Mutations in the rod outer segment membrane guanylate cyclase in a cone-rod dystrophy cause defects in calcium signaling.

Rod outer segment guanylate cyclase 1 (ROS-GC1) is a member of the subfamily of Ca(2+)-regulated membrane guanylate cyclases; and it is pivotal for vertebrate phototransduction. Two opposing regulatory modes control the activity of ROS-GC1. At nanomolar concentrations of Ca(2+), ROS-GC1 is activated by Ca(2+)-binding proteins named guanylate cyclase activating proteins (GCAPs). However, at micromolar concentrations of Ca(2+), ROS-GC1 is stimulated by S100beta [also named calcium-dependent (CD) GCAP]. This mode is not linked with phototransduction; instead, it is predicted to be involved in retinal synaptic activity. Two point mutations, E786D and R787C, in ROS-GC1 have been connected with cone-rod dystrophy (CORD6), with only one type of point mutation occurring in each family. The present study shows that the E786D mutation has no effect on the basal catalytic activity of ROS-GC1 and on its activation by GCAP1 and S100beta; however, the mutated cyclase becomes more activated by GCAP2. The R787C mutation has three consequences: (1) it causes major damage to the basal cyclase activity, (2) it makes the cyclase 5-fold more sensitive to activation by GCAP1; and 3) converts the cyclase into a form that is less sensitive to activation by GCAP2 and S100beta. Thus, the two CORD6-linked mutations in ROS-GC1, which occur at adjacent positions, result in vastly different biochemical phenotypes, and they are connected with very specific molecular defects in the Ca(2+) switching components of the cyclase. These defects, in turn, are proposed to have a profound effect on both the machinery of phototransduction and the retinal synapse. The study for the first time defines the biochemistry of CORD6 pathology in precise molecular terms.     

Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium

Many mutations in rpsL cause resistance to, or dependence on, streptomycin and are restrictive (hyperaccurate) in translation. Dependence on streptomycin and hyperaccuracy can each be reversed phenotypically by mutations in either rpsD or rpsE . Such compensatory mutations have been shown to have a ram phenotype (ribosomal ambiguity), increasing the level of translational errors. We have shown recently that restrictive rpsL alleles are also associated with a loss of virulence in Salmonella typhimurium . To test whether ram mutants could reverse this loss of virulence, we have isolated a set of rpsD alleles in Salmonella typhimurium . We found that the rpsD alleles restore the virulence of strains carrying restrictive rpsL alleles to a level close to that of the wild type. Unexpectedly, three out of seven mutant rpsD alleles tested have phenotypes typical of restrictive alleles of rpsL , being resistant to streptomycin and restrictive (hyperaccurate) in translation. These phenotypes have not been previously associated with the ribosomal protein S4. Furthermore, all seven rpsD alleles (four ram and three restrictive) can phenotypically reverse the hyperaccuracy associated with restrictive alleles of rpsL . This is the first demonstration that such compensations do not require that the compensating rpsD allele has a ribosomal ambiguity ( ram ) phenotype.     

Behavioral observations of codling moth, Cydia pomonella, in orchards permeated with synthetic pheromone

Mating disruption of codling moth, Cydia pomonella, was studied in apple orchards treated with the main pheromone compound codlemone, (E,E)-8,10-dodecadienol, and a blend of codlemone and codlemone acetate, (E,E)-8,10-dodecadienyl acetate, a strong pheromone antagonist. Codlemone alone and the pheromone/antagonist-blend had a similar effect on the behavior of males emerging into air-permeated orchards. Male flights within tree canopy and upwind orientation along tree rows were strongly enhanced by both formulations, compared to untreated plots. However, the codlemone/codlemone acetate-blend increased the rate of cross-wind and downwind flights within the orchard, compared to codlemone alone. The major difference between these two formulations was that males from nearby, untreated orchards were attracted towards orchards treated with codlemone, but not towards treatments with codlemone/codlemone acetate. Additional tests were done with an equilibrium blend of codlemone and its geometric isomers. Aerial pheromone concentrations in the orchards were recorded by the field electroantennogram technique. Decreasing pheromone concentrations towards the upper part of the tree canopy, together with the stimulation of male flight activity by synthetic pheromone explains failures to control codling moth at high population densities with current dispenser formulations.     

Biochemical and structural characterization of recombinant copper-metallothionein from Saccharomyces cerevisiae.

Methods were developed for large-scale purification of recombinant Cu-metallothionein (Cu-MT) for structural investigations and the determination of Cu-binding stoichiometry. Cu-MT of Saccharomyces cerevisiae overexpressed in Escherichia coli was purified using a procedure based on ion exchange and gel filtration chromatography followed by reversed-phase HPLC. The purified protein was fully characterized by electrophoresis, amino acid analysis, atomic absorption spectroscopy and elemental analysis, and was shown to contain 10 +/- 2 Cu(I) per molecule of protein. Small angle X-ray scattering measurements yielded a radius of gyration of 1.2 nm for the recombinant protein, indicating a more extended structure in solution than that derived from the recent NMR data [Peterson, C.W., Narula, S.S. & Armitage, I.A. (1996) FEBS Lett. 379, 85-93].     

The Origins of Hypertrophic Cardiomyopathy–Causing Mutations in Two South African Subpopulations: A Unique Profile of Both Independent and Founder Events

Hypertrophic cardiomyopathy (HCM) is an autosomal dominantly inherited disease of the cardiac sarcomere, caused by numerous mutations in genes encoding protein components of this structure. Mutation carriers are at risk of sudden cardiac death, mostly as adolescents or young adults. The reproductive disadvantage incurred may explain both the global occurrence of diverse independent HCM-associated mutations and the rare reports of founder effects within populations. We have investigated whether this holds true for two South African subpopulations, one of mixed ancestry and one of northern-European descent. Previously, we had detected three novel mutations-Ala797Thr in the beta-myosin heavy-chain gene (betaMHC), Arg92Trp in the cardiac troponin T gene (cTnT), and Arg645His in the myosin-binding protein C gene (MyBPC)-and two documented betaMHC mutations (Arg403Trp and Arg249Gln). Here we report three additional novel mutations-Gln499Lys in betaMHC and Val896Met and Deltac756 in MyBPC-and the documented betaMHC Arg719Gln mutation. Seven of the nine HCM-causing mutations arose independently; no conclusions can be drawn for the remaining two. However, the betaMHC Arg403Trp and Ala797Thr and cTnT Arg92Trp mutations were detected in another one, eight, and four probands, respectively, and haplotype analysis in families carrying these recurring mutations inferred their origin from three common ancestors. The milder phenotype of the betaMHC mutations may account for the presence of these founder effects, whereas population dynamics alone may have overridden the reproductive disadvantage incurred by the more lethal, cTnT Arg92Trp mutation.     

The three-for-one model for Gram-negative wall growth: a problem and a possible solution

The murein wall in Gram-negative bacteria is so thin that the mechanism of growth is necessarily complicated. From analytical data of murein components, Höltje suggested a model for the growth mechanism that would lead to safe wall enlargement. The model depended on the formation of trimers of peptidoglycan disaccharides linked via their pentapeptides. In the `three-for-one' model three oligopeptidoglycan chains are linked to each other in the usual linkages between the carboxyl group of d-alanine residues and the ϵ-amino group of diaminopimelic acid residues; these are designated `tail-to-tail' linkages. This three-chained raft is then linked to the stress-bearing wall via the formation of trimers, defined as three peptide chains linked together by tail-to-tail linkages. Then by autolyzing the oldest bonds in each trimer, the old chain is excised and the raft becomes part of the stress-bearing wall and the wall is enlarged. There is a problem with the three-for-one model in that it demands a precise fitting of the prefabricated raft of three crosslinked chains to a stress-bearing chain in the wall fabric to allow the series of trimer linkages to form. Because the wall, when bearing stress, must be pulled into a `honeycomb' structure, the end-to-end distance would be shortened. The possibility is raised here that the glycan chains in the stress-bearing wall are stretched to a sufficient degree by the cell's turgor pressure to compensate for its zig-zag structure; this could allow the model to function. A calculation is presented that assumes that the area of the pores in the fabric, called tessera, is maximized by the cell's turgor pressure. In this case the glycan chain must stretch 10% (and the end-to-end distance of peptide strands stretch 28%) so that the end-to-end distance of a glycan chain in the stress-bearing wall and the unstretched nascent wall can be the same and permit indefinite stable growth.     

On the Reversible Conjugation of [17-D2]GA20 in Seedlings of Phaseolus coccineus L.

After administering [17-D₂]GA₂₀ to Phaseolus coccineus L. cv. Preisgewinner seedlings, [17-D₂]GA₂₀-O-glucoside was identified by liquid chromatography (LC)/ESI-tandem mass spectrometry (MS). Likewise, by LC/ESI-tandem MS the metabolic formation of [17-D₂]GA₂₀ glucosyl ester was established. The application of both [17-D₂]-labeled GA₂₀ 13-O-glucoside and GA₂₀ glucosyl ester to Phaseolus coccineus L. seedlings resulted in free [17-D₂]GA₂₀ by gas chromatography/MS. The results demonstrate that conjugation of GA₂₀ and the reconversion of the glucosyl conjugates are concomitant processes in plants. Received October 27, 1998; accepted August 12, 1999     

Attachment of the chromosome to the cell poles: the strategy for the growth of bacteria in two and three dimensions.

Bacteria such as Staphylococcus, Lampropedia, and Sarcina develop in characteristic two-or three-dimensional groups of cells. We propose here a model of how bacteria may generate such groupings by an extension of an earlier model for rod-shaped bacteria. No other mechanism for forming two- or three-dimensional structures of groups of cells has been proposed. Our earlier model for division of rod-shaped bacteria into nearly equal-sized daughters assumed that the origin and terminus DNA were attached at a critical time to polar wall sites. While such binding was speculative 20 years ago, it has now been established that the DNA for the origin of replication, at least during some part of the cell cycle is located in the pole for several different bacteria. Evidence is also building showing that the terminus DNA region is sometimes located at a position in the cell that will develop into two new poles. Here, a new extension of the concept that polar sites bind specifically origin and terminus DNA of the chromosome is presented that can explain how division takes place in one and then in another dimension to form two-dimensional tablets of four cells or large planar arrays. A further possible extension to three dimensions to generate octets of cells is proposed.