Cloning and expression of the bioluminescent photoprotein pholasin from the bivalve mollusc Pholas dactylus.

Pholasin is the photoprotein responsible for luminescence in the bivalve Pholas dactylus and consists of a luciferin tightly bound to a glycosylated protein. It is a sensitive indicator of reactive oxygen species. A full-length clone encoding apopholasin was isolated from a P. dactylus light organ cDNA library. The unprocessed apoprotein contained 225 amino acids, starting with a signal peptide of 20 amino acids, 3 predicted N-linked glycosylation sites, 1 O-linked site, no histidines, and 7 cysteines. The recombinant apoprotein was expressed in cell extracts and insect cells. The size of the apoprotein expressed in cell extracts and the cytosol of insect cells was 26 kDa but that of the fully processed protein was 34 kDa, as was native pholasin. Both the processed and unprocessed recombinant apoproteins were recognized by a polyclonal antibody raised against native pholasin. Acid methanol extracts from Pholas added to recombinant apoprotein resulted in chemiluminescence triggered by sodium hypochlorite but not photoprotein formation. These results have important implications in understanding the molecular evolution of bioluminescence and will allow the development of recombinant pholasin as an intracellular indicator of reactive oxygen species.

A technique for determining convergence in human subjects undergoing rotational acceleration using a binocular eye-tracking system.

BACKGROUND: This report describes the measurement of convergent eye movements and calculation of the spatial convergence point from the angular eye position data. These measurements were made in the dark while the subject experienced inertial motion aboard a centrifuge. This was an exploratory experiment with the goal of evaluating the eyes' convergence in the dark, and to see if this convergence point is dependent on inertial motion. METHODS: The subject was rotated in the dark on NAMRL's Coriolis Acceleration Platform in Pensacola, FL. The pupil positions were tracked by two helmet-mounted infrared cameras connected to a computer-controlled data acquisition system. We used the position data to calculate the angles through which the eyes rotated, and then applied trigonometric principles to construct the line of sight for each eye for any instant in time. The intersection of these two lines of sight was the convergence point. RESULTS: With the binocular eye-tracking system, we could accurately determine an accelerating subject's convergence point to within 10%, if the point was less than 1.5 m away from the subject. At convergence distances greater than 1.5 m, the angular movements of the two eyes became so small that determining a convergence point was difficult.