This study describes regeneration of the neural retina in juvenile goldfish. The retina was destroyed with an intraocular injection of ouabain, a technique introduced by Wolburg and colleagues (Maier and Wolburg, 1979; Kurz-Isler and Wolburg, 1982). We confirmed their observation that the level of damage produced by the toxin was graded, in that neurons in the inner retinal layers were preferentially destroyed, and only in the more severely affected retinas were cells in the outer nuclear layer (i.e., photoreceptor cells) damaged. Evidence of retinal regeneration could be seen beginning about 2 weeks after the injection of ouabain. In contrast to previous studies (Maier and Wolburg, 1979), we found that regeneration took place only in those retinas in which photoreceptors had been destroyed. In cases in which the outer nuclear layer was spared, no regeneration of inner layers occurred, even after 6 months. Thymidine autoradiography was used to document the regeneration of new retinal neurons and to show that rod precursors, like other dividing cells, were not destroyed by the ouabain, but in contrast showed an increased mitotic activity. Regeneration did not proceed uniformly, but was initiated at neurogenic foci scattered across the retina. These foci consisted of clusters of dividing neuroepithelial-like cells. The evidence is consistent with the proposal that these cells were derived from rod precursors. These results imply that rod precursors are capable of a wider range of developmental fates than they normally express.

In photosystem II (PS II), chlorophyll, β-carotene, and cytochrome b 559 are alternate electron donors that may be involved in a photoprotection mechanism. The present study describes the use of high-field EPR spectroscopy to characterize the low-temperature photooxidation of Chl Z and Car cofactors in PS II. The EPR signals of the individual species, previously not resolved at X-band frequency (9 GHz), are resolved at higher D-band frequency (130 GHz) in deuterated Synechococcus liVidus PS II. Deuteration of PS II results in significant narrowing of the EPR lines, yielding well-resolved EPR spectra of the Car + and Chl Z + radicals at 130 GHz. The g tensors of the individual species were determined by EPR spectral simulations. The g tensor determined for the Car + radical (g xx ) 2.00335, g yy ) 2.00251, g zz ) 2.00227) is similar to that previously observed for a canthaxanthin cation radical but with a slightly rhombic tensor. The Chl Z + g tensor (g xx ) 2.00312, g yy ) 2.00263, g zz ) 2.00202) is similar to that of a chlorophyll a cation radical. This study shows that both the carotenoid and chlorophyll radicals are generated in PS II by illumination at temperatures from 6 to 190 K and that there is no interconversion of Car + and Chl Z + radicals upon dark annealing at temperatures up to 160 K. This study also establishes the feasibility of using deuteration and high-field EPR to resolve previously unresolvable cofactor signals in PS II.

The O 2 -evolving complex (OEC) of photosystem II (PSII) contains a tetramanganese (Mn 4 ) cluster, a redox-active tyrosine, and Ca 2+ /Clions, but its molecular structure has not been determined. Vibrational spectroscopy has the potential of providing new structural information for the OEC, particularly the Mn 4 cluster. Toward this goal, the vibrational characteristics of the OEC of PSII were examined using near-infrared (NIR) excitation Raman spectroscopy. NIR excitation decreases the background contribution from chlorophyll emission/ Raman scattering and affords the opportunity of probing selectively low-energy electronic transitions of the Mn 4 cluster. The primary emphasis of the Raman study was on the low-frequency range of the spectrum (220-620 cm -1 ) where Mn-ligand vibrational modes are expected to occur. The low-frequency region was examined for both the S 1 and S 2 oxidation states of the Mn 4 cluster. A particular effort was made to probe a NIR transition of the S 2 state that has been reported to mediate photoconversion from the multiline to the g ) 4.1 form of the S 2 state [Boussac et al. Biochemistry 1996, 35, 6984-6989]. The Raman studies revealed the following: (1) the Raman spectra of Mn-depleted PSII and PSII in the S 2 state are nearly identical; (2) the Raman spectrum of PSII in the S 1 state displays several unique low-frequency bands not present in the S 2 state that can be assigned as Mn-ligand vibrational modes and appear to maximize in intensity at λ ex ∼ 820 nm; and (3) several of the S 1 state Raman bands are shifted by D 2 O/H 2 O exchange. Collectively, these results indicate that the S 1 state of the Mn 4 cluster (1) has a NIR electronic transition from which resonance enhanced Raman scattering can be induced and ( 2) is coordinated by at least two H 2 O or OHgroups. The studies reported herein also demonstrate the potential of NIR-excitation Raman techniques for probing selectively the OEC in PSII and, in particular, for characterizing the coordination environment of the Mn 4 cluster.