Comparison of the average structures,from molecular dynamics,of complexes of GTPase activating protein (GAP) with oncogenic and wild-type ras-p21: identification of potential effector domains

GTPase activating protein (GAP) is a known regulator of ras-p21 activity and is a likely target of ras-induced mitogenic signaling. The domains of GAP that may be involved in this signaling are unknown. In order to infer which domains of GAP may be involved, we have performed molecular dynamics calculations of GAP complexed to wild-type and oncogenic (Val 12–containing) ras-p21, both bound to GTP. We have computed and superimposed the average structures for both complexes and find that there are four domains of GAP that undergo major changes in conformation: residues 821–851, 917–924, 943–953, and 1003–1020. With the exception of the 943–953 domain, none of these domains is involved in making contacts with ras-p21, and all of them occur on the surface of the protein, making them good candidates for effector domains. In addition, three ras-p21 domains undergo major structural changes in the oncogenic p21-GAP complex: 71–76 from the switch 2 domain; 100–108, which interacts with SOS, jun and jun kinase (JNK); and residues 122–138. The change in conformation of the 71–76 domain appears to be induced by changes in conformation in the switch 1 domain (residues 32–40) and in the adjacent domain involving residues 21–31. In an accompanying paper, we present results from microinjection of peptides corresponding to each of these domains into oocytes induced to undergo maturation by oncogenic ras-p21 and by insulin-activated wild-type cellular p21 to determine whether these domain peptides may be involved in ras signaling through GAP.     

Geographic structure of lineage associations in a plant-bacterial mutualism

Two species linked by a mutualistic relationship may evolve correlated population differentiation if there is long-term continuity of interactions between specific partners. This phenomenon was analyzed by multilocus enzyme electrophoresis on the annual legume Amphicarpaea bracteata and its nitrogen-fixing bacterial symbionts (Bradyrhizobium sp.) sampled from >20 sites over a 1000 km area. Three analyses indicated that genetic differentiation was correlated in the two organisms. First, the genetic distance between bacterial populations at each pair of sites was significantly positively related to the genetic distance between their host plant populations, as evaluated by the Mantel test. Second, a cluster analysis revealed that several divergent lineages were present both among plants and among bacteria. Bacterial lineages showed a highly nonrandom distribution across plant lineages that was consistent in each of two regions sampled. Finally, there were numerous cases where populations of the same plant lineage 1000 km apart harbored bacterial isolates with an identical multilocus genotype. Thus, despite recurrent opportunities for partner switching, particular genotypes of these two organisms associate consistently across multiple habitats throughout their geographic range.     

Oxytocin and pair compatibility in adult male rhesus macaques (Macaca mulatta)

Pair housing is considered one of the best ways of promoting psychological wellbeing for caged macaques. However, incompatible partnerships can result in stress or aggression. Though previous studies have analyzed the role of variables such as age, weight, gender, and temperament on pair compatibility, few have examined the relationship between physiological parameters and pair compatibility. Oxytocin is known to promote prosocial nonsexual behavior in various primate species and may serve as an indicator of pair compatibility. In this study, we examined the association between peripheral oxytocin levels and prosocial behaviors in isosexual pairs of male rhesus macaques. We hypothesized that animals that demonstrated high levels of prosocial behaviors would have higher oxytocin levels than those showing low levels of the behavior. In addition, to elucidate the relationship between oxytocin and compatibility, we compared peripheral oxytocin between the highly affiliative animals and single‐housed males identified as having multiple unsuccessful pair attempts with multiple partners. We collected plasma oxytocin on 40 pairs of monkeys that had lived together for at least 1 month and 20 single‐housed animals. Further, we simultaneously collected behavioral data on the pairs, recording prosocial interactions (e.g., groom, play). Oxytocin varied among individuals, but was highly correlated between members of a pair (r = 0.58, p < .001). Additionally, prosocial behavior was positively correlated with plasma oxytocin (r = 0.38, p < .02). However, contrary to our expectations, oxytocin did not differ between single and highly affiliative pair‐housed animals (F_(1,38) = 0.71, p = .40). Our results suggest that oxytocin may be associated with the quality of isosexual pairs of male macaques. More work is needed to determine the nature of this relationship.     

Evolution of protamine P1 genes in primates

Protamine P1 genes have been sequenced by PCR amplification and direct DNA sequencing from 9 primates representing 5 major families, Cebidae (new world monkeys), Cercopithecidae (old world monkeys), Hylobatidae (gibbons), Pongidae (gorilla, orangutan, and chimpanzee), and Hominidae (human). In this recently diverged group of primates these genes are clearly orthologous but very variable, both at the DNA level and in their expressed amino acid sequences. The rate of variation amongst the protamine Pls indicates that they are amongst the most rapidly diverging polypeptides studied. However, some regions are conserved both in primates and generally in other placental mammals. These are the 13 N-terminal residues (including a region of alternating serine and arginine residues (the motif SRSR, res. 10–13) susceptible to Ser phosphorylation), a tract of six Arg residues (res. 24–29) in the center of the molecule, and a six-residue region (RCCRRR, res. 39–44), consisting of a pair of cysteines flanked by arginines. Detailed consideration of nearest neighbor matrices and trees based on maximum parsimony indicates that PI genes from humans, gorillas, and chimpanzees are very similar. The amino acid and nucleotide differences between humans and gorillas. are fewer than those between humans and chimpanzees. This finding is at variance with data from DNA-DNA hybridization and extensive globin and mitochondrial DNA sequences which place human and chimpanzee as closest relatives in the super family, Hominoidea. This may be related to the fact that protamine Pls are expressed in germ line rather than somatic cells. In contrast to the variability of the exon regions of the protamine P1 genes, the sequence of the single intron is highly conserved.     

Comparison of protein structure and genomic structure of human, rabbit, and limulus C-reactive proteins: Possible implications for function and evolution

The primary structures of human, rabbit, and Limulus C-reactive proteins (CRPs) have been compared by a computer program. Based on these data, a PAMs matrix (accepted point mutation per 100 residues) was constructed to generate topologies for the three proteins. Five trees with the shortest absolute length were generated, but only one positive tree was found. Using the relatively well-established distance between human and rabbit of 150 million years, we calculate that human and Limulus CRPs diverged at least 500 million years ago. The data indicate that the amino acid sequence indentity between Limulus CRPs and their mammalian counterparts is about 25%, strongly suggesting that human CRP, rabbit CRP, and Limulus CRPs share common ancestral genes. There are two highly conserved regions in the primary structures among the CRPs. Residues 52–67 in Limulus CRP and residues 51–66 in human CRP show identity in 10 of 16 positions, with 3 additional conservative replacements. This region of the molecule is thought to be involved in the binding of phosphorylcholine ligand. Residues 139–153 in Limulus CRP and residues 133–147 in human CRP show identity in 9 of 15 positions, with 5 additional conservative replacements. The biological function of this stretch of amino acid sequence is thought to be associated with the CA²⁺ binding of the CRPs.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

MICROSATELLITE MARKERS REVEAL POPULATION GENETIC STRUCTURE OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM TAMARENSE (DINOPHYCEAE) IN JAPANESE COASTAL WATERS1

This is the first report to explore the fine‐scale diversity, population genetic structure, and biogeography of a typical planktonic microbe in Japanese and Korean coastal waters and also to try to detect the impact of natural and human‐assisted dispersals on the genetic structure and gene flow in a toxic dinoflagellate species. Here we present the genetic analysis of Alexandrium tamarense (Lebour) Balech populations from 10 sites along the Japanese and Korean coasts. We used nine microsatellite loci, which varied widely in number of alleles and gene diversity across populations. The analysis revealed that Nei's genetic distance correlated significantly with geographic distance in pair‐wise comparisons, and that there was genetic differentiation in about half of 45 pair‐wise populations. These results clearly indicate genetic isolation among populations according to geographic distance and restricted gene flow via natural dispersal through tidal currents among the populations. On the other hand, high P‐values in Fisher's combined test were detected in five pair‐wise populations, suggesting similar genetic structure and a close genetic relationship between the populations. These findings suggest that the genetic structure of Japanese A. tamarense populations has been disturbed, possibly by human‐assisted dispersal, which has resulted in gene flow between geographically separated populations.     

Predicting the Structure of the Flavodoxin from Eschericia coli by Homology Modeling,Distance Geometry and Molecular Dynamics

Abstract

As part of our on-going development of a method, based upon distance geometry calculations, for predicting the structures of proteins from the known structures of their homologues, we have predicted the structure of the 176 residue Flavodoxin from Escherichia coli. This prediction was based upon the crystal structures of the homologous Flavodoxins from Anacystis nidulans, Chondrus crispus, Desulfovibrio vulgaris and Clostridium beijerinckii, whose sequence identities with Escherichia coli were 44%, 33%, 23% and 16%, respectively. A total of 13,043 distance constraints among the alpha-carbons of the Escherichia coli structure were derived from the sequence alignments with the known structures, together with 8,893 distance constraints among backbone and sidechain atoms of adjacent residues, 978 between the alpha-carbons and selected atoms of the flavin mononucleotide cofactor, 116 constraints to enforce conserved hydrogen bonds, and 452 constraints on the torsion angles in conserved residues. An ensemble of ten random Escherichia coli structures was computed from these constraints, with an average root mean square coordinate deviation (RMSD) among the alpha carbons of 0.85 Ångstroms (excluding the first 1 and last 6 residues, which have no corresponding residues in any of the homologues and hence were unconstrained); the corresponding average heavy-atom RMSD was 1.60 Å.

Since the distance geometry calculations were performed without hydrogen atoms, protons were added to the resulting structures and these structures embedded in a 50 × 50 × 40 Å solvent box with periodic boundary conditions. They were then subjected to a 20 picosecond dynamical simulated annealing procedure, starting at 300 K and gradually reduced to 10K, in which all the distance and torsion angle constraints were maintained by means of harmonic restraint functions. This was followed up by 1000 iterations of unrestrained conjugate gradients minimization. The goal of this energy refinement procedure was not to drastically modify the structures in an attempt at a priori prediction, but merely to improve upon the predictions obtained from the geometric constraints, particularly with regard to their local backbone and sidechain conformations and their hydrogen bonds. The resulting structures differed from the respective starting structures by an average of 1.52 Å in their heavy atom RMSD's, while the average RMSD among the heavy atoms of residues 2-170 increased slightly to 1.66 Å. We hope these structures will be good enough to enable the phase problem to be solved for the crystallographic data that is now being collected on this protein.     

Amino acid composition and the evolutionary rates of protein-coding genes

Summary Based on the rates of amino acid substitution for 60 mammalian genes of 50 codons or more, it is shown that the rate of amino acid substitution of a protein is correlated with its amino acid composition. In particular, the content of glycine residues is negatively correlated with the rate of amino acid substitution, and this content alone explains about 38% of the total variation in amino acid substitution rates among different protein families. The propensity of a polypeptide to evolve fast or slowly may be predicted from an index or indices of protein mutability directly derivable from the amino acid composition. The propensity of an amino acid to remain conserved during evolutionary times depends not so much on its being features prominently in active sites, but on its stability index, defined as the mean chemical distance [R. Grantham (1974) Science 185862–864] between the amino acid and its mutational derivatives produced by single-nucleotide substitutions. Functional constraints related to active and binding sites of proteins play only a minor role in determining the overall rate of amino acid substitution. The importance of amino acid composition in determining rates of substitution is illustrated with examples involving cytochrome c, cytochrome b₅,ras-related genes, the calmodulin protein family, and fibrinopeptides.     

Phylogenetic Relationships of Xylella fastidiosa Strains Based on 16S–23S rDNA Sequences

In spite of the lack of resolution of Xylella fastidiosa phylogenetic relationships, parsimony analysis of the 16S–23S rDNA sequence from a wide range of hosts has been evaluated in this research. In order to establish an easier method for sequencing the spacer region completely, a new primer pair was designed. The sequences obtained revealed a higher level of variation than that found in 16S gene sequences, with similarity values ranging from 0.80 to 1.00. The cladogram constructed allowed the clustering of two major clades. From these results it has been possible to recognize the monophyletic grouping of some strains belonging to the same host, possibly representing only one infection process. However, for other hosts there is paraphyletic and polyphyletic grouping. This methodology followed from promising results regarding strain–host clustering. With the parsimony approach, hypothetical genealogical relationship among Xylella strains may be inferred.     

Structure-based method for analyzing protein–protein interfaces

Hydrogen bond, hydrophobic and vdW interactions are the three major non-covalent interactions at protein–protein interfaces. We have developed a method that uses only these properties to describe interactions between proteins, which can qualitatively estimate the individual contribution of each interfacial residue to the binding and gives the results in a graphic display way. This method has been applied to analyze alanine mutation data at protein–protein interfaces. A dataset containing 13 protein–protein complexes with 250 alanine mutations of interfacial residues has been tested. For the 75 hot-spot residues (G1.5 kcal mol^-1), 66 can be predicted correctly with a success rate of 88%. In order to test the tolerance of this method to conformational changes upon binding, we utilize a set of 26 complexes with one or both of their components available in the unbound form. The difference of key residues exported by the program is 11% between the results using complexed proteins and those from unbound ones. As this method gives the characteristics of the binding partner for a particular protein, in-depth studies on protein–protein recognition can be carried out. Furthermore, this method can be used to compare the difference between protein–protein interactions and look for correlated mutation. Figure Key interaction grids at the interface between barnase and barstar. Key interaction grid for barnase and barstar are presented in one figure according to their coordinates. In order to distinguish the two proteins, different icons were assigned. Crosses represent key grids for barstar and dots represent key grids for barnase. The four residues in ball and stick are Asp40 in barstar and Arg83, Arg87, His102 in barnase.     

Visualization of the nature of protein folding by a study of a distance constraint approach in two-dimensional models

A distance constraint approach is applied to two-dimensional models of proteins in order to visualize the nature of protein folding and to examine the relative roles of different ranges of interaction. Three different native structures (I, II, and III) are considered; they have two different kinds of residues, viz., hydrophobic and hydrophilic, and different sequences of these residues. We examine how the distance constraint approach functions in the prediction of protein folding when we know the sequence of the residues, the (fixed) bond lengths, the mean distances between residues i and i + 2, and i and i + 3, and the mean distances for hydrophobic–hydrophobic, hydrophobic–hydrophilic, and hydrophilic–hydrophilic contacts between residues i and i + j, where j ≥ 4. This approach involves optimization of an object function with respect to 98 variables and is not free of the multiple-minimum problem. The optimization is always terminated if the chain is entangled and/or the segments (residues) are packed too compactly to move. In order to escape from such situations and to take the excluded-volume effect into account, a Monte Carlo method is used after the optimization is trapped in local minima. Success in the prediction of folding is found to depend on the starting conformations and on the native conformations. Fair success is obtained in predicting the helix-like structure in protein I and the overall structure of protein III, but not the β-like structures of proteins I and II. Insofar as the prediction of the structure of protein III is reasonable, it appears that some sequences of residues produce greater constraints on their conformations than others, if one considers only the hydrophobic and hydrophilic nature of the residues. These results imply that, in the folding of real proteins in three dimensions, the competition for hydrophobic (and hydrophilic) residues for inside (outside) positions in the molecule probably constitutes a necessary but not a sufficient condition to form and stabilize the native structure. The failure to predict the structure of protein II, and part of that of protein I, suggests that there are two types of long-range interactions. One (which we considered here) is nonspecific (i.e., is defined only in terms of contacts between residues of the same or different polarity) and acts at any stage of protein folding; the other (which we did not consider here) is a specific interaction between residues in pairs and contributes only when the residues in the specific pair take on the native conformation. Presumably, incorporation of such specific long-range interactions, together with the nonspecific ones, is necessary for successful protein folding, using the distance constraint approach.     

Geographic and genetic isolation in spring-associated Eurycea salamanders endemic to the Edwards Plateau region of Texas

Populations of neotenic, spring-associated salamanders of the genus Eurycea occupy discontinuous sites throughout the Edwards Plateau of central Texas and many warrant conservation attention. Here we used DNA sequence data from a nuclear (rag1) and a mitochondrial (ND4) gene to determine (1) the extent of genetic isolation among seven Edwards Plateau Eurycea populations and (2) the relationship between genetic distance and both geographic distance and hydrogeological features. Coalescent-based methods detected little gene flow among the sampled Eurycea populations, and we were unable to reject a model of complete isolation for any pair of populations. These findings were consistent with the relatively high genetic distances we detected among the sampled Eurycea populations (pairwise ϕ_ST ranged from 0.249 to 0.924). We detected a positive correlation between genetic distance and geographic distance, which is consistent with a pattern of isolation by distance. However, while controlling for geographic distance, we did not detect a positive relationship between genetic distance and aquifer or river distance. Thus, we found no evidence that aquifers and/or rivers serve as dispersal corridors among isolated Eurycea populations. Based on these results, we have no evidence that re-colonization of spring sites by migrant salamanders following local extirpation would be likely. Our findings indicate that spring-associated Eurycea salamander populations occupying the Edwards Plateau region are genetically isolated, and that each of these populations should be considered a distinct management unit.     

Use of Accelerometers to Measure Stress Levels in Shelter Dogs

Stress can compromise welfare in any confined group of nonhuman animals, including those in shelters. However, an objective and practical method for assessing the stress levels of individual dogs housed in a shelter does not exist. Such a method would be useful for monitoring animal welfare and would allow shelters to measure the effectiveness of specific interventions for stress reduction. In this pilot study, activity levels were studied in 13 dogs using accelerometers attached to their collars. Behavioral stress scores as well as urinary and salivary cortisol levels were measured to determine if the dogs' activity levels while confined in the kennel correlated with behavioral and physiological indicators of stress in this population. The results indicated that the accelerometer could be a useful tool to study stress-related activity levels in dogs. Specific findings included a correlation between the salivary cortisol and maximum activity level (r = .62, p = .025) and a correlation between the urine cortisol-to-creatinine ratio and average activity level (r = .61, p = .028) among the study dogs. Further research is needed to better understand the complex relationship between stress and activity level among dogs in a kennel environment.     

Phosphorus regulation of nitrogen fixation in a traditional Mexican agroecosystem

Although nitrogen is considered to be the nutrient that most commonly limits production of natural and managed terrestrial ecosystems, I propose that phosphorus may regulate productivity in many continuously cultivated agroecosystems that do not receive applications of synthetic fertilizers. One way P may limit agroecosystem productivity is by controlling nitrogen fixation of legume crops, thus affecting nitrogen availability in the overall agroecosystem. I tested this hypothesis in two studies by examining the effect of phosphorus nutrition on nitrogen fixation of alfalfa in traditional Mexican agroecosystems. All farms used in the research relied on alfalfa as the primary nitrogen source for maize cultivation and other crops, and had minimal or no reliance on synthetic fertilizers.In one study, I used the natural abundance of¹⁵N to estimate nitrogen fixation in five alfalfa plots with soils representing a wide range of P fertility. I found a correlation of r = 0.85 between foliage P concentrations and nitrogen fixation in the alfalfa plots. Mean nitrogen fixation in alfalfa plots ranged between 232–555 kg ha^–1 yr^–1 as estimated by the¹⁵N-natural abundance method.In a second study, I sampled soils from alfalfa plots on traditional farms located in 5 different physiographic regions of Mexico. Half of each soil sample was augmented with phosphorus in a greenhouse experiment. I grew alfalfa on the fertilized and unfertilized soils from each site and then determined nitrogenase activity (acetylene reduction) of the Rhizobium on the plant roots. Nitrogenase activity increased in the alfalfa grown on all soils with added phosphorus, with two of the five differences being statistically significant at P < 0.01, 0 and one at P < 0.05. Foliage P concentrations and nitrogenase activity were 0 positively correlated (r = 0.81,P < 0.01).0     

A Ribokinase Family Conserved Monovalent Cation Binding Site Enhances the MgATP-induced Inhibition in E.?coli Phosphofructokinase-2

The enzymatic activity of secreted phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes is associated with bacterial virulence. Although the PI-PLC active site has no obvious lid, molecular-dynamics simulations suggest that correlated loop motions may limit access to the active site, and two Pro residues, Pro²⁴⁵ and Pro²⁵⁴, are associated with these correlated motions. Whereas the region containing both Pro residues is quite variable among PI-PLCs, it shows high conservation in virulence-associated, secreted PI-PLCs that bind to the surface of cells. These regions of the protein are also associated with phosphatidylcholine binding, which enhances PI-PLC activity. In silico mutagenesis of Pro²⁴⁵ disrupts correlated motions between the two halves of Bacillus thuringiensis PI-PLC, and Pro²⁴⁵ variants show significantly reduced enzymatic activity in all assay systems. PC still enhanced activity, but not to the level of wild-type enzyme. Mutagenesis of Pro²⁵⁴ appears to stiffen the PI-PLC structure, but experimental mutations had minor effects on activity and membrane binding. With the exception of P245Y, reduced activity was not associated with reduced membrane affinity. This combination of simulations and experiments suggests that correlated motions between the two halves of PI-PLC may be more important for enzymatic activity than for vesicle binding.     

Karyotype analysis of Eucinostomus argenteus, E. gula, E. harengulus, and Eugerres plumieri (Teleostei, Gerreidae) from Florida and Puerto Rico

The karyotypes of four gerreids of the western Atlantic Ocean are documented. A diploid chromosome complement of 48 telocentric chromosomes was found in the four species (2N=48t, fundamental number FN=48). No differences were detected either in the number of chromosomes of the standard karyotype, in their karyotype size, or between the karyotypes derived from male or female specimens of any of the species. Chromosome length decreased progressively and slightly from pair 1 to pair 24. The Ag–NOR karyotypes of E. argenteus and E. harengulus were characterized by the position of the nucleolar organizer regions next to the centromere in chromosome pair 1, whereas in E. gula and E. plumieri Ag–NORs were detected in pair 4. The other 46 chromosomes showed a light staining of the centromere with no terminal or intermediate heterochromatic blocks. All Eucinostomus species showed Ag–NORs of similar size, while Eugerres plumieri showed Ag–NORs 10–20% larger than Eucinostomus species. A combination of size and position of the Ag–NORs identified E. gula, while size alone identified E. plumieri. However, the ancestral state for size and position of Ag–NORs could not be established. There was no differential staining of the chromosomes by G-banding. The karyotype of the gerreids appears similar to the hypothetical ancestral karyotype of fish. The phylogenetic relationships among these species could not be established because of the lack of chromosome G-bands. Most likely this indicates a homogeneous distribution of GC nucleotides in the chromosomes.     

THE UTILITY OF PROTEOMICS IN ALGAL TAXONOMY: BOSTRYCHIA RADICANS/B. MORITZIANA (RHODOMELACEAE,RHODOPHYTA) AS A MODEL STUDY1

A comparison of the proteome of eight genetically well‐characterized isolates of the Bostrychia radicans (Mont.) Mont./B. moritziana (Sond. ex Kütz.) J. Agardh species complex was undertaken to establish if genetic relationships among them can be determined using proteome data. Genetic distances were calculated on the basis of common and distinct spots in two‐dimensional gel electrophoresis (2‐DE). Proteomes of the male and female plants of each population were compared to analyze the range of genetic difference within an isolate. Haploid male and female plants of the same species had 3.7%–7.1% sex‐specific proteins. The degree of similarity of the proteome was consistent with previous DNA sequence data and sexual compatibility studies between the isolates. Two sexually compatible isolates from Venezuela showed a pair‐wise distance ranging from 0.14 to 0.21. The isolates from Mexico and Venezuela, which were partially compatible, showed a maximum pair‐wise distance of 0.26. A high level of genetic difference was found among isolates that were sexually incompatible. The isolate from Brazil was reproductively isolated from the Mexico and Venezuela isolates and showed a maximum pair‐wise distance of 0.65 and 0.58, respectively. Comparative proteomics may be helpful for studying genetic distances among algal samples, if intraisolate variation (gene expression) can be minimized or tested.     

Effects of <Emphasis Type="Italic">N</Emphasis>- and <Emphasis Type="Italic">C</Emphasis>-terminal truncation of HP (2-20) from <Emphasis Type="Italic">Helicobacter pylori</Emphasis> ribosomal protein L1 (RPL1) on its anti-microbial activity

HP (2-20) [derived from the N-terminal region of Helicobacter pylori Ribosomal Protein L1 (RPL1)], a 19-mer peptide, possesses broad-spectrum anti-microbial activity. As the N- (residues 2–3) and C-terminal (residues 14–20) residues can be deleted without affecting antimicrobial activity, we have now determined the minimum chain length necessary for the retention of antimicrobial activity, and its mode of action. The N- (residues 2–3) and C-terminal (residues 17–20) truncated fragments [HP (4–16)] induce increased antibiotic activity against several bacterial strains without hemolysis. Flow cytometric analysis, scanning electron microscopy and fluorescence confocal microscopy revealed that HP (4–16) acted rapidly on the plasma membranes of the fungal cells in a salt- and energy-independent manner.Revisions requested 16 September 2004/1 November 2004; Revisions received 29 October 2004/8 December 2004     

Unique Reconstruction of Tree-Like Phylogenetic Networks from Distances Between Leaves

In this paper, a class of rooted acyclic directed graphs (called TOM-networks) is defined that generalizes rooted trees and allows for models including hybridization events. It is argued that the defining properties are biologically plausible. Each TOM-network has a distance defined between each pair of vertices. For a TOM-network N, suppose that the set X consisting of the leaves and the root is known, together with the distances between members of X. It is proved that N is uniquely determined from this information and can be reconstructed in polynomial time. Thus, given exact distance information on the leaves and root, the phylogenetic network can be uniquely recovered, provided that it is a TOM-network. An outgroup can be used instead of a true root.     

Crystal structure of deltarhodopsin‐3 from Haloterrigena thermotolerans

Deltarhodopsin, a new member of the microbial rhodopsin family, functions as a light‐driven proton pump. Here, we report the three‐dimensional structure of deltarhodopsin (dR3) from Haloterrigena thermotolerans at 2.7 Å resolution. A crystal belonging to space group R32 (a, b = 111.71 Å, c = 198.25 Å) was obtained by the membrane fusion method. In this crystal, dR3 forms a trimeric structure as observed for bacteriorhodopsin (bR). Structural comparison of dR with bR showed that the inner part (the proton release and uptake pathways) is highly conserved. Meanwhile, residues in the protein–protein contact region are largely altered so that the diameter of the trimeric structure at the cytoplasmic side is noticeably larger in dR3. Unlike bR, dR3 possesses a helical segment at the C‐terminal region that fills the space between the AB and EF loops. A significant difference is also seen in the FG loop, which is one residue longer in dR3. Another peculiar property of dR3 is a highly crowded distribution of positively charged residues on the cytoplasmic surface, which may be relevant to a specific interaction with some cytoplasmic component.Proteins 2013; © 2013 Wiley Periodicals, Inc.