Structure-activity relationship study of ProxyPhos chemosensors for the detection of proximal phosphorylation and other phosphate species.

Chemosensors for the detection of phosphate-containing biological species are in high need. Detection of proximally phosphorylated sites of PPi and those found in peptides and proteins has been demonstrated using chemosensors containing pyrene, as a fluorescent reporter, and a Zn2+-chelate, as a phosphate-binding group. Using these sensors, detection of proximal phosphate groups is afforded by binding of at least two of the sensor molecules to the adjacent phosphates, via the Zn2+ centres, leading to excimer formation between the pyrene groups and the corresponding shift in emission from 376 to 476 nm. Although several reports of this chemosensor class have been made, no detailed studies of selectivity of these sensors among major phosphate targets have been reported. In this study, a library of this class of chemosensors, termed ProxyPhos, which contained various linkers and Zn2+-chelating groups (i.e. DPA, cyclen and cyclam), was prepared and the effects of structural variation on the sensing efficiency and selectivity were evaluated among proximally phosphorylated peptides, proteins, nucleotides, Pi and PPi. As a result of this study, we have identified ProxyPhos library members that are most suitable for the detection of proximally phosphorylated peptides, PPi, UTP, and a DpYD peptide motif, and have generally provided a foundation for the selection of ProxyPhos chemosensors for further development of specific biologically relevant assays. The broad utility of ProxyPhos is further supported by the demonstrated lack of these sensors' cytotoxicity, ability to rapidly permeate into live and fixed cells and compatibility with gel staining methods.

Retinal transformation at metamorphosis in the winter flounder (Pseudopleuronectes americanus).

Winter flounder (Pseudopleuronectes americanus) are hatched as bilaterally symmetric larvae which live near the ocean surface. At metamorphosis, they become laterally compressed, one eye migrates to the opposite side of the head, and they live the remainder of their lives lying on their blind side on the ocean floor. The present study characterizes and quantifies retinal cell distribution throughout the larval period and contrasts it with the adult retina. Based on light- and electron-microscopic analyses, retinas of larval flounder contain only a single cone-like photoreceptor type, arranged in a hexagonal array. In contrast, after metamorphosis, the adult retina has three types of photoreceptors: rods, single cones, and double cones. Rod photoreceptors are numerous in the ventral retina and decrease in density dorsad. The cone photoreceptor density, in contrast to rods, is higher in the dorsal retina decreasing ventrad. Adult cone photoreceptors are arranged in a square mosaic with four double cones surrounding one single cone. The differences in larval and adult retinal morphology reflect the distinctly different habitat each occupies.

Photoreceptor spectral absorbance in larval and adult winter flounder (Pseudopleuronectes americanus).

The habitat occupied by larval winter flounder (Pseudopleuronectes americanus) differs considerably in light regime from that of the adult. To understand how the visual system has adapted to such changes, photoreceptor spectral absorbance was measured microspectrophotometrically in premetamorphic and postmetamorphic specimens of winter flounder. Before metamorphosis, larval flounder retinas contain only one kind of photoreceptor which is morphologically cone-like with peak absorbance at 519 nm. After metamorphosis, the adult retina has three types of photoreceptors: single cones, double cones, and rods. The visual pigment in single cones has a peak absorbance at lambda max = 457 nm, the double cones at lambda max = 531 and 547 nm, and the rod photoreceptors at lambda max = 506 nm. Double cones were morphologically identical, but the two members contained either different (531/547 nm) or identical pigments (531/531 nm). The latter type were found only in the dorsal retina. The measured spectral half-bandwidths (HBW) were typical of visual pigments with chromophores derived from vitamin A1 with the possible exception of the long-wavelength absorbing pigment in double cones which appeared slightly broader. Because the premetamorphic pigment absorbance has a different lambda max than those of the postmetamorphic pigments, different opsin genes must be expressed before and after metamorphosis.

Metamorphosis and fish vision.

Many species of fish exhibit metamorphosis in which dramatic external transformations occur as a consequence of coordinated changes in gene expression within an organism. Because postembryonic development and change appears to be the rule rather than the exception in teleost fish species, we view metamorphosis as one of many developmental strategies in fish which have continued plasticity as a common theme. Metamorphic changes are manifested in the visual system by modification of photoreceptor peak sensitivity, rod photoreceptor cell addition, and retinal reorganization. These changes correspond to significant changes in the natural habitat of the animal and in its visual capabilities as demonstrated behaviorally. Thyroxine is the main metamorphic hormone as has also been found in amphibia. The sequence of metamorphic events occur in all teleosts, but they are compressed in time in direct developing animals suggesting that such animals might prove useful for understanding the evolution of metamorphosis in fish. It seems likely that rod photoreceptors may have evolved in conjunction with the change from larval to juvenile stage through metamorphosis in indirect developing fishes. During evolution, the contraction and/or loss of the larval stage has resulted in earlier appearance of rod photoreceptors during development although they always arise later than cone photoreceptors. This ontogenetic developmental sequence supports Walls's (1942) proposal that cones are phylogenetically older than rods and suggests that rods may have evolved several times.

Correlation between histological and behavioral measures of visual acuity in a zooplanktivorous fish, the white crappie (Pomoxis annularis).

Estimates of visual acuity in a pelagic freshwater zooplanktivorous fish, the white crappie (Pomoxis annularis, Centrarchidae), were made using a behavioral measure, the maximum observed prey pursuit distance (MxPD), and a histological measure, the density of cone cells in the retina. The greatest number of pursuits occurs in the 0-30 degrees wedge of the visual field; 87% of all pursuits occur in the first 40 degrees. The longest pursuits (200 mm) also occur in this area and generally get shorter from 0 to 180 degrees (from forward-directed) in the visual field. Consistent with the behavioral results, the largest number of cone photoreceptors (13,000/mm2) is found in the far temporal retina along the eye's horizontal meridian. Cone cell densities in the corresponding region of the nasal retina are approximately half this value. The number of cones decreases dorsally and ventrally from the horizontal meridian. Although the absolute values of visual acuity calculated from cone cell topography (i.e. MxPDs of 500 mm) are 2-3 times greater than those observed behaviorally (i.e. MxPDs of 200 mm), the trends in visual acuity across the visual field obtained from both measures are consistent. We suggest that overestimates of visual acuity obtained from cone cell counts alone result from this measure's not accounting for, among other properties of the nervous system, cone cell convergence onto ganglion cells and higher brain centers. Behavioral measures of visual acuity are, therefore, likely to yield a more accurate estimate of an animal's visual abilities.

Flexible search tactics and efficient foraging in saltatory searching animals.

Foraging is one of the most important endeavors undertaken by animals, and it has been studied intensively from both mechanistic-empirical and optimal foraging perspectives. Planktivorous fish make excellent study organisms for foraging studies because they feed frequently and in a relatively simple environment. Most optimal foraging studies of planktivorous fish have focused, either on diet choice or habitat selection and have assumed that these animals used a cruise search foraging strategy. We have recently recognized that white crappie do not use a cruise search strategy (swimming continuously and searching constantly) while foraging on zooplankton but move in a stop and go pattern, searching only while paused. We have termed thissaltatory search. Many other animals move in a stop and go pattern while foraging, but none have been shown to search only while paused. Not only do white crappie search in a saltatory manner but the components of the search cycle change when feeding on prey of different size. When feeding on large prey these fish move further and faster after an unsuccessful search than when feeding on small prey. The fish also pause for a shorter period to search when feeding on large prey. To evaluate the efficiency of these alterations in the search cycle, a net energy gain simulation model was developed. The model computes the likelihood of locating 1 or 2 different size classes of zooplankton prey as a function of the volume of water scanned. The volume of new water searched is dependent upon the dimensions of the search volume and the length of the run. Energy costs for each component of the search cycle, and energy gained from the different sized prey, were assessed. The model predicts that short runs produce maximum net energy gains when crappie feed on small prey but predicts net energy gains will be maximized with longer runs when crappie feed on large prey or a mixed assemblage of large and small prey. There is an optimal run length due to high energy costs of unsuccessful search when runs are short and reveal little new water, and high energy costs of long runs when runs are lengthy. The model predicts that if the greater search times observed when crappie feed on small prey are assessed when they feed on a mixed diet of small and large prey, net energy gained is less than if small prey are deleted from the diet. We believe the model has considerable generality. Many animals are observed to move in a saltatory manner while foraging and some are thought to search only while stationary. Some birds and lizards are, known to modify the search cycle in a manner similar to white crappie.