Binding of iodomercurates to sulfhydryl-blocked beta-lactoglobulin-A, -B, and -C

Sulfhydryl-blocked beta-lactoglobulins (beta-LG-S-SCH2CH2OH)-A, -B, and -C bind only one iodomercurate species, HgI3-, at only one site, with a dissociation constant of 4.0 X 10(-5) M at 25 degrees, pH 5.0, 0.10 ionic strength. (Binding to native beta-LG-SH-A, -B, and -C is more complex, involving the sulfhydryl and two other sites and several iodomercurates.) The red shift of the HgI3- spectrum on binding would ordinarily suggest a hydrophobic site, but the HgI3- site is distinct from, and independent of, the alkane-binding site of native and blocked beta-LG; HgI3- may bind a group that shifts its trigonal planar structure toward the tetrahedron of HgI4(2-). Binding of HgI3- to blocked beta-LG interferes with the well-known association of beta-LG-A to octamers at pH 4.6 and low temperature. The relation of the HgI3- site to the crystallographic iodomercurate-binding sites of beta-LG-SH is examined. To facilitate these and future studies of iodomercurate binding, the 200-400 nm spectra of HgI2, HgI3-, and HgI4(2-) in aqueous solutions and the thermodynamic formation constants at 25 degrees for the equilibria HgI2 + I- = HgI3- (4.9 X 10(3) M-1) and HgI3- + I- = HgI4(2-) (0.118 X 10(3) M-1) were obtained.     

Maintenance of rat taste buds in primary culture

The differentiated taste bud is a complex end organ consisting of multiple cell types with various morphological, immunocytochemical and electrophysiological characteristics. Individual taste cells have a limited lifespan and are regularly replaced by a proliferative basal cell population. The specific factors contributing to the maintenance of a differentiated taste bud are largely unknown. Supporting isolated taste buds in culture would allow controlled investigation of factors relevant to taste bud survival. Here we describe the culture and maintenance of isolated rat taste buds at room temperature and at 37 degrees C. Differentiated taste buds can be sustained for up to 14 days at room temperature and for 3-4 days at 37 degrees C. Over these periods individual cells within the cultured buds maintain an elongated morphology. Further, the taste cells remain electrically excitable and retain various proteins indicative of a differentiated phenotype. Despite the apparent health of differentiated taste cells, cell division occurs for only a short period following plating, suggesting that proliferating cells in the taste bud are quickly affected by isolation and culture.     

Short-term changes of fish assemblages observed in the near-pristine reefs of the Phoenix Islands

Climate change-related disturbances are increasingly recognized as critical threats to biodiversity and species abundance. On coral reefs, climate disturbances have known consequences for reef fishes, but it is often difficult to isolate the effect of coral bleaching from preceding or simultaneous disturbances such as fishing, pollution, and habitat loss. In this study, pre-bleaching surveys of fish family assemblages in the remote Phoenix Islands in 2002 are compared to post-bleaching in 2005, following severe thermal stress. Post-bleaching, total coral cover decreased substantially, as did the combined abundance of all fish families. Yet, changes in abundance for specific fish families were not uniform, and varied greatly from site to site. Of the 13 fish families examined, 3 exhibited significant changes in abundance from 2002 to 2005, regardless of site (Carangidae, Chaetodontidae, and serranid subfamily Epinephelinae). For these families, we explored whether changes in abundance were related to island type (island vs atoll) and/or declining coral cover (percent change). Carangidae on islands experienced larger changes in abundance than those on atolls, though declines in abundance over time were not associated with changes in live coral cover. In contrast, for Chaetodontidae, declines in abundance over time were most dramatic on atolls, and were also associated with changes in live coral cover. The remoteness of the Phoenix Islands excludes many typical local anthropogenic stressors as drivers of short-term changes; observed changes are instead more likely attributed to natural variation in fish populations, or associated with coral loss following the 2002–2003 major thermal stress event.     

Distinct CDR3 conformations in TCRs determine the level of cross-reactivity for diverse antigens, but not the docking orientation

T cells are known to cross-react with diverse peptide MHC Ags through their alphabeta TCR. To explore the basis of such cross-reactivity, we examined the 2C TCR that recognizes two structurally distinct ligands, SIY-K(b) and alloantigen QL9-L(d). In this study we characterized the cross-reactivity of several high-affinity 2C TCR variants that contained mutations only in the CDR3alpha loop. Two of the TCR lost their ability to cross-react with the reciprocal ligand (SIY-K(b)), whereas another TCR (m67) maintained reactivity with both ligands. Crystal structures of four of the TCRs in complex with QL9-L(d) showed that CDR1, CDR2, and CDR3beta conformations and docking orientations were remarkably similar. Although the CDR3alpha loop of TCR m67 conferred a 2000-fold higher affinity for SIY-K(b), the TCR maintained the same docking angle on QL9-L(d) as the 2C TCR. Thus, CDR3alpha dictated the affinity and level of cross-reactivity, yet it did so without affecting the conserved docking orientation.     

Qualitative analysis of proteins rapidly transported in ventral horn motoneurons and bidirectionally from dorsal root ganglia

Two-dimensional electrophoresis has allowed a higher-resolution comparison of rapid transport in ventral horn motoneurons and bidirectionally in dorsal root sensory neurons. Dorsal root ganglia 8 and 9, or hemisected spinal cords, from frog were selectively exposed in vitro to 35S-methionine. Transported, labelled proteins that accumulated in 3 mm segments proximal to ligatures on dorsal roots and spinal nerves or sciatic nerves were subjected to two-dimensional gel electrophoresis. Comparisons were made of fluorographic patterns from dried gels. Sixty-five species of proteins were found to be rapidly transported in both bifurcations of dorsal root sensory neurons. No abundant species of protein was rapidly transported in dorsal roots that was not also found in spinal nerves. A comparison of proteins rapidly transported in the sciatic nerve from ventral horn motoneurons with those from dorsal root sensory neurons yielded 50 common species of polypeptides. At most four minor species were possibly transported only in ventral horn motoneurons. An overall comparison indicates that at least 45 species of proteins, including all of the more abundantly transported ones, were consistently common to both dorsal root bifuractions and to ventral horn motoneurons. This appears to be the case despite the very different functions carried out by motoneurons and sensory neurons.     

Actin surface structure revealed by antibody imprints: evaluation of phage-display analysis of anti-actin antibodies

Phage-display peptide library analysis of an anti-F actin polyclonal antibody identified 12 amino acid residues of actin that appear, in its X-ray crystal structure, to be grouped together in a surface accessible conformational epitope. Phage epitope mapping was carried out by isolating immune complexes containing members of the J404 nonapeptide phage-display library formed in diluted antiserum and isolated on a protein A affinity matrix. Immunoreactive clones were grown as plaques, replica plated onto nitrocellulose, and labeled with anti-actin immune serum. One hundred and forty-four positively staining clones identified in this way were sequenced. Of these, 54 displayed peptides with sequence similarities. When the most abundantly selected sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on Western blots and inhibited the binding of the antibody to immobilized F-actin by 60%. A scrambled version of this sequence WQDK WLQTQ, when coupled to ovalbumin, was not recognized by the antiserum and minimally inhibited binding of antiserum to immobilized F-actin by 10%. KQTWQQLWD contained four residues that corresponded, in frame, to a highly conserved six residue region of the chicken beta-actin sequence 351TFQQMW356 (identical residues are shown in bold). Examination of the rabbit skeletal muscle X-ray crystal structure suggested that within a 15 A radius of W356, nine additional residues were arranged on the actin surface in such a way that they could be mimicked by several of the selected phage sequences with root-mean-square deviation fits of 2.1-2.5 A. We conclude that phage-display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.     

Enzymatic properties of proteolytic derivatives of human alpha-thrombin

The use of derivatives of alpha-thrombin obtained by limited proteolysis, that have only a single peptide bond cleaved, allowed the unequivocal correlation between the change in covalent structure and alteration of the enzymatic properties. beta T-Thrombin contains a single cleavage in the surface loop corresponding to residues 65-83 of alpha-chymotrypsin [Birktoft, J. J., & Blow, D. M. (1972) J. Mol. Biol. 68, 187-240]. Compared with alpha-thrombin, this modification had a minor effect on the following: (1) The Michaelis constant (Km) for two tripeptidyl p-nitroanilide substrates increased 2-3 fold, whereas the catalytic constant (k cat) remained unaltered. (2) A 2-3 fold increase in the binding constant (KI) of a tripeptidyl chloromethane inhibitor was observed, but the inactivation rate constant (k i) was the same, which indicated that the nucleophilicity of the active-site histidyl residue had not changed. (3) The second-order rate constant for the inhibition by antithrombin III decreased 2-fold. Heparin accelerated the inactivation, and the degree of acceleration was similar to that obtained with alpha-thrombin. Pronounced effects of the cleavage of this loop were found. (1) The cleavage of fibrinogen was approximately 80-fold slower than that with alpha-thrombin. This was mainly due to a 40-fold decrease in k cat. In contrast, only a 1.9-fold increase in the Michaelis constant was observed. (2) The affinity for thrombomodulin had decreased 39-fold compared to alpha-thrombin. epsilon-Thrombin contains a single cleaved peptide bond in the loop corresponding to residues 146-150 in alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)