Functional Demonstration of Connexin—Protein Binding Using Surface Plasmon Resonance

Surface plasmon resonance (SPR) allows examination of protein-protein interactions in real time, from which both binding affinities and kinetics can be directly determined. We have used the SPR technique to search for proteins in heart tissue that would be candidate binding partners for the cardiac gap junction protein, connexin43 (Cx43). Heart lysate showed a strong, pH-dependent binding to the carboxyl terminus (CT) of Cx43 (amino acids 254-382) covalently linked to an SPR cuvette. Binding was inhibited by the presence of v-src transfected 3T3 cell lysate, suggesting that binding partners in these two lysates may compete for overlapping epitopes on Cx43CT. The combined application of proteomic and functional studies is expected to identify which proteins within heart tissue interact with Cx43 and what roles they may play in gap junction function.     

Visualization of an Endophytic Streptomyces Species in Wheat Seed

Endophytic filamentous actinobacteria were isolated from surface-sterilized roots of wheat plants. Endophytic colonization of germinating wheat seed was examined using one of these endophytes, Streptomyces sp. strain EN27, tagged with the egfp gene. Endophytic colonization was observed from a very early stage of plant development with colonization of the embryo, endosperm, and emerging radicle.     

A quantitative system of age analysis for the dog-whelk, Nucella lapillus

Past attempts to age classify dog-whelks are revised briefly. The most satisfactory method is a system which relies upon familarity of morphological characteristics with which to separate population samples into five different classes. An attempt has been made to quantitate this system. Definite standards have been suggested as the criteria with which to separate the five age classes, and a series of simple methods have been demonstrated which, when followed give finite values for each of these standards. The standards suggested are: (i) the lower limits for shell-length in mature individuals; (ii) the lip-thickness coinciding with maturity; (iii) the upper limits for shell-length in juvenile crevice dwellers. The methods of estimate for values for these standards are illustrated in practice with population samples from Blackrock, Sussex. The values thus obtained and the methods used in each estimation are discussed with view to their validity. It is reasonable to suspect that the quantitative age classification system can be used to classify dog-whelks from any shore provided large enough population samples are collected to estimate the values for each standard. Use of the system is further demonstrated by classification of animals from the shore at Martin's Haven, Pembrokeshire.     

Dipole source localization by the mottled sculpin II. The role of lateral line excitation patterns

Extracellular, single unit recording techniques were used to measure the responses of posterior lateral line nerve fibers to a 50-Hz dipole source that slowly changed its location along the length of the fish. The flow-field equations for a dipole source were used to model the pressure gradient pattern and thus, the expected excitation pattern along a linear array of lateral line receptor organs for different source locations. Finally, excitation patterns were similarly modeled along the left and right side of the fish's head for actual steps taken by sculpin in approach pathways to the 50-Hz dipole source. Spatial histograms of posterior lateral line nerve fiber responses to different locations of the dipole source could be predicted from pressure gradient patterns modeled from the flow-field equations, confirming that the modeling approach applied to behavioral results was a good predictor of excitation patterns likely to be encoded by the lateral line periphery. An examination of how modeled excitation patterns changed from one position to the next in typical approach pathways and how patterns differed between positions from which successful and unsuccessful strikes were launched suggests that approach and strike strategies can indeed be explained by the information available in excitation patterns. In particular, changes in the spatial distribution of pressure gradient directions (polarities), available only when the source is lateral (as opposed to directly in front of the fish), appear to enhance the ability of sculpin to determine source distance. Without such information, misses are more likely to occur and successful strikes are more likely to be launched from short distances only. Accepted: 23 October 1996     

Density gradient fractionation by piston displacement.

A method for the fractionation of centrifuged density gradients is described. A piston forced into the centrifuge tube from above displaces the gradient, while its tip collects liquid from the small volume immediately below it without disturbing the unfractionated part of the gradient. The centrifuge tube holder permits sensitive visual detection of bands of light scattering particles which may then be rapidly and precisely extracted. Continuous fractionation of the entire gradient is also possible.     

New Findings on Evolution of Metal Homeostasis Genes: Evidence from Comparative Genome Analysis of Bacteria and Archaea

In order to examine the natural history of metal homeostasis genes in prokaryotes, open reading frames with homology to characterized P_IB-type ATPases from the genomes of 188 bacteria and 22 archaea were investigated. Major findings were as follows. First, a high diversity in N-terminal metal binding motifs was observed. These motifs were distributed throughout bacterial and archaeal lineages, suggesting multiple loss and acquisition events. Second, the CopA locus separated into two distinct phylogenetic clusters, CopA1, which contained ATPases with documented Cu(I) influx activity, and CopA2, which contained both efflux and influx transporters and spanned the entire diversity of the bacterial domain, suggesting that CopA2 is the ancestral locus. Finally, phylogentic incongruences between 16S rRNA and P_IB-type ATPase gene trees identified at least 14 instances of lateral gene transfer (LGT) that had occurred among diverse microbes. Results from bootstrapped supported nodes indicated that (i) a majority of the transfers occurred among proteobacteria, most likely due to the phylogenetic relatedness of these organisms, and (ii) gram-positive bacteria with low moles percent G+C were often involved in instances of LGT. These results, together with our earlier work on the occurrence of LGT in subsurface bacteria (J. M. Coombs and T. Barkay, Appl. Environ. Microbiol. 70:1698-1707, 2004), indicate that LGT has had a minor role in the evolution of P_IB-type ATPases, unlike other genes that specify survival in metal-stressed environments. This study demonstrates how examination of a specific locus across microbial genomes can contribute to the understanding of phenotypes that are critical to the interactions of microbes with their environment.