Genomic organization and sequences of immunoglobulin light chain genes in a primitive vertebrate suggest coevolution of immunoglobulin gene organization.

The genomic organization and sequence of immunoglobulin light chain genes in Heterodontus francisci (horned shark), a phylogenetically primitive vertebrate, have been characterized. Light chain variable (VL) and joining (JI) segments are separated by 380 nucleotides and together with the single constant region exon (CI), occupy less than 2.7 kb, the closest linkage described thus far for a rearranging gene system. The VL segment is flanked by a characteristic recombination signal sequence possessing a 12 nucleotide spacer; the recombination signal sequence flanking the JL segment is 23 nucleotides. The VL genes, unlike heavy chain genes, possess a typical upstream regulatory octamer as well as conserved enhancer core sequences in the intervening sequence separating JL and CL. Restriction mapping and genomic Southern blotting are consistent with the presence of multiple light chain gene clusters. There appear to be considerably fewer light than heavy chain genes. Heavy and light chain clusters show no evidence of genomic linkage using field inversion gel electrophoresis. The findings of major differences in the organization and functional rearrangement properties of immunoglobulin genes in species representing different levels of vertebrate evolution, but consistent similarity in the organization of heavy and light chain genes within a species, suggests that these systems may be coevolving.     

Reduced G protein-coupled signaling efficiency in retinal rod outer segments in response to n-3 fatty acid deficiency

The fatty acid (FA) docosahexaenoic acid (DHA, 22: 6n-3) is highly enriched in membrane phospholipids of the central nervous system and retina. Loss of DHA because of n-3 FA deficiency leads to suboptimal function in learning, memory, olfactory-based discrimination, spatial learning, and visual acuity. G protein-coupled receptor (GPCR) signal transduction is a common signaling motif in these neuronal pathways. Here we investigated the effect of n-3 FA deficiency on GPCR signaling in retinal rod outer segment (ROS) membranes isolated from rats raised on n-3-adequate or -deficient diets. ROS membranes of second generation n-3 FA-deficient rats had approximately 80% less DHA than n-3-adequate rats. DHA was replaced by docosapentaenoic acid (22:5n-6), an n-6 FA. This replacement correlated with desensitization of visual signaling in n-3 FA-deficient ROS, as evidenced by reduced rhodopsin activation, rhodopsin-transducin (G(t)) coupling, cGMP phosphodiesterase activity, and slower formation of metarhodopsin II (MII) and the MII-G(t) complex relative to n-3 FA-adequate ROS. ROS membranes from n-3 FA-deficient rats exhibited a higher degree of phospholipid acyl chain order relative to n-3 FA-adequate rats. These findings reported here provide an explanation for the reduced amplitude and delayed response of the electroretinogram a-wave observed in n-3 FA deficiency in rodents and nonhuman primates. Because members of the GPCR family are widespread in signaling pathways in the nervous system, the effect of reduced GPCR signaling due to the loss of membrane DHA may serve as an explanation for the suboptimal neural signaling observed in n-3 FA deficiency.     

Novel Immune-type Receptor Genes and the Origins of Adaptive and Innate Immune Recognition

The prototypic forms of teleost novel immune-type receptors(NITRs) consist of a variable (V) region, a unique V-like C2(V/C2) domain, a transmembrane region and a cytoplasmic tailcontaining immunoreceptor tyrosine-based inhibition motifs (ITIMs).NITRs encode diversified V regions in large multigene familiesbut do not undergo somatic rearrangement. Studies in four differentbony fish model systems have identified a number of differentorganizational forms of NITRs. Specifically, NITR genes encodeN-terminal ectodomains of the V-type but otherwise vary in the:total number of extracellular immunoglobulin domains, numberand location of joining (J) region-like motifs, presence oftransmembrane regions, presence of charged residues within transmembraneregions, presence of cytoplasmic tails, and/or distributionof ITIM(s) within the cytoplasmic tails. V region-containingNITRs constitute a far more complex family than recognized originallyand currently include individual members that potentially functionthrough inhibitory as well as activating mechanisms. The genomicorganization of the NITR gene cluster as well as the structuraldiversity and overall architecture of the NITR proteins is reminiscentof genes encoded at the mammalian leukocyte receptor cluster(LRC); however, there presently is no functional evidence tosupport an orthologous relationship between NITR and LRC geneproducts. Comparisons of the predicted structures of the NITRshave identified several short regions of sequence identity anda novel cloning strategy has been devised that selects for secretoryand transmembrane proteins that encode these short motifs. Usingthis approach, related genes termed immune-type receptors (ITRs)have been identified in cartilaginous fish. Taken together,these studies indicate that leukocyte regulatory receptors,including those that mediate natural killer function, mighthave emerged early in vertebrate evolution and that the NITR/ITRgenes represent a new and potentially highly significant linkbetween innate and adaptive immune responses.     

Effect of cholesterol on molecular order and dynamics in highly polyunsaturated phospholipid bilayers.

The effect of cholesterol on phospholipid acyl chain packing in bilayers consisting of highly unsaturated acyl chains in the liquid crystalline phase was examined for a series of symmetrically and asymmetrically substituted phosphatidylcholines (PCs). The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic structural properties of bilayers containing 30 mol % cholesterol. The bilayers were composed of symmetrically substituted PCs with acyl chains of 14:0, 18:1n9, 20:4n6, or 22:6n3, containing 0, 1, 4, or 6 double bonds, respectively, and mixed-chain PCs with a saturated 16:0 sn-1 chain and 1, 4, or 6 double bonds in the sn-2 chain. DPH excited-state lifetime was fit to a Lorentzian lifetime distribution, the center of which was increased 1-2 ns by 30 mol % cholesterol relative to the cholesterol-free bilayers. Lifetime distributions were dramatically narrowed by the addition of cholesterol in all bilayers except the two consisting of dipolyunsaturated PCs. DPH anisotropy decay was interpreted in terms of the Brownian rotational diffusion model. The effect of cholesterol on both the perpendicular diffusion coefficient D perpendicular and the orientational distribution function f(theta) varied with acyl chain unsaturation. In all bilayers, except the two dipolyunsaturated PCs, 30 mol % cholesterol dramatically slowed DPH rotational motion and restricted DPH orientational freedom. The effect of cholesterol was especially diminished in di-22:6n3 PC, suggesting that this phospholipid may be particularly effective at promoting lateral domains, which are cholesterol-rich and unsaturation-rich, respectively. The results are discussed in terms of a model for lipid packing in membranes containing cholesterol and PCs with highly unsaturated acyl chains.     

A bioactive somatostatin analog without a type II' beta-turn: synthesis and conformational analysis in solution.

A cyclic somatostatin analog [structure: see text] (1) has been synthesized. Biological assays show that this compound has strong binding affinities to somatostatin hsst2 and hsst5 receptor subtypes (5.2 and 1.2 nM, respectively, and modest affinity to hsst4 (41.1 nM)). Our conformational analysis carried out in DMSO-d6 indicates that this compound exists as two structures arising from the trans and cis configurations of the peptide bond between Phe7 and N-alkylated Gly8. However, neither conformer exhibits a type II' beta-turn. This is the first report of a potent bioactive somatostatin analog that does not exhibit a type II' beta-turn in solution. Molecular dynamics simulations (500 ps) carried out at 300 K indicate that the backbone of compound 1 is more flexible than other cyclic somatostatin analogs formed by disulfide bonds.     

Light-activated calcium release from sonicated bovine retinal rod outer segment disks.

Calcium trapped within sonicated and resealed bovine rod outer segment disks is released upon light exposure with a stoichiometry of 0.75 +/- 0.05 calcium for each rhodopsin bleached. The amount of calcium liberated is proportional to the amount of bleaching in the range of 20 to 100% bleaching and is relatively insensitive to the internal trapped calcium concentration. The results are obtained using a flow system in which the disk membrane vesicles are adsorbed on glass particle supported by a filter. The external calcium is washed away and subsequent calcium release is monitored by collecting fractions of the effluent before, during, and after light exposure. Disks that are sonicated and allowed to reseal prior to incubation with 45Ca show no change in calcium efflux upon bleaching. The light-activated calcium release is also eliminated if disks sonicated in the presence of 45Ca are treated with a calcium ionophore prior to bleaching. The results demonstrate that the light-released calcium comes from the disks and not from the external disk surface. Lowering temperature to 3--4 degrees C surpresses the light-stimulated release, implicating a transition after the formation of metarhodopsin I in the transport process. The resluts suggest a model for the disk in which each bleached rhodopsin functions as a "one-shot carrier" to transport a single calcium ion across the membrane.