Different dark conformations function in color-sensitive photosignaling by the sensory rhodopsin I-HtrI complex.

The haloarchaeal phototaxis receptor sensory rhodopsin I (SRI) in complex with its transducer HtrI delivers an attractant signal from excitation with an orange photon and a repellent signal from a second near-UV photon excitation. Using a proteoliposome system with purified SRI in complex with its transducer HtrI, we identified by site-directed fluorescence labeling a site (Ser(155)) on SRI that is conformationally active in signal relay to HtrI. Using site-directed spin labeling of Ser(155)Cys with a nitroxide side chain, we detected a change in conformation following one-photon excitation such that the spin probe exhibits a splitting of the outer hyperfine extrema (2A'(zz)) significantly smaller than that of the electron paramagnetic resonance spectrum in the dark state. The dark conformations of five mutant complexes that do not discriminate between orange and near-UV excitation show shifts to lower or higher 2A'(zz) values correlated with the alterations in their motility behavior to one- and two-photon stimuli. These data are interpreted in terms of a model in which the dark complex is populated by two conformers in the wild type, one that inhibits the CheA kinase (A) and the other that activates it (R), shifted in the dark by mutations and shifted in the wild-type SRI-HtrI complex in opposite directions by one-photon and two-photon reactions.

Generalized fuzzy clustering for segmentation of multi-spectral magnetic resonance images.

An integrated approach for multi-spectral segmentation of MR images is presented. This method is based on the fuzzy c-means (FCM) and includes bias field correction and contextual constraints over spatial intensity distribution and accounts for the non-spherical cluster's shape in the feature space. The bias field is modeled as a linear combination of smooth polynomial basis functions for fast computation in the clustering iterations. Regularization terms for the neighborhood continuity of intensity are added into the FCM cost functions. To reduce the computational complexity, the contextual regularizations are separated from the clustering iterations. Since the feature space is not isotropic, distance measure adopted in Gustafson-Kessel (G-K) algorithm is used instead of the Euclidean distance, to account for the non-spherical shape of the clusters in the feature space. These algorithms are quantitatively evaluated on MR brain images using the similarity measures.

High-sensitivity rod photoreceptor input to the blue-yellow color opponent pathway in macaque retina.

Small bistratified cells (SBCs) in the primate retina carry a major blue-yellow opponent signal to the brain. We found that SBCs also carry signals from rod photoreceptors, with the same sign as S cone input. SBCs exhibited robust responses under low scotopic conditions. Physiological and anatomical experiments indicated that this rod input arose from the AII amacrine cell-mediated rod pathway. Rod and cone signals were both present in SBCs at mesopic light levels. These findings have three implications. First, more retinal circuits may multiplex rod and cone signals than were previously thought to, efficiently exploiting the limited number of optic nerve fibers. Second, signals from AII amacrine cells may diverge to most or all of the approximately 20 retinal ganglion cell types in the peripheral primate retina. Third, rod input to SBCs may be the substrate for behavioral biases toward perception of blue at mesopic light levels.

Volume and shape in feature space on adaptive FCM in MRI segmentation.

Intensity non-uniformity (bias field) correction, contextual constraints over spatial intensity distribution and non-spherical cluster's shape in the feature space are incorporated into the fuzzy c-means (FCM) for segmentation of three-dimensional multi-spectral MR images. The bias field is modeled by a linear combination of smooth polynomial basis functions for fast computation in the clustering iterations. Regularization terms for the neighborhood continuity of either intensity or membership are added into the FCM cost functions. Since the feature space is not isotropic, distance measures, other than the Euclidean distance, are used to account for the shape and volumetric effects of clusters in the feature space. The performance of segmentation is improved by combining the adaptive FCM scheme with the criteria used in Gustafson-Kessel (G-K) and Gath-Geva (G-G) algorithms through the inclusion of the cluster scatter measure. The performance of this integrated approach is quantitatively evaluated on normal MR brain images using the similarity measures. The improvement in the quality of segmentation obtained with our method is also demonstrated by comparing our results with those produced by FSL (FMRIB Software Library), a software package that is commonly used for tissue classification.

Molecular and biochemical analysis of the {\it Drosophila\/} segmentation gene {\it runt\/}

Genetic evidence has indicated that the segmentation gene runt plays a key role in regulating gene expression of the pair-rule genes hairy, even-skipped, and fushi tarazu. In contrast to other pair-rule genes, sequence data of the runt open reading frame did not reveal homologies to DNA-binding motifs of known transcriptional regulatory proteins. This thesis project examined several properties of the runt gene based on the sequence of the transcription unit, including the subcellular localization of the protein in vivo, its ability to bind DNA, and the functionality of a putative nucleotide binding domain.^ A runt-specific antibody was generated and used to demonstrate that runt is localized in the nucleus. Since the precise overlap of the pair-rule stripes is thought to be critical for the determination of cellular identity along the anterior-posterior axis, phasing of early runt expression in the blastoderm was examined with regard to the segmentation genes hairy, even-skipped, and fushi tarazu. runt was also expressed at later stages of embryogenesis, including expression in neuroblasts, and ganglion mother cells of the developing nervous system. Expression at this stage was required for the subsequent formation of specific neurons and runt was extensively expressed in the central and peripheral nervous systems.^ Several experiments were done to address the biochemical function of the runt protein. A direct interaction of runt with DNA was first examined. Although bacterial expressed runt was found to bind dsDNA-cellulose, subsequent experiments failed to detect sequence-specific interactions with DNA. Inter-species conservation of the putative nucleotide binding domain suggested that this region was functionally important, and runt protein bound a labeled ATP analog with high affinity in vitro. Finally, the effect of substitution of a critical residue of the nucleotide binding domain on runt activity was examined in vivo. Ectopic expression of the mutant protein indicated that this conserved substitution altered, but did not eliminate, runt activity as evaluated by segmentation phenotype and viability. ^