Treatment of Propionibacterium acnes endophthalmitis.

Propionibacterium acnes is a well-known cause of delayed endophthalmitis following cataract surgery. A white intracapsular plaque, keratic precipitates, and hypopyon may be present. Although there is no consensus on the treatment approach, recent studies have reviewed the outcomes of relatively large numbers of patients. Reasonable treatment options include pars plana vitrectomy and intra-ocular antibiotics with either partial or total capsulectomy/lens exchange. A good visual outcome is possible.

Opsin localization and rhodopsin photochemistry in a transgenic mouse model of retinitis pigmentosa.

The VPP mouse is a transgenic strain carrying three mutations (P23H, V20G, P27L) near the N-terminus of opsin, the apoprotein of rhodopsin, the rod photopigment. These animals exhibit a slowly progressive degeneration of the rod photoreceptors, and concomitant changes in retinal function that mimic those seen in humans with autosomal dominant retinitis pigmentosa resulting from a point mutation (P23H) in opsin. In the present study we attempted to determine whether the disease process prevents the translocation of mutant opsin to the rod outer segments of transgenic mice, and whether it affects the photochemical properties of the rhodopsin present within their rod outer segments. Immunocytochemistry with a monoclonal antibody against a region of the C-terminus that recognizes epitopes common to both normal and mutant opsin (monoclonal antibody-1D4), and a polyclonal antibody that reacts preferentially with the mutant opsin (anti-VPP), were used to identify the opsin present in the rods of three-week-old VPP mice and normal littermates. Absorbance spectra, photosensitivity, and regeneration kinetics of rhodopsin in rod outer segment disc membranes were analysed by spectrophotometry. Western blot analysis with anti-VPP antibody indicated the specific binding of this antibody to the mutant opsin. Immunolocalization with monoclonal antibody-1D4 and anti-VPP antibodies suggested a normal translocation of the mutant protein to the outer segments. Aside from a small disparity in the absorbance spectra of rhodopsin obtained from normal and VPP retinas, there were no significant differences in either the ability of opsin to bind 11-cis retinal chromophore, or in the photic sensitivity of rhodopsin. The results indicate that mutant opsin is translated and incorporated into the rod outer segment disc membranes of VPP mice, and that the photochemical properties of rhodopsin in the rods of VPP retinas are similar to those of rhodopsin in normal retinas.

Reduced light-dependent phosphorylation of an analog visual pigment containing 9-demethylretinal as its chromophore.

9-Demethyl rhodopsin (9dR), an analog of vertebrate rhodopsin, consists of opsin and a covalently attached chromophore of 11-cis 9-demethylretinal. Electrophysiological evidence that photoactivated 9dR (9dR*) undergoes abnormally slow deactivation in salamander rods (Corson, D. W., Cornwall, M. C., and Pepperberg, D. R. (1994) Visual Neurosci. 11, 91-98) raises the possibility that opsin phosphorylation, a reaction involved in visual pigment deactivation, operates abnormally on 9dR*. This possibility was tested by measuring the light-dependent phosphorylation of 9dR in preparations obtained from bovine rod outer segments. Outer segment membranes containing 9dR or regenerated rhodopsin were flash-illuminated in the presence of [gamma-32P]ATP and rhodopsin kinase, further incubated in darkness, and then analyzed for opsin-bound [32P]Pi. [32P]Pi incorporation by 9dR* increased with both incubation period and bleaching extent but, under all conditions tested, was less than that measured in rhodopsin controls. Results obtained with 30-s incubation periods indicated that the maximal initial rate of incorporation by 9dR* is about 25% of that by photoactivated rhodopsin. The results imply that the low incorporation of Pi by 9dR* results from a reduced rate of phosphorylation by rhodopsin kinase and are consistent with the prolonged lifetime of 9dR* determined electrophysiologically.

Regulation of retinal cGMP cascade by phosducin in bovine rod photoreceptor cells. Interaction of phosducin and transducin.

Photoexcitation of retinal rod photoreceptor cells involves the activation of cGMP enzyme cascade in which sequential activation of rhodopsin, transducin, and the cGMP phosphodiesterase in the rod outer segment constitutes the signal amplification mechanism. Phosducin, a 33-kDa phosphoprotein, has been shown to form a tight complex with the T beta gamma subunit of transducin. In this study, we examined the interaction of phosducin-T beta gamma and the possible regulatory role of phosducin on the cGMP cascade. Addition of phosducin to photolyzed rod outer segment (ROS) membrane reduced the GTP hydrolysis activity of transducin as well as the subsequent activation of the cGMP phosphodiesterase. Phosducin also inhibited the pertussis toxin-catalyzed ADP-ribosylation of transducin, indicating that the interaction between the T alpha and T beta gamma subunits of transducin was interrupted upon binding of phosducin. The inhibitory effects of phosducin were reversed by the addition of exogenous T beta gamma. These results suggest that phosducin is capable of regulating the amount of T beta gamma available to interact with T alpha to form the active transducin complex and thereby functions as a negative regulator of the cGMP cascade. The phosducin-induced alteration of the subunit organization of transducin was examined by chemical cross-linking method using para-phenyl dimaleimide as cross-linker. It was found that the cross-linking among T alpha and T beta gamma was blocked in the presence of phosducin. This result implies that T beta gamma may undergo a conformational change upon phosducin binding which leads to the release of T alpha. Since phosducin is a soluble protein, the interaction with transducin only occurs when transducin is dissociated from ROS disc membrane. Indeed, phosducin failed to dissociate membrane-bound transducin and did not inhibit the initial cycle of transducin activation as measured by the presteady state GTP hydrolysis. However, phosducin interacts effectively with transducin released into solution after the initial activation and blocks the re-binding of T alpha. T beta gamma to ROS membrane by forming a tight complex with T beta gamma. This interaction may play an important role in regulating the turnover of the cGMP cascade in photoreceptor cells.

Molecular mechanism of GTP hydrolysis by bovine transducin: pre-steady-state kinetic analyses.

During the visual transduction process in rod photoreceptor cells, transducin (T) mediates the flow of information from photoexcited rhodopsin (R*) to the cGMP phosphodiesterase (PDE) via a cycle of GTP binding and hydrolysis. The pre-steady-state kinetics of GTP hydrolysis by T was studied by rapid quenching and filtration techniques in a reconstituted system containing purified R* and T. Kinetic analyses have shown that the turnover of T-bound GTP can be dissected into four partial reactions: (1) the R*-catalyzed GTP binding via a GDP/GTP exchange reaction, (2) the on-site hydrolysis of bound GTP, which leads to the formation of a T-GDP.Pi complex, (3) the release of the tightly bound inorganic phosphate (Pi) from T-GDP.Pi, and (4) the recycling of T-GDP. The R*-catalyzed GTP binding was estimated to occur in less than 1 s. In rapid acid quenching experiments, the rate of Pi formation due to GTP hydrolysis exhibited biphasic characteristics. An initial burst of Pi formation occurred between 1 and 4 s, which was followed by a slow steady-state rate. Increasing T concentration yielded a proportional increase in the burst and steady-state rate. The addition of Gpp(NH)p decreased both parameters. D2O decreased the rise of the initial burst with a kinetic isotope effect of approximately 1.7 but has no effect on the steady-state rate of Pi formation. These results indicate that the burst represents the fast hydrolysis of GTP at the binding site of T, which results in the accumulation of T-GDP.Pi complexes. The steady-state rate represents the slow release of Pi. This finding was further supported by rapid filtration experiments that monitored the formation of free Pi in solution. An initial lag time in the formation of free Pi was observed before a steady-state rate was established, indicating that the initially formed Pi was tightly bound to T. Finally, the release of GDP from T-GDP.Pi was not detected. This suggests that another cycle of GTP exchange catalyzed by R* should occur before the release of bound GDP. The rate of Pi release from T-GDP.Pi was measured under single-turnover conditions and had a half life of approximately 20 s, which was identical with the rate of deactivation of the PDE due to GTP hydrolysis by T.(ABSTRACT TRUNCATED AT 400 WORDS)