The solution structure of C1-T1, a two-domain proteinase inhibitor derived from a circular precursor protein from Nicotiana alata

A two-domain portion of the proteinase inhibitor precursor from Nicotiana alata (NaProPI) has been expressed and its structure determined by NMR spectroscopy. NaProPI contains six almost identical 53 amino acid repeats that fold into six highly similar domains; however, the sequence repeats do nut coincide with the structural domains. Five of the structural domains comprise the C-terminal portion of one repeat and the N-terminal portion of the next. The sixth domain contains the C-terminal portion of the sixth repeat and the N-terminal portion of the first repeat. Disulphide bonds link these C and N-terminal fragments to generate the clasped-bracelet fold of NaProPI. The three-dimensional structure of NaProPI is not known, but it is conceivable that adjacent domains in NaProPI interact to generate the circular bracelet with the N and C termini in close enough proximity to facilitate formation of the disulphide bonds that form the clasp The expressed protein, examined in the current study, comprises residues 25-135 of NaProPI and encompasses the first two contiguous structural domains, namely the chymotrypsin inhibitor C1 and the trypsin inhibitor T1, joined by a five-residue linker, and is referred to as C1-T1. The tertiary structure of each domain in C1-T1 is identical to that found in the isolated inhibitors. However, no nuclear Overhauser effect contacts are observed between the two domains and the five-residue linker adopts an extended conformation. The absence of interactions between the domains indicates that adjacent domains do not specifically interact to drive the circularisation of NaProPI. These results are in agreement with recent data which describe similar PI precursors from other members of the Solanaceae having two, three, or four repeats. The lack of strong interdomain association is likely to be important for the function of individual inhibitors by ensuring that there is no masking of reactive sites upon release from the precursor. (C) 2001 Academic Press.

Molecular motion in nanocomposites of poly(ethylene oxide) and montmorillonite

Motion of chains of poly(ethylene oxide) within the interlayer spacing of 2:1 phyllosilicate/montmorillonite was studied with H-1 and C-13 NMR spectroscopy. Measurements of the H-1 NMR line widths and relaxation times across a large temperature range were used to determine the effect of bulk thermal transitions on polymer chain motion within the nanocomposites. The results were consistent with previous reports of low apparent activation energies of motion. Details of the frequency and geometry of motion were obtained from a comparison of the C-13 cross-polarity/magic-angle spinning spectra and relaxation times of the nanocomposite with those of the pure polymer. (C) 2001 John Wiley & Sons, Inc.

Primal joints and hind-leg cuts of entire and castrated Javan Rusa (Cervus timorensis russa) stags

The effects of castration on the primal joints and the cuts of the leg joint of Javan rusa (Cervus timorensis russa) stag carcases was investigated at three slaughter ages (13, 19 and 25 months). Castration reduced the weights of some primal joints in the 19- and 25-months age groups, but not at 13 months. At 19 months, the neck, and neck plus chuck, were heavier by 35 and 17% respectively in entires (P < 0.05), and at 25 months entires had heavier carcases, shoulder, forequarter and hindquarter (P < 0.05). The leg and saddle joints were approximately 39 and 18% of the side, respectively, for both treatments and all ages. The proportions of the neck, and neck plus chuck, were higher (P < 0.05) in 19-month old entires than castrates. There were few significant differences between treatments in the weight or proportion of the hind leg cuts at any slaughter age, but in the 25-months group the silverside was 8% (P < 0.05) heavier in entires. In both castrates and entires, there appeared to be an increase in the percentage of the rump as the animals grew from 13 to 19 months of age. (C) 2001 Elsevier Science Ltd. All rights reserved.

Assessment of the ability to model proteins with leucine-rich repeats in light of the latest structural information

The three-dimensional structures of leucine-rich repeat (LRR) -containing proteins from five different families were previously predicted based on the crystal structure of the ribonuclease inhibitor. using an approach that combined homology-based modeling, structure-based sequence alignment of LRRs, and several rational assumptions. The structural models have been produced based on very limited sequence similarity, which, in general. cannot yield trustworthy predictions. Recently, the protein structures from three of these five families have been determined. In this report we estimate the quality of the modeling approach by comparing the models with the experimentally determined structures. The comparison suggests that the general architecture, curvature, interior/exterior orientations of side chains. and backbone conformation of the LRR structures can be predicted correctly. On the other hand. the analysis revealed that, in some cases. it is difficult to predict correctly the twist of the overall super-helical structure. Taking into consideration the conclusions from these comparisons, we identified a new family of bacterial LRR proteins and present its structural model. The reliability of the LRR protein modeling suggests that it would be informative to apply similar modeling approaches to other classes of solenoid proteins.

Crystal structure of yeast acetohydroxyacid synthase: A target for herbicidal inhibitors

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) catalyzes the first step in branched-chain amino acid biosynthesis. The enzyme requires thiamin diphosphate and FAD for activity, but the latter is unexpected, because the reaction involves no oxidation or reduction. Due to its presence in plants, AHAS is a target for sulfonylurea and imidazolinone herbicides. Here, the crystal structure to 2.6 A resolution of the catalytic subunit of yeast AHAS is reported. The active site is located at the dimer interface and is near the proposed herbicide-binding site. The conformation of FAD and its position in the active site are defined. The structure of AHAS provides a starting point for the rational design of new herbicides. (C) 2002 Elsevier Science Ltd.

Synthesis and structural properties of patellamide a derivatives and their copper(II) compounds

The synthesis, characterization and copper(II) coordination chemistry of three new cyclic peptide ligands, PatJ(1) (cyclo-(Ile -Thr- (Gly)Thz-lle-Thr(Gly)Thz)), PatJ(2) (cyclo-(Ile-Thr(Gly)Thz-(D)-Ile-Thr-(Gly)Thz)), and PatL (cyclo-(Ile-Ser-(Gly)Thz-Ile-Ser(Gly)Thz)) are reported. All of these cyclic peptides and PatN (cyclo-(Ile-Ser(Gly)Thz-Ile-Thr-(Gly)Thz)) are derivatives of patellamide A and have a [24]azacrown-8 macrocyclic structure. All four synthetic cyclic peptides have two thiazole rings but, in contrast to patellamide A, no oxazoline rings. The molecular structure of PatJ1, determined by X-ray crystallography, has a saddle conformation with two close-to-co-parallel thiazole rings, very similar to the geometry of patellamide D. The two coordination sites of PatJ1 with thiazole-N and amide-N donors are each well preorganized for transition metal ion binding. The coordination of copper(II) was monitored by UV/Vis spectroscopy, and this reveals various (meta)stable mono- and dinuclear copper(II) complexes whose stoichiometry was confirmed by mass spectra. Two types of dinuclear copper(II) complexes, [Cu-2(H4L)(OH2)(n)](2+) (n = 6, 8) and [Cu-2(H4L)(OH2)(n)] (n=4, 6; L=PatN, PatL, PatJ1, PatJ2) have been identified and analyzed structurally by EPR spectroscopy and a combination of spectra simulations and molecular mechanics calculations (MM-EPR). The four structures are similar to each other and have a saddle conformation, that is, derived from the crystal structure of PatJ(1) by a twist of the two thiozole rings. The small but significant structural differences are characterized by the EPR simulations.

Theory of mind and peer acceptance in preschool children

Two studies tested the hypothesis that preschool children's theory of mind ability is related to their levels of peer acceptance. In Study 1, 78 children between the ages of 4 and 6 provided peer nominations that allowed determination of social preference and social impact scores, and classification in one of five peer status groups (following Coie & Dodge, 1983). Children were also tested on five different theory of mind tasks. The results showed that theory of mind scores were significantly related to social preference scores in a subsample of children who were over 5 years old. Further, popular children were found to score higher on theory of mind tasks than children classified as rejected. Study 2 replicated and extended the first study with a new sample of 87 4- to 6-year-old children. Study 2 included measures of peer acceptance, theory of mind ability and verbal intelligence, as well as teacher ratings of prosocial and aggressive behaviours. The results of Study 2 showed that for the total group of children, prosocial behaviour was the best predictor of social preference scores. When the Study 2 sample was split into older and younger children, theory of mind ability was found to be the best predictor of social preference scores for the older children (over age 5), while aggressive and prosocial behaviours were the best predictors of peer acceptance in the younger children. Overall, the pattern of results suggests that the impact of theory of mind ability on peer acceptance is modest but increases with children's age.

Children's body knowledge: Understanding 'life' as a biological goal

Two studies assessed the development of children's understanding of life as a biological goal of body functioning. In Study 1, 4-to-10-year-old children were given an interview consisting of a series of structured questions about the location and function of various body organs. Their responses were coded both for factual correctness and for appeals to the goal of maintaining life. The results showed a gradual increase in children's factual knowledge across this age range but an abrupt increase in appeals to life between the ages of 4 and 6. Analyses of the 4-year-olds' responses suggested that appeals to life were associated with increased knowledge of organ function, but not of organ location. Study 2 was designed to replicate the pattern found in Study I. A continuous sample of 4-to 5-year-old children was administered an abbreviated version of the interview from Study 1. Children's understanding of life as a biological goal was again found to be predictive of their knowledge of organ function, but not of organ location. These results indicate a reorganization in children's understanding of the body between the ages of 4 and 6, which coincides with children's discovery of 'life' as a biological goal for bodily function.

Development of preferences for the human body shape in infancy

Two studies investigated the development of infants' visual preferences for the human body shape. In Study 1, infants of 12,15 and 18 months were tested in a standard preferential looking experiment, in which they were shown paired line drawings of typical and scrambled bodies. Results indicated that the 18-month-olds had a reliable preference for the scrambled body shapes over typical body shapes, while the younger infants did not show differential responding. In Study 2, 12- and 18-month-olds were tested with the same procedure, except that the typical and scrambled body stimuli were photographic images. The results of Study 2 again indicated that only the 18-month-olds had a reliable preference for the scrambled body shapes. This finding contrasts sharply with infants' precocious preferences for human faces, suggesting that infants' learning about human faces and human bodies follow different developmental trajectories. (C) 2002 Elsevier Science B.V. All rights reserved.

Characterization of redox centers in dimethyl sulfide dehydrogenase from Rhodovulum sulfidophilum

Dimethyl sulfide dehydrogenase from the purple phototrophic bacterium Rhodovulum sulfidophilum catalyzes the oxidation of dimethyl sulfide to dimethyl sulfoxide. Recent DNA sequence analysis of the ddh operon, encoding dimethyl sulfide dehydrogenase (ddhABC), and biochemical analysis (1) have revealed that it is a member of the DMSO reductase family of molybdenum enzymes and is closely related to respiratory nitrate reductase (NarGHI). Variable temperature X-band EPR spectra (120122 K) of purified heterotrimeric dimethyl sulfide dehydrogenase showed resonances arising from multiple redox centers, Mo(V), [3Fe-4S](+), [4Fe-4S](+), and a b-type heme. A pH-dependent EPR study of the Mo(V) center in (H2O)-H-1 and (H2O)-H-2 revealed the presence of three Mo(V) species in equilibrium, Mo(V)-OH2, Mo(v)-anion, and Mo(V)-OH. Above pH 8.2 the dominant species was Mo(V)-OH. The maximum specific activity occurred at pH 9.27. Comparison of the rhombicity and anisotropy parameters for the Mo(V) species in DMS dehydrogenase with other molybdenum enzymes of the DMSO reductase family showed that it was most similar to the low-pH nitrite spectrum of Escherichia coli nitrate reductase (NarGHI), consistent with previous sequence analysis of DdhA and NarG. A sequence comparison of DdhB and NarH has predicted the presence of four [Fe-S] clusters in DdhB. A [3Fe-4S](+) cluster was identified in dimethyl sulfide dehydrogenase whose properties resembled those of center 2 of NarH. A [4Fe-4S](+) cluster was also identified with unusual spin Hamiltonian parameters, suggesting that one of the iron atoms may have a fifth non-sulfur ligand. The g matrix for this cluster is very similar to that found for the minor conformation of center 1 in NarH [Guigliarelli, B., Asso, M., More, C., Augher, V., Blasco, F., Pommier, J., Giodano, G., and Bertrand, P. (1992) Eur. J. Biochem. 307,63-68]. Analysis of a ddhC mutant showed that this gene encodes the b-type cytochrome in dimethyl sulfide dehydrogenase. Magnetic circular dichroism studies revealed that the axial ligands to the iron in this cytochrome are a histidine and methionine, consistent with predictions from protein sequence analysis. Redox potentiometry showed that the b-type cytochrome has a high midpoint redox potential (E-o = +315 mV, pH 8).

A study of "white spotted kidneys" in cattle

A study was performed at an abattoir in Australia, in an attempt to correlate focal chronic interstitial nephritis (FCIN) producing the so-called white spotted kidney, with Leptospira spp. and other pathogens in cattle. Samples of kidneys, urine and blood were collected immediately after slaughter from 46 two-year-old heifers, and 72 cows and bulls with gross lesions consistent with FCIN. The same samples were also collected from nine heifers and 12 cows with no gross kidney lesions. Aqueous humour was also collected from the eye of 17 of the adult animals. The sera were processed by a microscopic agglutination test for leptospira antibodies, while all the other samples were cultured for Leptospira spp. and also processed for routine aerobic and anaerobic culture for other pathogens. Sub-samples from all the kidneys were fixed in 10% buffered formalin and processed histologically. Antibody titers of 1:400 or higher for Lepstospira borgpeterseni serovar hardjo were found in six adult animals with FCIN and in one adult animal with no gross kidney changes, while antibody titers of 1:400 to L borgpeterseni serovar tarassovi were found in only one animal with FCIN. L. borgpeterseni serovar hardjo was isolated from the urine and kidney of one adult animal and from the urine of another adult animal, both with FCIN. No pathogens were isolated from any of the other samples. The histological lesions were consistent in most cases with FCIN. The results suggest that neither Leptospira spp. nor active infection by other bacteria are associated with c the so-called white spotted kidneys. (C) 2002 Elsevier Science B.V. All rights reserved.

Conformational changes in monolayers of a cationic amphiphilic porphyrin on saline subphases

Langmuir monolayer films of the tetracationic porphyrin tetrakis(octadecyl-4-pyridin ium)porphyrinatozinc(II) bromide on various salt containing subphases were analyzed using surface pressure-area isotherms and X-ray reflectivity. The use of these complementary techniques showed that the porphyrin molecules undergo changes in conformation upon compression. Two main phases were identified, one in which the porphyrin moiety is parallel to the subphase and one in which the porphyrin moiety is tilted out of the plane. The addition of different salts into the subphase brought about changes in film behaviour, which are explained in terms of a lyotropic series. Copyright (C) 2002 Society of Porphyrins,& Phthalocyanines.

beta-strand mimicking macrocyclic amino acids: Templates for protease inhibitors with antiviral activity

New amino acids are reported in which component macrocycles are constrained to mimic tripeptides locked in a beta-strand conformation. The novel amino acids involve macrocycles functionalized with both an N- and a C-terminus enabling addition of appendages at either end to modify receptor affinity, selectivity, or membrane permeability. We show that the cycles herein are effective templates within inhibitors of HIV-1 protease. Eleven compounds originating from such bifunctionalized cyclic templates are potent inhibitors of HIV-1 protease (Ki 0.3-50 nM; pH 6.5, I = 0.1 M). Unlike normal peptides comprising amino acids, five of these macrocycle-containing compounds are potent antiviral agents with sub-micromolar potencies (IC50 170-900 nM) against HIV-1 replication in human MT2 cells. The most active antiviral agents are the most lipophilic, with calculated values of LogD(6.5) greater than or equal to 4. All molecules have a conformationally constrained 17-membered macrocyclic ring that has been shown to structurally mimic a tripeptide segment (Xaa)-(Val/Ile)-(Phe/Tyr) of a peptide substrate in the extended conformation. The presence of two trans amide bonds and a para-substituted aromatic ring prevents intramolecular hydrogen bonds and fixes the macrocycle in the extended conformation. Similarly constrained macrocycles may be useful templates for the creation of inhibitors for the many other proteins and proteases that recognize peptide beta-strands.

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NAIR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

Towards a taxonomic revision of the genus Echinococcus

Echinococcus remains a significant public health problem worldwide and, in several regions, the aetiological agents of cystic hydatid disease/echinococcosis are extending their range. The taxonomy of Echinococcus has been a controversial issue for decades, but the outcome of recent molecular epidemiological studies has served to reinforce proposals made ten years ago to revise the taxonomy of Echinococcus. A formal nomenclature is essential for effective communication, and provides the stability that underpins epidemiological investigations. It will also serve to recognize the contribution of early taxonomists.