GFP is a selective non-linear optical sensor of electrophysiological processes in Caenorhabditis elegans.

Electrophysiology of the nematode Caenorhabditis elegans has the potential to bridge the wealth of information on the molecular biology and anatomy of this organism with the responses of selected cells and cellular neural networks associated with a behavioral response. In this paper we report that the nonlinear optical phenomenon of second harmonic generation (SHG) can be detected using green fluorescent protein (GFP) chimeras expressed in selected cells of living animals. Alterations in the SHG signal as a result of receptor ligand interactions and mechanical stimulation of the mechanosensory cells indicate that this signal is very sensitive to membrane potential. The results suggest that this approach to membrane potential measurements in C. elegans and in other biological systems could effectively couple data on selective locations within specific cells with functional responses that are associated with behavioral and sensory processes.

Near-field optical imaging of unstained bacteria: comparison with normal atomic force and far-field optical microscopy in air and aqueous media.

Simultaneous near-field scanning optical and atomic force imaging of bacteria is presented. The bacteria imaged in these studies were unstained. The near-field optical images had excellent signal-to-noise and showed excellent contrast even in these unstained specimens. The images obtained were interpreted in terms of the images that have been obtained by transmission electron microscopy and X-ray imaging. The results show that bacterial near-field optical imaging is going to be a very important tool in the arsenal of the bacteriologist both in terms of understanding the fundamental processes in the life cycle of bacteria with and without cytochemical staining and in terms of clinical diagnostic applications.

Active site lysine backbone undergoes conformational changes in the bacteriorhodopsin photocycle.

Results are presented demonstrating that the backbone of the active site lysine of bacteriorhodopsin undergoes light-induced structural alterations during bacteriorhodopsin-mediated light-induced proton pumping. This conclusion is based on difference Fourier transform infrared spectroscopy of isotopically labeled bacteriorhodopsin. The data demonstrate that the backbone carbonyl of lysine achieves an extremely low vibrational frequency during M412 intermediate formation. This is preceded by a structural transition in the lysine backbone that leads to an active site lysine carbonyl with the observed low vibrational frequency, probably due to a high degree of solvation.

Hydroxyl radical generation in the cat retina during reperfusion following ischemia.

There is increasing evidence that oxygen-derived free radicals are generated during the early phase of reperfusion, and account for part of the damage caused by transient ischemia in various tissues. To study this in the retina, cats were injected intravenously with sodium salicylate (100 mg kg-1), which reacts as a hydroxyl radical trap to form 2,3- and 2,5-dihydroxybenzoic acids (DHBA). Thirty minutes following injection, the retina of one eye of each animal was subjected to ischemia by intraocular pressure elevation via cannulation of the anterior chamber, while the fellow eye served as a sham-operated control. Ischemia was induced for 60 min (six eyes) and 90 min (eight eyes) followed by 5 min of reperfusion. In six other eyes, ischemia was induced for 90 min without reperfusion. After enucleation, the retinas were immediately removed, placed in ice-cold buffer and the retinal levels of 2,3- and 2,5-DHBA were quantitated by high pressure liquid chromatography, coupled with electrochemical detection. Results were normalized and expressed as ng DHBA microgram-1 salicylate mg-1 retinal protein. After 60 min of ischemia followed by reperfusion the normalized levels of 2,3- and 2-5-DHBA were no different in the experimental and control retinas. However, the levels of both 2,3- and 2,5-DHBA were significantly higher in the retinas subjected to 90 min ischemia followed by reperfusion than in the control tissues (P = 0.012 and P = 0.036, n = 8 respectively). Following 90 min ischemia without reperfusion, the normalized dihydroxybenzoate levels in the retinas were no higher than in their controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Participation of bacteriorhodopsin active-site lysine backbone in vibrations associated with retinal photochemistry.

Bacteriorhodopsin (bR) has been biosynthetically prepared with lysine deuterated at its alpha carbon (C alpha--H). The labeled membranes containing bR were investigated by difference Fourier transform infrared (FTIR) spectroscopy. It has been derived from K/bR and M/bR difference spectra (K and M are photocycle intermediates) that several bands previously assigned to the retinal chromophore are coupled to the C alpha--H. The vibrational modes that exhibit this coupling are principally associated with C15--H and N--H vibrations. [C alpha--2H]Lysine-labeled bR was fragmented enzymatically, and bR structures were regenerated with the C alpha--2H label either on lysine-216 and -172 or on the remaining five lysine residues of the protein. FTIR studies of the regenerated bR system, together with methylation of all lysines except the active-site lysine, reveal that the changes observed due to backbone labeling arise from the active-site lysine. The intensity of the C15--H out-of-plane wag is interpreted as a possible indication of a twist around the C15 = N bond.