right-hand panel, isomeric compositions of retinal before and after light irradiation. (F) Absorption spectra of Rh7-Cap purified after it was regenerated with all-trans-retinal. Spectra were measured in the dark (black curve), after yellow light (>500 nm) irradiation (red curve), and after subsequent UV light irradiation (blue curve). Inset, Difference spectra calculated by subtracting the spectrum before from that after yellow light irradiation (red curve) and the spectrum after yellow light irradiation from that after subsequent UV light irradiation (blue curve). (G) Time courses of G protein activation ability of Rh7-Cap reconstituted with 11-cis-retinal. Gq activation abilities were measured in the dark (black line), after UV light irradiation (red line), and after subsequent yellow light irradiation (green line). Data are presented as the means ± S.E. of three independent experiments.

Absorption spectra, retinal configurations and G protein activation of cOpn5L2. A, Absorption spectra of cOpn5L2 purified after incubation with 11-cis-retinal. Spectra were recorded in the dark (curve 1), after UV light irradiation (curve 2), after subsequent yellow light (>500 nm) irradiation (curve 3), and after UV light re-irradiation (curve 4). (inset) The calculated absorption spectra of cOpn5L2 in the dark (curve 1) and after UV light irradiation (curve 2). The calculation procedures are described in the text. B, Spectral changes caused by UV light irradiation (curve 1), subsequent yellow light irradiation (curve 2), and UV light re-irradiation (curve 3). C, Absorption spectra of cOpn5L2 purified after incubation with all-trans-retinal. Spectra were recorded in the dark (curve 1), after yellow light irradiation (curve 2), after subsequent UV light irradiation (curve 3), after yellow light re-irradiation (curve 4), and after UV light re-irradiation (curve 5). D, Spectral changes caused by yellow light irradiation (curve 1), subsequent UV light irradiation (curve 2), yellow light re-irradiation (curve 3), and UV light re-irradiation (curve 4). E, Retinal configurations in cOpn5L2 purified after incubation with all-trans-retinal. (left-hand panel) The retinal isomers before irradiation, after yellow light irradiation, and after subsequent UV light irradiation were analyzed with HPLC after extraction of the chromophore as retinal oximes (syn and anti forms of 9-cis-, 11-cis-, 13-cis-, and all-trans-retinal oximes). (right-hand panel) Isomeric compositions of retinal before and after light irradiation of cOpn5L2. F, Gi-type G protein activation ability by cOpn5L2 purified after incubation with 11-cis-retinal. The time-dependent change of the activity was measured in the dark (open circle), after UV light irradiation (open square), after subsequent yellow light irradiation (open triangle), and after UV light re-irradiation (open diamond). G, The Gi activation ability of cOpn5L2 purified after incubation with all-trans-retinal. The activity was measured in the dark (open circle), after yellow light irradiation (open square), after subsequent UV light irradiation (open triangle), and after yellow light re-irradiation (open diamond). G protein activation assay in (F) and (G) was performed at 0°C, and data are presented as the means ± S.D. of three independent experiments.

In vivo antitumor effects of T–IR700-mediated NIR light irradiation and T–DM1–IR700-mediated NIR light irradiation. (A) Large-tumor experiments: strong antitumor effects were observed in the groups of T–IR700 plus NIR light irradiation and T–DM1–IR700 plus NIR light irradiation compared to the untreated control group. T–DM1–IR700 plus NIR light irradiation treatment tended to reduce the tumor volume compared to T–IR700 plus NIR light treatment. Data are presented as means ± SEM (n = 10 in each group, 11 days after initial treatment; ***, P = 0.001: T–IR700 plus NIR light irradiation vs untreated control; ****, P < 0.0001: T–DM1–IR700 plus NIR light irradiation vs untreated control; Mann–Whitney U test). (B) Large-tumor experiments: survival was prolonged significantly when mice were treated with T–DM1–IR700 plus NIR light irradiation compared to T–IR700 plus NIR light irradiation. (n = 10 in each group; **, P = 0.0077: T–DM1–IR700 plus NIR light irradiation vs T–IR700 plus NIR light irradiation; log-rank test). (C) Small-tumor experiments: strong antitumor effects were observed in the groups of T–IR700 plus NIR light irradiation and T–DM1–IR700 plus NIR light irradiation compared to the untreated control group. There was no difference in the tumor volume and no significant difference in survival between the 2 NIR-PIT groups. Data are presented as means ± SEM (n = 10 in each group, 11 days after initial treatment; ****, P < 0.0001: T–IR700 plus NIR light irradiation vs untreated control; ****, P < 0.0001: T–DM1–IR700 plus NIR light irradiation vs untreated control; Mann–Whitney U test). (D) Small-tumor experiments: survival was prolonged significantly when mice were treated with T–IR700 plus NIR light irradiation and T–DM1–IR700 plus NIR light irradiation. However, there was no significant difference in survival between the 2 NIR-PIT groups (n = 10 in each group; log-rank test).

Absorption spectra of wild-type (A) or G188C mutant (B) of bovine rhodopsin purified after the incubation with 11-cis retinal at 0°C. Spectra were recorded in the dark (curve 1), after yellow light (>500 nm) irradiation (curve 2), after subsequent UV light (360 nm) irradiation (curve 3) and after yellow light reirradiation (curve 4). (Inset) Spectral changes of wild-type (A) or G188C mutant (B) induced by yellow light irradiation (curve 1), subsequent UV light (curve 2) irradiation and yellow light reirradiation (curve 3). Difference spectra were calculated based on the spectra shown in (A) and (B). Isomeric compositions of retinal of wild-type (C) and G188C mutant (D). The retinal configurations were analyzed by high-performance liquid chromatography (HPLC) after extraction of the chromophore from the samples before light irradiation, after yellow light irradiation, after subsequent UV light irradiation and after yellow light reirradiation at 0°C as shown in . (E) Gi-type of G protein activation ability of wild-type. The activation ability was measured in the dark (closed circle) and after yellow light irradiation (open circle). (F) Gi-type of G protein activation ability of G188C mutant. The activation ability was measured in the dark (closed circles), after yellow light irradiation (open circles), after subsequent UV light irradiation (open triangles) and after yellow light reirradiation (open diamonds). Data shown in (E) and (F) were obtained at 0°C and are presented as the means ± SEM of three independent experiments. (G) Absorption spectrum of G188C/N2C/D282C mutant purified after incubation with 11-cis retinal at 0°C. Spectra were recorded in the dark (curve 1), after yellow light (>500 nm) irradiation (curve 2), after subsequent UV light (360 nm) irradiation (curve 3), after yellow light reirradiation (curve 4) and after UV light reirradiation (curve 5). (Inset) Spectral changes induced by yellow light irradiation (curve 1), subsequent UV light (curve 2) irradiation, yellow light reirradiation (curve 3), and UV light reirradiation (curve 4). Difference spectra were calculated based on the spectra shown in (G).