2, ?,3,3, ?,4),4), we then evaluated the effects of long-term administration of 9- em cis /em -R-Ac. propane (pH 7.5) Nutlin carboxylic acid containing 500 mM NaCl and 0.1% dodecyl–maltoside at room temperature. The purified rhodopsin concentration was determined at 500 nm and the total amount of opsin and rhodopsin at 280 nm with a Hewlett-Packard 8452A UV-visible spectrophotometer. 25 The purity of eluted rhodopsin was evaluated by SDS-PAGE. Rhodopsin and isorhodopsin coeluted from the immunoaffinity 1D4-column. The vast majority of opsin molecules were regenerated with 11-an expanded scale of the chromatogram shows peaks 3 and 4 corresponding to levels Nutlin carboxylic acid of 0.01, one-way ANOVA) under both photopic and scotopic conditions except for a-waves noted under photopic conditions (Fig. 5A, B, C, D). Open in a separate window Open in a separate window Fig. 5 ERG responses of 10- and 14-month-old mice treated monthly with 9-The a- and b-wave amplitudes are plotted as a function of light intensity and error bars are shown (n = 10) and gray colored background areas represent responses from C0 mice. Compared to control 10-month-old mice (C1), responses of 10-month-old mice treated monthly for 6 months with 9- 0.001, one-way ANOVA). To indicate statistical differences between C0 and other plots, the C0 data are shown as a gray colored background area representing the C0 mean +/? 1 SD. Retinoid content in the eyes of each group was quantitatively evaluated at four time points during dark adaptation (0, 10, 30, and 60 min) after the 3 min bleach. The amount of regenerated 11-isomer. Analyses of rhodopsin, A2E and retinoid acids in control and long-term treated mice with 9-cis-R-Ac In long-term experiments, regeneration ratios (rhodopsin/opsin) and total amounts of purified rhodopsin showed no significant differences between control and treated groups of mice at both 10 and 14 months of age. Amounts of purified protein recovered from the treated groups at 14 months of age (N2 and N3) were significantly lower than those from control mice (C2), whereas the corresponding amounts were only slightly less in 10-month-old treated as compared to control mice (N1 versus C1) (data not shown). A2E and Magnified elution profiles of A2E and em iso /em -A2E are highlighted. Spectra of these peaks (I and II) represent A2E and em iso /em -A2E, respectively (top right). (B) Amounts of A2E (black bars) and em iso /em -A2E (gray bars) from different experimental groups are shown. Amounts of A2E did not differ significantly among all groups of mice with the exception of N3, where they were slightly decreased (p 0.05). em Iso /em -A2E levels were similar Rabbit Polyclonal to CRP1 among all groups. Neither compound was detected at significant levels in young 4-month-old untreated mice (C0). Means S.D. are indicated (n = 5), ND, not determined. Health Nutlin carboxylic acid status and retinal morphology of long-term treated micewith 9-cis-R-Ac Animals were evaluated weekly for activity and changes in coat and skin appearance. No changes in these parameters were observed during the experimental period aside from those due to natural aging. Body weights obtained before treatment and at 10 months and 14 months of age showed no significant differences between vehicle control (C1C2) and treated groups of mice (N1C3) (data not shown). Light microscopy revealed no major abnormalities in the retinas of vehicle, 9- em Nutlin carboxylic acid cis- /em R-Ac or all- em trans /em -R-Ac (n = 2) treated mice at 10 and 14 months of age. Moreover, retinas from the two 9- em cis- /em R-Ac treated groups were indistinguishable. Lengths of rod outer segments (ROS) were similar in control and treated groups at 10- and 14 months of age (Fig. 8B; n = 5) but were significantly decreased as compared to ROS lengths of 4-month-old mice (C0) whose retinal histology is shown in Fig. 8A. The thickness of each major layer in the retina also was similar in control and treated groups. TEM analysis of the outer retina and RPE layer revealed no gross differences between control and treated mice nor did higher resolution of the interface between the RPE and ROS reveal any Nutlin carboxylic acid abnormalities (data not shown). Open in a separate window Fig. 8 Retinal morphology of 9- em cis /em -R-Ac gavaged mice(A) A representative cross- section of retina from a 4-month-old untreated mouse (C0) is shown. RPE, retinal pigment epithelium; PR, photoreceptors, ROS, rod outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; and GCL, ganglion cell layer. (B) Thicknesses of the ROS and IS and numbers of ONL nuclei in retinas of control and 9- em cis- /em R-Ac treated mice of various ages are shown. Data points are plotted as a function of the distance from the optic nerve head (ONH). Lengths.
