Three-dimensional solution structure of alpha-conotoxin MII by NMR spectroscopy: Effects of solution environment on helicity

alpha-Conotoxin MII, a 16-residue polypeptide from the venom of the piscivorous cone snail Conus magus, is a potent and highly specific blocker of mammalian neuronal nicotinic acetylcholine receptors composed of alpha 3 beta 2 subunits. The role of this receptor type in the modulation of neurotransmitter release and its relevance to the problems of addiction and psychosis emphasize the importance of a structural understanding of the mode of interaction of MII with the alpha 3 beta 2 interface. Here we describe the three-dimensional solution structure of MIT determined using 2D H-1 NMR spectroscopy. Structural restraints consisting of 376 interproton distances inferred from NOEs and 12 dihedral restraints derived from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimization in the program X-PLOR. The final set of 20 structures is exceptionally well-defined with mean pairwise rms differences over the whole molecule of 0.07 Angstrom for the backbone atoms and 0.34 Angstrom for all heavy atoms. MII adopts a compact structure incorporating a central segment of alpha-helix and beta-turns at the N- and C-termini. The molecule is stabilized by two disulfide bonds, which provide cross-links between the N-terminus and both the middle and C-terminus of the structure. The susceptibility of the structure to conformational change was examined using several different solvent conditions. While the global fold of MII remains the same, the structure is stabilized in a more hydrophobic environment provided by the addition of acetonitrile or trifluoroethanol to the aqueous solution. The distribution of amino acid side chains in MII creates distinct hydrophobic and polar patches on its surface that may be important for the specific interaction with the alpha 3 beta 2 neuronal nAChR. A comparison of the structure of MII with other neuronal-specific alpha-conotoxins provides insights into their mode of interaction with these receptors.

Structure-activity studies of conantokins as human N-methyl-D-aspartate receptor modulators

The activities of conantokin-G (con-G), conantokin-T (con-T), and several novel analogues have been studied using polyamine enhancement of [H-3]MK-801 binding to human glutamate-N-methyl-D-aspartate (NMDA) receptors, and their structures have been examined using CD and H-1 NMR spectroscopy. The potencies of con-G[A7], con-G, and con-T as noncompetitive inhibitors of spermine-enhanced [H-3]MK-801 binding to NMDA receptor obtained from human brain tissue are similar to those obtained using rat brain tissue. The secondary structure and activity of con-G are found to be highly sensitive to amino acid substitution and modification. NMR chemical shift data indicate that con-G, con-G[D8,D17], and con-G[A7] have similar conformations in the presence of Ca2+. This consists of a helix for residues 2-16, which is kinked in the vicinity of Gla10. This is confirmed by 3D structure calculations on con-G[A7]. Restraining this helix in a linear form (i.e., con-G[A7,E10-K13]) results in a minor reduction in potency. Incorporation of a 7-10 salt-bridge replacement (con-G[K7-E10]) prevents helix formation in aqueous solution and produces a peptide with low potency. Peptides with the Leu5-Tyr5 substitution also have low potencies (con-G[Y5,A7] and con-G[Y5,K7]) indicating that Leu5 in con-G is important for full antagonist behavior. We have also shown that the Gla-Ala7 substitution increases potency, whereas the Gla-Lys7 substitution has no effect. Con-G and con-G[K7] both exhibit selectivity between NMDA subtypes from mid-frontal and superior temporal gyri, but not between sensorimotor and mid-frontal gyri. Asn8 and/or Asn17 appear to be important for the ability of con-G to function as an inhibitor of polyamine-stimulated [3H]MK-801 binding, but not in maintaining secondary structure. The presence of Ca2+ does not increase the potencies of con-G and con-T for NMDA receptors but does stabilize the helical structures of con-G, con-G[D8,D17], and, to a lesser extent, con-G[A7]. The NMR data support the existence of at least two independent Ca2+-chelating sites in con-G, one involving Gla7 and possibly Gla3 and the other likely to involve Gla10 and/or Gla14.

Crystal structure of mammalian purple acid phosphatase

Background: Mammalian purple acid phosphatases are highly conserved binuclear metal-containing enzymes produced by osteoclasts, the cells that resorb bone. The enzyme is a target for drug design because there is strong evidence that it is involved in bone resorption. Results: The 1.55 Angstrom resolution structure of pig purple acid phosphatase has been solved by multiple isomorphous replacement. The enzyme comprises two sandwiched beta sheets flanked by or-helical segments. The molecule shows internal symmetry, with the metal ions bound at the interface between the two halves. Conclusions: Despite less than 15% sequence identity, the protein fold resembles that of the catalytic domain of plant purple acid phosphatase and some serine/threonine protein phosphatases. The active-site regions of the mammalian and plant purple acid phosphatases differ significantly, however. The internal symmetry suggests that the binuclear centre evolved as a result of the combination of mononuclear ancestors. The structure of the mammalian enzyme provides a basis for antiosteoporotic drug design.

Structure of a putative ancestral protein encoded by a single sequence repeat from a multidomain proteinase inhibitor gene from Nicotiana alata

Background: The ornamental tobacco Nicotiana alata produces a series of proteinase inhibitors (Pls) that are derived from a 43 kDa precursor protein, NaProPl. NaProPl contains six highly homologous repeats that fold to generate six separate structural domains, each corresponding to one of the native Pls. An unusual feature of NaProPl is that the structural domains lie across adjacent repeats and that the sixth Pl domain is generated from fragments of the first and sixth repeats. Although the homology of the repeats suggests that they may have arisen from gene duplication, the observed folding does not appear to support this. This study of the solution structure of a single NaProPl repeat (aPl1) forms a basis for unravelling the mechanism by which this protein may have evolved, Results: The three-dimensional structure of aPl1 closely resembles the triple-stranded antiparallel beta sheet observed in each of the native Pls. The five-residue sequence Glu-Glu-Lys-Lys-Asn, which forms the linker between the six structural domains in NaProPl, exists as a disordered loop in aPl1. The presence of this loop in aPl1 results in a loss of the characteristically flat and disc-like topography of the native inhibitors. Conclusions: A single repeat from NaProPl is capable of folding into a compact globular domain that displays native-like Pl activity. Consequently, it is possible that a similar single-domain inhibitor represents the ancestral protein from which NaProPl evolved.

Habitat structure and egg distributions in the processionary caterpillar Ochrogaster lunifer: lessons for conservation and pest management

1. The spatial and temporal distribution of eggs laid by herbivorous insects is a crucial component of herbivore population stability, as it influences overall mortality within the population. Thus an ecologist studying populations of an endangered butterfly can do little to increase its numbers through habitat management without knowledge of its egg-laying patterns across individual host-plants under different habitat management regimes. At the other end of the spectrum, a knowledge of egg-laying behaviour can do much to control pest outbreaks by disrupting egg distributions that lead to rapid population growth. 2. The distribution of egg batches of the processionary caterpillar Ochrogaster lunifer on acacia trees was monitored in 21 habitats during 2 years in coastal Australia. The presence of egg batches on acacias was affected by host-tree 'quality' (tree size and foliar chemistry that led to increased caterpillar survival) and host-tree 'apparency' (the amount of vegetation surrounding host-trees). 3. In open homogeneous habitats, more egg batches were laid on high-quality trees, increasing potential population growth. In diverse mixed-species habitats, more egg batches were laid on low-quality highly apparent trees, reducing population growth and so reducing the potential for unstable population dynamics. The aggregation of batches on small apparent trees in diverse habitats led to outbreaks on these trees year after year, even when population levels were low, while site-wide outbreaks were rare. 4. These results predict that diverse habitats with mixed plant species should increase insect aggregation and increase population stability. In contrast, in open disturbed habitats or in regular plantations, where egg batches are more evenly distributed across high-quality hosts, populations should be more unstable, with site-wide outbreaks and extinctions being more common. 5. Mixed planting should be used on habitat regeneration sites to increase the population stability of immigrating or reintroduced insect species. Mixed planting also increases the diversity of resources, leading to higher herbivore species richness. With regard to the conservation of single species, different practices of habitat management will need to be employed depending on whether a project is concerned with methods of rapidly increasing the abundance of an endangered insect or concerned with the maintenance of a stable, established insect population that is perhaps endemic to an area. Suggestions for habitat management in these different cases are discussed. 6. Finally, intercropping can be highly effective in reducing pest outbreaks, although the economic gains of reduced pest attack may be outweighed by reduced crop yields in mixed-crop systems.

Morphology and histology of the spermathecal sac, a novel structure in the female reproductive system of Therevidae (Diptera : Asiloidea)

The reproductive system of many female Therevidae has a sac-like structure associated with the spermathecae. This structure, termed the spermathecal sac, has not been recorded previously from any other Diptera and appears unique to certain members of the Therevidae. There is enormous variety in spermathecal sac size and shape, with greatest development in the Australasian Therevidae. A histological examination of the reproductive system of two;Australian therevids, Agapophytus albobasalis Mann and Ectinorhynchus variabilis (Macquart) (Diptera: Asiloidea), reveals that the spermathecal sacs are cuticle-lined and that the intima is frequently highly folded. In some mated individuals, sperm was found within the spermathecal sac, suggesting that sperm and perhaps male accessory gland material is deposited there during copulation. (C) 2000 Elsevier Science Ltd. All rights reserved.

Crystal structure of a heptameric Sm-like protein complex from archaea: Implications for the structure and evolution of snRNPs

The Sm/Lsm proteins associate with small nuclear RNA to form the core of small nuclear ribonucleoproteins, required for processes as diverse as pre-mRNA splicing, mRNA degradation and telomere formation. The Lsm proteins from archaea are likely to represent the ancestral Sm/Lsm domain. Here, we present the crystal structure of the Lsm alpha protein from the thermophilic archaeon Methanobacterium thermoautrophicum at 2.0 Angstrom resolution. The Lsm alpha protein crystallizes as a heptameric ring comprised of seven identical subunits interacting via beta -strand pairing and hydrophobic interactions. The heptamer can be viewed as a propeller-like structure in which each blade consists of a seven-stranded antiparallel beta -sheet formed from neighbouring subunits. There are seven slots on the inner surface of the heptamer ring, each of which is lined by Asp, Asn and Arg residues that are highly conserved in the Sm/Lsm sequences. These conserved slots are likely to form the RNA-binding site. In archaea, the gene encoding Lsm alpha is located next to the L37e ribosomal protein gene in a putative operon, suggesting a role for the Lsm alpha complex in ribosome function or biogenesis. (C) 2001 Academic Press.

Effect of a highly concentrated lipopeptide extract of Bacillus subtilis on fungal and bacterial cells

Lipopeptides produced by Bacillus subtilis are known for their high antifungal activity. The aim of this paper is to show that at high concentration they can damage the surface ultra-structure of bacterial cells. A lipopeptide extract containing iturin and surfactin (5 mg mL-1) was prepared after isolation from B. subtilis (strain OG) by solid phase extraction. Analysis by atomic force microscope (AFM) showed that upon evaporation, lipopeptides form large aggregates (0.1-0.2 mu m2) on the substrates silicon and mica. When the same solution is incubated with fungi and bacteria and the system is allowed to evaporate, dramatic changes are observed on the cells. AFM micrographs show disintegration of the hyphae of Phomopsis phaseoli and the cell walls of Xanthomonas campestris and X. axonopodis. Collapses to fungal and bacterial cells may be a result of formation of pores triggered by micelles and lamellar structures, which are formed above the critical micelar concentration of lipopeptides. As observed for P. phaseoli, the process involves binding, solubilization, and formation of novel structures in which cell wall components are solubilized within lipopeptide vesicles. This is the first report presenting evidences that vesicles of uncharged and negatively charged lipopeptides can alter the morphology of gram-negative bacteria.

Superior electric double layer capacitors using ordered mesoporous carbons

This paper reports for the first time superior electric double layer capacitive properties of ordered mesoporous carbon (OMCs) with varying ordered pore symmetries and mesopore structure. Compared to commercially used activated carbon electrode, Maxsorb, these OMC carbons have superior capacitive behavior, power output and high-frequency performance in EDLCs due to the unique structure of their mesopore network, which is more favorable for fast ionic transport than the pore networks in disordered microporous carbons. As evidenced by N-2 sorption, cyclic voltammetry and frequency response measurements, OMC carbons with large mesopores, and especially with 2-D pore symmetry, show superior capacitive behaviors (exhibiting a high capacitance of over 180 F/g even at very high sweep rate of 50 mV/s, as compared to much reduced capacitance of 73 F/g for Maxsorb at the same sweep rate). OMC carbons can provide much higher power density while still maintaining good energy density. OMC carbons demonstrate excellent high-frequency performances due to its higher surface area in pores larger than 3 nm. Such ordered mesoporous carbons (OMCs) offer a great potential in EDLC capacitors, particularly for applications where high power output and good high-frequency capacitive performances are required. (C) 2005 Elsevier Ltd. All rights reserved.

Improved comparison plot method for pore structure characterization of MCM-41

MCM-41 samples of various pore dimensions are synthesized. Plotting of nitrogen adsorption data at 77 K versus the statistical film thickness (comparison plot) reveals three distinct stages, with a characteristic of two points of inflection. The steep intermediate stage caused by capillary condensation occurred in the highly uniform mesopores. From the slopes of the sections before and after the condensation, the surface area of the mesopores is calculated. The linear portion of the last section is extrapolated to the adsorption axis of the comparison plot, and this intercept is used to obtain the volume of the mesopores. From the surface area and pore volume, average mesopore diameter is calculated, and the value thus obtained is in good agreement with the pore dimension obtained from powder X-ray diffraction measurements. The principle of the calculation as well as problems associated are discussed in detail.

Family advocacy roles and highly dependent residents in nursing homes

A developing critique has questioned the practical utility of user rights policy initiatives for highly dependent residents of nursing homes. This paper seeks to extend this critique to the advocacy roles that families have been accorded within the policy initiatives, The discussion is based on a qualitative research study of family participation in six aged care units, The paper argues that the capacity of families to act as advocates for highly dependent nursing home residents is is limited by the their weak position within the organisations and the complexity of their relations with staff It questions both the applicability and the appropriateness of rights models which do not take sufficient account of the structure and meaning of care.

Crystal structure of Escherichia coli xanthine phosphoribosyltransferase

Xanthine phosphoribosyltransferase (XPRT; EC 2.4.2.22) from Escherichia coil is a tetrameric enzyme having 152 residues per subunit. XPRT catalyzes the transfer of the phosphoribosyl group from 5-phospho-alpha-D-ribosyl l-pyrophosphate (PRib-PP) to the 6-oxopurine bases guanine, xanthine, and hypoxanthine to form GMP, XMP, and IMP, respectively. Crystals grown in the absence of substrate or product were used to determine the structure of XPRT at a resolution of 1.8 Angstrom by multiple isomorphous replacement. The core structure of XPRT includes a five-stranded parallel B-sheet surrounded by three or-helices, which is similar to that observed in other known phosphoribosyltransferase (PRTase) structures. The XPRT structure also has several interesting features. A glutamine residue in the purine binding site may be responsible for the altered 6-oxopurine base specificity seen in this enzyme compared to other 6-oxopurine PRTases. Also, we observe both a magnesium ion and a sulfate ion bound at the PRib-PP binding site of XPRT. The sulfate ion interacts with Arg-37 which has a cis-peptide conformation, and the magnesium ion interacts with Asp-89, a highly conserved acidic residue in the PRib-PP binding site motif. The XPRT structure also incorporates a feature which has not been observed in other PRTase structures. The C-terminal 12 residues of XPRT adopt an unusual extended conformation and make interactions with a neighboring subunit. The very last residue, Arg-152, could form part of the active site of a symmetry-related subunit in the XPRT tetramer.

Cell-wall polysaccharides from Australian red algae of the family Solieriaceae (Gigartinales, Rhodophyta): Novel, highly pyruvated carrageenans from the genus Callophycus

Cell-wall polysaccharides from six species of red algae of the genus Callophycus were mainly galactans comprised predominantly of galactose (Gal) and 3,6-anhydrogalactose (AnGal), and were rich in pyruvate and sulfate. The Fourier Transform Infrared (FTIR) spectra of the polysaccharides superficially resembled that of alpha-carrageenan (composed of the repeating disaccharide carrabiose 2-sulfate), with major bands of absorption indicative of if-linked AnGal, axial 2-sulfate on 4-linked AnGal, and unsulfated, 3-linked Gal. The FTIR spectra of solutions of Callophycus polysaccharides in D2O-phosphate buffer displayed absorption, corresponding to the carboxylate anion of the pyruvate acetal substituent. Methylation analysis showed that 3,4,6-linked Galp (interpreted as 4,6-pyruvated, 3-linked Galp) and 2,4-linked AnGalp (interpreted as 4-linked AnGalp 2-sulfate) were the dominant links, together with significant quantities of 3-linked Galp. Proton-decoupled C-13 nuclear magnetic resonance (NMR) spectroscopy showed the polysaccharides to be composed predominantly of pyruvated carrageenans. The C-13 NMR spectra were completely assigned by a J-modulated spin-echo pulse sequence and 2D experiments employing gradient Heteronuclear Multiple Bond Correlation (HMBC), C-13/H-1 Heteronuclear Multiple Quantum Coherence (HMQC), and HMQC Total Correlation Spectroscopy (HMQC-TOCSY). The Callophycus galactans thus consist predominantly of the novel repeating disaccharide 4',6'-O-(1-carboxyethylidene)carrabiose 2-sulfate and minor amounts of the alpha-carrageenan repeating unit (carrabiose 2-sulfate), and other structural variations. (C) 1997 Elsevier Science Ltd.

Brain structure and symptom dimension relationships in obsessive-compulsive disorder: A voxel-based morphometry study

Background: Obsessive-compulsive disorder (OCD) is a clinically heterogenous disorder characterized by temporally stable symptom dimensions. Past inconsistent results from structural neuroimaging studies of OCD may have resulted from the effects of these specific symptom dimensions as well as other socio-demographic and clinical variables upon gray matter (GM) volume. Methods: GM volume was measured in 25 adult OCD patients and 20 adult healthy controls using voxel-based morphometry (VBM), controlling for age and total brain GM volume. Univariate and multivariate regression analyses were carried out between regions of GM difference and age, age of onset, medication load, OCD severity, depression severity, and separate symptom dimension scores. Results: Significant GM volumetric differences in OCD patients relative to controls were found in dorsal cortical regions, including bilateral BA6, BA46, BA9 and right BA8 (controls > patients), and bilateral midbrain (patients > controls). Stepwise regression analyses revealed highly significant relationships between greater total OCD symptom severity and smaller GM volumes in dorsal cortical regions and larger GM volumes in bilateral midbrain. Greater age was independently associated with smaller GM volumes in right BA6, left BA9, left BA46 and larger GM volumes in right midbrain. Greater washing symptom severity was independently associated with smaller GM volume in right BA6, while there was a trend association between greater hoarding symptom severity and lower GM volume in left BA6. Limitations: The sample was relatively small to examine the relationship between symptom scores and GM volumes. Multiple patients were taking medication and had comorbid disorders. Conclusions: These analyses suggest dorsal prefrontal cortical and bilateral midbrain GM abnormalities in OCD that appear to be primarily driven by the effects of total OCD symptom severity. The results regarding the relationship between GM volumes and symptom dimension scores require examination in larger samples. (C) 2008 Elsevier B.V. All rights reserved.

Population structure and mouse-virulence of Toxoplasma gondii in Brazil

Recent studies found that isolates of Toxoplasma gondii from Brazil were biologically and genetically different from those in North America and Europe. However, to date only a small number of isolates have been analysed from different animal hosts in Brazil. In the present study DNA samples of 46 T. gondii isolates from cats in 11 counties in Sao Paulo state, Brazil were genetically characterised using 10 PCR restriction fragment length polymorphism markers including SAG1, SAG2, SAG3, STUB, GRA6, c22-8, c29-2, L358, PKI and Apico. An additional marker, CS3, that locates on chromosome VIIa and has previously been shown to be linked to acute virulence of T. gondii was also used to determine its association to virulence in mice. Genotyping of these 46 isolates revealed a high genetic diversity with 20 genotypes but no clonal Type I, II or III lineage was found. Two of the 46 isolates showed mixed infections. Combining genotyping data in this study with recent reported results from chickens, dogs and cats in Brazil (total 125 isolates) identified 48 genotypes and 26 of these genotypes had single isolates. Four of the 48 genotypes with multiple isolates identified from different hosts and locations are considered the common clonal lineages in Brazil. These lineages are designated as Types BrI, BrII, BrIII and BrIV. These results indicate that the T. gondii population in Brazil is highly diverse with a few successful clonal lineages expanded into wide geographical areas. In contrast to North America and Europe, where the Type II clonal lineage is overwhelmingly predominant, no Type II strain was identified from the 125 Brazil isolates. Analysis of mortality rates in infected mice indicates that Type BrI is highly virulent, Type BrIII is non-virulent, whilst Type BrII and BrIV lineages are intermediately virulent. In addition, allele types at the CS3 locus are strongly linked to mouse-virulence of the parasite. Thus, T. gondii has an epidemic population structure in Brazil and the major lineages have different biological traits. (C) 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.