D-amino acid residue in the C-type natriuretic peptide from the venom of the mammal, Ornithorhynchus anatinus, the Australian platypus

The C-type natriuretic peptide from the platypus venom (OvCNP) exists in two forms, OvCNPa and OvCNPb, whose amino acid sequences are identical. Through the use of nuclear magnetic resonance, mass spectrometry, and peptidase digestion studies, we discovered that OvCNPb incorporates a D-amino acid at position 2 in the primary structure. Peptides containing a D-amino acid have been found in lower forms of organism, but this report is the first for a D-amino acid in a biologically active peptide from a mammal. The result implies the existence of a specific isomerase in the platypus that converts an L-amino acid residue in the protein to the D-configuration. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Characterization of two HKT1 homologues from Eucalyptus camaldulensis that display intrinsic osmosensing capability

Plants have multiple potassium (K+) uptake and efflux mechanisms that are expressed throughout plant tissues to fulfill different physiological functions. Several different classes of K+ channels and carriers have been identified at the molecular level in plants. K+ transporters of the HKT1 superfamily have been cloned from wheat (Triticum aestivum), Arabidopsis, and Eucalyptus camaldulensis. The functional characteristics as well as the primary structure of these transporters are diverse with orthologues found in bacterial and fungal genomes. In this report, we provide a detailed characterization of the functional characteristics, as expressed in Xenopus laevis oocytes, of two cDNAs isolated from E. camaldulensis that encode proteins belonging to the HKT1 superfamily of K+/Na+ transporters. The transport of K+ in EcHKT-expressing oocytes is enhanced by Na+, but K+ was also transported in the absence of Na+. Na+ is transported in the absence of K+ as has been demonstrated for HKT1 and AtHKT1. Overall, the E. camaldulensis transporters show some similarities and differences in ionic selectivity to HKT1 and AtHKT1. One striking difference between HKT1 and EcHKT is the sensitivity to changes in the external osmolarity of the solution. Hypotonic solutions increased EcHKT induced currents in oocytes by 100% as compared with no increased current in HKT1 expressing or uninjected oocytes. These osmotically sensitive currents were not enhanced by voltage and may mediate water flux. The physiological function of these osmotically induced increases in currents may be related to the ecological niches that E. camaldulensis inhabits, which are periodically flooded. Therefore, the osmosensing function of EcHKT may provide this species with a competitive advantage in maintaining K+ homeostasis under certain conditions.

NmlR of Neisseria gonorrhoeae: a novel redox responsive transcription factor from the MerR family

A MerR-like regulator (NmlR -Neisseria merR-like Regulator) identified in the Neisseria gonorrhoeae genome lacks the conserved cysteines known to bind metal ions in characterized proteins of this family. Phylogenetic analysis indicates that NmlR defines a subfamily of MerR-like transcription factors with a distinctive pattern of conserved cysteines within their primary structure. NmlR regulates itself and three other genes in N. gonorrhoeae encoding a glutathione-dependent dehydrogenase (AdhC), a CPx-type ATPase (CopA) and a thioredoxin reductase (TrxB). An nmlR mutant lacked the ability to survive oxidative stress induced by diamide and cumene hydroperoxide. It also had > 50-fold lower NADH-S-nitrosoglutathione oxidoreductase activity consistent with a role for AdhC in protection against nitric oxide stress. The upstream sequences of the NmlR regulated genes contained typical MerR-like operator/promoter arrangements consisting of a dyad symmetry located between the -35 and -10 elements of the target genes. The NmlR target operator/promoters were cloned into a beta-galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans. Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress P-AdhC (or P-CopA) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)-dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Site-directed mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.

Predicting structural disruption of proteins caused by crossover

We present a machine learning model that predicts a structural disruption score from a protein’s primary structure. SCHEMA was introduced by Frances Arnold and colleagues as a method for determining putative recombination sites of a protein on the basis of the full (PDB) description of its structure. The present method provides an alternative to SCHEMA that is able to determine the same score from sequence data only. Circumventing the need for resolving the full structure enables the exploration of yet unresolved and even hypothetical sequences for protein design efforts. Deriving the SCHEMA score from a primary structure is achieved using a two step approach: first predicting a secondary structure from the sequence and then predicting the SCHEMA score from the predicted secondary structure. The correlation coefficient for the prediction is 0.88 and indicates the feasibility of replacing SCHEMA with little loss of precision. ©2005 IEEE

A study of pyramidal cell structure in the cingulate cortex of the macaque monkey with comparative notes on inferotemporal and primary visual cortex

Recent studies have revealed a marked degree of variation in the pyramidal cell phenotype in visual, somatosensory, motor and prefrontal cortical areas in the brain of different primates, which are believed to subserve specialized cortical function. In the present study we carried out comparisons of dendritic structure of layer III pyramidal cells in the anterior and posterior cingulate cortex and compared their structure with those sampled from inferotemporal cortex (IT) and the primary visual area (V1) in macaque monkeys. Cells were injected with Lucifer Yellow in flat-mounted cortical slices, and processed for a light-stable DAB reaction product. Size, branching pattern, and spine density of basal dendritic arbors was determined, and somal areas measured. We found that pyramidal cells in anterior cingulate cortex were more branched and more spinous than those in posterior cingulate cortex, and cells in both anterior and posterior cingulate were considerably larger, more branched, and more spinous than those in area V1. These data show that pyramidal cell structure differs between posterior dysgranular and anterior granular cingulate cortex, and that pyramidal neurons in cingulate cortex have different structure to those in many other cortical areas. These results provide further evidence for a parallel between structural and functional specialization in cortex.

Structure of Caribbean ciguatoxin isolated from Caranx latus

Caribbean ciguatoxins (C-CTXs) are responsible for the widespread occurrence of ciguatera in the Caribbean Sea. The structure and configuration of C-CTX-1 (1), the major ciguatoxin isolated from the horse-eye jack (Caranx latus), has been determined from DQF-COSY, E-COSY, TOCSY, NOESY, POESY, ge-HSQC. and HMQC experiments performed at 750 MHz and 500 MHz on a 0.13 pmol sample. C-CTX-1 ([M + H](+) m/z 1141.6 Da, molecular formula C62H92O19) has a ciguatoxin/breveroxin ladder structure comprising 14 trans-fused, ether-linked rings (7/6/6/7/8/9/7/6/8/6/7/6/7/6) assembled fi um 6 protonated fragments. The relative stereochemistry and ring configuration of 1 was determined from an analysis of coupling constant and NOE data. Like ciguatoxins in the Pacific Ocean (P-CTX), C-CTX-1 possesses a flexible nine-membered ring which may be a conserved feature among ciguatoxins. However, C-CTX-1 has a longer contiguous carbon backbone (57 vs 55 carbons for P-CTX-1), one extra ring, and a hemiketal in ring N but no spiroketal as found in P-CTX. C-CTX-1 possesses a primary hydroxyl which may allow selective derivatization. A minor analogue, C-CTX-2, was also isolated from fish and assigned the structure 56 epi-C-CTX-1 (2). since it slowly rearranged to C-CTX-1 in solution. Given the structural similarities between Caribbean and Pacific ciguatoxins, we propose that C-CTX-1 and C-CTX-2 arise from a Caribbean strain of the benthic dinoflagellate, Gambierdiscus toxicus.

Metal-directed synthesis routes to a 16-membered tetraazamacrocycle with two pendant primary amine groups

The reaction of the bis(propane-1,3-diamine)copper(II) ion with paraformaldehyde and nitroethane in dry methanol under basic conditions produces a macrocyclic product, (cis-3,11-dimethyl-3,11-dinitro-1,5,9,13-tetraazacyclohexadecane)copper(II) perchlorate, in low yield, compared with the good yield obtained in the parallel chemistry possible even under aqueous conditions using palladium(II) as a template. The palladium complex was reduced with zinc amalgam in dilute aqueous acid to yield the metal-free 16-membered macrocyclic hexaamine, in this case re-complexed and characterised by an X-ray crystal structure as the (cis-3,11-dimethyl-1,5,9,13-tetraazacyclohexadecane-3,11-diamine)copper(II) perchlorate. The copper ion is found in a tetragonally elongated and trigonally-distorted octahedral environment, with all six of the ligand nitrogens coordinated, the two primary amine pendant groups occupying cis sites. (C) 2000 Elsevier Science S.A. All rights reserved.

The effect of solidification rate on the structure of magnesium-aluminium eutectic grains

A magnesium-aluminium alloy of eutectic composition was solidified under two different cooling conditions, producing a low and a high growth rate of the eutectic solid-liquid interface. The high growth rate specimen contained smaller eutectic grains and cells, with a smaller interphase spacing compared with the low growth rate specimen. The high growth rate specimen also contained some primary Mg17Al12 dendrites, suggesting that the coupled zone is skewed towards the Mg phase with increased undercooling, A lamellar eutectic morphology was observed in the low growth rate specimen, while the morphology was fibrous in the high growth rate specimen.

Three-dimensional structure of RK-1: A novel alpha-defensin peptide

NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of RK-1, an antimicrobial peptide from rabbit kidney recently discovered from homology screening based on the distinctive physicochemical properties of the corticostatins/defensins. RK-1 consists of 32 residues, including six cysteines arranged into three disulfide bonds. It exhibits antimicrobial activity against Escherichia coli and activates Ca2+ channels in vitro. Through its physicochemical similarity, identical cysteine spacing, and linkage to the corticostatins/defensins, it was presumed to be a member of this family. However, RK-1 lacks both a large number of arginines in the primary sequence and a high overall positive charge, which are characteristic of this family of peptides. The three-dimensional solution structure, determined by NMR, consists of a triple-stranded antiparallel beta -sheet and a series of turns and is similar to the known structures of other alpha -defensins. This has enabled the definitive classification of RK-1 as a member of this family of antimicrobial peptides. Ultracentrifuge measurements confirmed that like rabbit neutrophil defensins, RK-1 is monomeric in solution, in contrast to human neutrophil defensins, which are dimeric.

Tissue-specific expression of head-to-tail cyclized miniproteins in Violaceae and structure determination of the root cyclotide Viola hederacea root cyclotide1

The plant cyclotides are a family of 28 to 37 amino acid miniproteins characterized by their head-to-tail cyclized peptide backbone and six absolutely conserved Cys residues arranged in a cystine knot motif: two disulfide bonds and the connecting backbone segments form a loop that is penetrated by the third disulfide bond. This knotted disulfide arrangement, together with the cyclic peptide backbone, renders the cyclotides extremely stable against enzymatic digest as well as thermal degradation, making them interesting targets for both pharmaceutical and agrochemical applications. We have examined the expression patterns of these fascinating peptides in various Viola species (Violaceae). All tissue types examined contained complex mixtures of cyclotides, with individual profiles differing significantly. We provide evidence for at least 57 novel cyclotides present in a single Viola species (Viola hederacea). Furthermore, we have isolated one cyclotide expressed only in underground parts of V, hederacea and characterized its primary and three-dimensional structure. We propose that cyclotides constitute a new family of plant defense peptides, which might constitute an even larger and, in their biological function, more diverse family than the well-known plant defensins.

Genetic structure of a population of Ganoderma boninense on oil palm

Ganoderma boninense (the causal agent of basal stem rot of oil palm in Papua New Guinea) has a tetrapolar mating system with multiple alleles. Investigations into the population structure of G. boninense, using interfertility between isolates as a marker, revealed that the population on oil palm was comprised predominantly of genetically distinct individuals, although a number of isolates were found to share single mating alleles. No direct hereditary relationship was found between isolates on neighbouring or spatially separated diseased palms, indicating that outcrossing had probably occurred over several generations in the founder population prior to colonization of oil palm. In this study, a total of 81 A and 83 B mating type alleles (factors) were detected with 18 allelic repeats at the A locus and 17 at the B locus. Alleles appeared to be randomly dispersed throughout the population in each study block, although there was a significantly (P

Electronic structure methods applied to gas-carbon reactions

A review is given on the fundamental studies of gas-carbon reactions using electronic structure methods in the last several decades. The three types of electronic structure methods including semi-empirical, ab initio and density functional theory, methods are briefly introduced first, followed by the studies on carbon reactions with hydrogen and oxygen-containing gases (non-catalysed and catalysed). The problems yet to solve and possible promising directions are discussed. (C) 2002 Elsevier Science Ltd. All rights reserved.

Population structure of the peridomestic mosquito Ochlerotatus notoscriptus in Australia

Ochlerotatus notoscriptus (Skuse) (Diptera: Culicidae) is the predominant peridomestic mosquito in Australia where it is the primary vector of dog heartworm, Dirofilaria immitis (Leidy), and a potentially important vector of arboviruses (Barmah Forest, Ross River) with geographical variation of vector competence. Although widespread, Oc. notoscriptus has low dispersal ability, so it may have isolated subpopulations. The identification of gene flow barriers may assist in understanding arbovirus epidemiology and disease risk, and for developing control strategies for this species. We investigated the population structure of Oc. notoscriptus from 17 sites around Australia, using up to 31 putative allozyme loci, 11 of which were polymorphic. We investigated the effect of larval environment and adult morphology on genetic variation. At least five subpopulations were found, four in New South Wales (NSW) and one unique to Darwin. Perth samples appear to be a product of recent colonization from the Australian east coast. For NSW sites, a Mantel test revealed an isolation by distance effect and spatial autocorrelation analysis revealed an area of effective gene flow of 67 km, which is high given the limited dispersal ability of this species. No consistent difference was observed between 'urban' and 'sylvan' habitats, which suggests frequent movement between these sites. However, a finer-scaled habitat study at Darwin revealed small but significant allele frequency differences, including for Gpi. No fixed allozyme differences were detected for sex, size, integument colour or the colour of species-diagnostic pale scales on the scutum. The domestic habit of Oc. notoscriptus and assisted dispersal have helped to homogenize this species geographically but population structure is still detectable on several levels associated with geographical variation of vector competence.

The influence of substrate kinetics on the microbial community structure in granular anaerobic biomass

The development of a strong, active granular sludge bed is necessary for optimal operation of upflow anaerobic sludge blanket reactors. The microbial and mechanical structure of the granules may have a strong influence on desirable properties such as growth rate, settling velocity and shear strength. Theories have been proposed for granule microbial structure based on the relative kinetics of substrate degradation, but contradict some observations from both modelling and microscopic studies. In this paper, the structures of four granule types were examined from full-scale UASB reactors, treating wastewater from a cannery, a slaughterhouse, and two breweries. Microbial structure was determined using fluorescence in situ hybridisation probing with 16S rRNA-directed oligonucleotide probes, and superficial structure and microbial density (volume occupied by cells and microbial debris) assessed using scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The granules were also modelled using a distributed parameter biofilm model, with a previously published biochemical model structure, biofilm modelling approach, and model parameters. The model results reflected the trophic structures observed, indicating that the structures were possibly determined by kinetics. Of particular interest were results from simulations of the protein grown granules, which were predicted to have slow growth rates, low microbial density, and no trophic layers, the last two of which were reflected by microscopic observations. The primary cause of this structure, as assessed by modelling, was the particulate nature of the wastewater, and the slow rate of particulate hydrolysis, rather than the presence of proteins in the wastewater. Because solids hydrolysis was rate limiting, soluble substrate concentrations were very low (below Monod half saturation concentration), which caused low growth rates. (C) 2003 Elsevier Ltd. All rights reserved.

Accurate prediction of scorpion toxin functional properties from primary structures

Scorpion toxins are common experimental tools for studies of biochemical and pharmacological properties of ion channels. The number of functionally annotated scorpion toxins is steadily growing, but the number of identified toxin sequences is increasing at much faster pace. With an estimated 100,000 different variants, bioinformatic analysis of scorpion toxins is becoming a necessary tool for their systematic functional analysis. Here, we report a bioinformatics-driven system involving scorpion toxin structural classification, functional annotation, database technology, sequence comparison, nearest neighbour analysis, and decision rules which produces highly accurate predictions of scorpion toxin functional properties. (c) 2005 Elsevier Inc. All rights reserved.