Immunological evidence that anemone repair proteins include replacement linkages.

In response to damage to hair bundles caused by exposure to calcium free buffers, sea anemones secrete large protein complexes named 'repair proteins' that rapidly restore structural integrity and function to hair bundles. A specific chromatographic fraction of the repair protein mixture, named 'fraction beta', has biological activity comparable to the complete repair protein mixture (Watson et al., 1998, Hear. Res. 115, 119-128). In this study, we find that polyclonal antibodies raised against deglycosylated fraction beta specifically bind fraction beta on Western blots. Anti-fraction beta delays the normal recovery of vibration sensitivity in experimental animals (i.e., those with hair bundles damaged by calcium free buffers). Moreover, anti-fraction beta disrupts vibration sensitivity in control animals (i.e., those with healthy hair bundles). Experimentally damaged hair bundles subsequently exposed to repair protein and then processed for immunoelectron microscopy show labeled linkages interconnecting stereocilia of the hair bundle. Immunofluorescence microscopy confirms strong labeling of hair bundles treated with repair proteins and only weak labeling of tips of hair bundles from control animals. Immunofluorescence microscopy indicates stores of repair proteins in gland cells of the body column in control animals and in gland cells of the mouth in experimental animals. Repair biological activity is confirmed in column purified homogenates of these tissues. Apparently repair proteins are delivered to damaged hair bundles in mucus carried by beating cilia.

Gap junctional communication in the vibration-sensitive response of sea anemones.

Although gap junctions occur in auditory and vestibular systems, their function is unclear. Here we present evidence for gap junctional communication in transmitting mechanosensory signals in a sea anemone model system. Hair bundles on anemone tentacles are vibration-sensitive mechanoreceptors that regulate discharge of nematocyst from effector cells. We find that vibration-dependent nematocyst discharge is selectively and reversibly blocked by the gap junction uncouplers, heptanol and arachidonic acid. Epidermal cells within excised tentacles exhibit a low level of dye coupling which is significantly enhanced upon deflection of overlying hair bundles. Dye coupling is inhibited both by gap junction uncouplers and by agents that interfere with mechanotransduction, including streptomycin and elastase. Electrophysiological data suggest gap junctional communication between cells giving rise to different hair bundles. When hair bundles are stimulated with a sweep of vibrations, individual cells show responses to five to eight frequencies. The number of responsive frequencies is reduced to one or two by heptanol and essentially abolished with streptomycin treatment. Immunoreactivity to the gap junction protein, connexin 43, is abundant in the tentacle epidermis and localized to membranes at junctions between several cell types. Small areas of close membrane apposition are observed between these cell types with intermembrane clefts of 4-7 nm. Of the several membrane proteins isolated from tentacles, immunoreactivity to connexin 43 is observed in a single band with an apparent molecular weight of approximately 46 kDa.

Evaluating chiral separation interactions by use of diastereomeric polymeric dipeptide surfactants.

Poly sodium N-undecyl leucine-leucine (poly SULL) is used as a diagnostic tool to investigate chiral molecular interactions via electrokinetic chromatography (EKC). Poly SULL has two chiral centers which are defined by two asymmetric carbons. Each chiral center of poly SULL can have two possible configurations (D or L). Consequently, four different optical configurations are possible within the surfactant molecule (L-L, D-D, L-D, and D-L). In this study, five chiral analytes of various charge states and hydrophobicities were used to investigate the role of electrostatic interactions and hydrophobicity on chiral recognition with polymeric dipeptide surfactants. These studies lead to a proposed hypothesis for interaction of the analytes with dipeptide surfactants. The hypothesis was tested and the contribution of the double chiral centers to this interaction was evaluated by use of two dipeptide surfactants in which one chiral amino acid is replaced by an achiral amino acid glycine, i.e., poly sodium N-undecyl L-leucine-glycine (poly L-SULG) and poly sodium N-undecyl L-glycine-leucine (poly L-SUGL). The results reported here provide new insights into the mechanism for chiral recognition of select chiral analytes by use of polymeric chiral surfactants.

Amino acid order in polymeric dipeptide surfactants: effect on physical properties and enantioselectivity.

The effect of amino acid order on chiral selectivity in polymeric dipeptide surfactants, as well as the physical properties of the surfactants, is investigated. An understanding of enantioselectivity of such dipeptide surfactants is crucial to the design of more efficient polymeric surfactants and has implications in other areas of research such as enantioselective interactions of amino acid based compounds (i.e., enzymes, hemoglobin, antibodies, etc.). It should be noted that such polymeric surfactants are not easily crystallized. Therefore, in a manner similar to the study of proteins, fluorescence spectroscopy is a powerful tool used to study the structure-function relationship of these polymeric surfactants. The microenvironments inside the core of 18 polymeric surfactants were characterized using the environmentally sensitive probes pyrene and 6-propionyl-2-(dimethylamino)naphthalene (Prodan). The surfactants examined in this study include all possible dipeptide combinations of the L-form of alanine, valine, and leucine and the achiral amino acid glycine (except glycine-glycine) as well as the single amino acid surfactants of alanine, valine, and leucine. The results of the fluorescent probe studies led to a proposed structure of the polymeric dipeptide surfactants in solution. The implications of the proposed structure for chiral selectivity were tested with two model atropisomers, (+/-)1,1'-bi-2-naphthol and (+/-)1,1'-bi-2-naphthyl-2,2'-diyl hydrogen phosphate, using capillary electrokinetic chromatography.

Chiral separations using dipeptide polymerized surfactants: effect of amino acid order.

Chiral separations using various polymerized dipeptide surfactants in electrokinetic capillary chromatography (EKC) are investigated. The two main dipeptide surfactants used in this study were sodium N-undecylenyl-L-valine-L-leucine (L-SUVL), and sodium N-undecylenyl-L-leucine-L-valine (L-SULV). These studies were performed in order to determine if the order of amino acids in dipeptide surfactants is important in terms of chiral recognition and separations. Both the monomer and the polymer of these two surfactants were compared for the separation of two model atropisomers, (+/-)-1,1-bi-2-naphtol (BOH) and (+/-)-1,1'-bi-2-naphthyl-2,2'-diyl hydrogen phosphate (BNP). Some advantages and disadvantages of the polymer relative to the monomer are discussed. Four other surfactants, the polymers of sodium N-undecylenyl-L-leucine-L-leucine (L-SULL), sodium N-undecylenyl-L-valine-L-valine (L-SUVV), sodium N-undecylenyl-L-valine (L-SUV), and sodium N-undecylenyl-L-leucine (L-SUL), were also used in this study, and their performance was compared to that of poly(L-SULV). These data show conclusively that the order of amino acids in dipeptide surfactants has a dramatic effect on chiral recognition. Our investigations indicate that poly-(L-SULV) provides the best enantioselectivity among the four dipeptide and two single amino acid surfactants for the separation of BNP and BOH. The advantages of poly-(L-SULV) are demonstrated via the ultrafast separation of the enantiomers of BNP and BOH in less than 1 min.

Genetic dissection of the biochemical activities of RecBCD enzyme.

RecBCD enzyme of Escherichia coli is required for the major pathway of homologous recombination following conjugation. The enzyme has an ATP-dependent DNA unwinding activity, ATP-dependent single-stranded (ss) and double-stranded (ds) DNA exonuclease activities, and an activity that makes a ss DNA endonucleolytic cut near Chi sites. We have isolated and characterized ten mutations that reduced recombination proficiency and inactivated some, but not all, activities of RecBCD enzyme. One class of mutants had weak ds DNA exonuclease activity and lacked Chi-dependent DNA cleavage activity, a second class lacked only Chi-dependent DNA cleavage activity, and a third class retained all activities tested. The properties of these mutants indicate that the DNA unwinding and ss DNA exonuclease activities of the RecBCD enzyme are not sufficient for recombination. Furthermore, they suggest that the Chi-dependent DNA cleavage activity or another, as yet unidentified activity or both are required for recombination. The roles of the RecBCD enzymatic activities in recombination and exclusion of foreign DNA are discussed in light of the properties of these and other recBCD mutations.