Solution structures by H-1 NMR of the novel cyclic trypsin inhibitor SFTI-1 from sunflower seeds and an acyclic permutant

SFTI-1 is a recently discovered cyclic peptide trypsin inhibitor from sunflower seeds comprising 14 amino acid residues. It is the most potent known Bowman-Birk inhibitor and the only naturally occurring cyclic one. The solution structure of SFTI-1 has been determined by H-1-NMR spectroscopy and compared with a synthetic acyclic permutant. The solution structures of both are remarkably similar. The lowest energy structures from each family of 20 structures of cyclic and acyclic SFTI-1 have an rmsd over the backbone and heavy atoms of 0.29 Angstrom and 0.66 Angstrom, respectively. The structures consist of two short antiparallel beta -strands joined by an extended loop containing the active site at one end. Cyclic SFTI-1 also has a hairpin turn completing the cycle. Both molecules contain particularly stable arrangements of cross-linking hydrogen bonds between the beta -strands and a single disulfide bridge, making them rigid and well defined in solution. These stable arrangements allow both the cyclic and acyclic variants of SFTI-1 to inhibit trypsin with very high potencies (0.5 nM and 12.1 nM, respectively). The cyclic nature of SFTI-1 appears to have evolved to provide higher trypsin inhibition as well as higher stability. The solution structures are similar to the crystal structure of the cyclic inhibitor in complex with trypsin. The lack of a major conformational change upon binding suggests that the structure of SFTI-1 is rigid and already pre-organized for maximal binding due to minimization of entropic losses compared to a more flexible ligand. These properties make SFTI-1 an ideal platform for the design of small peptidic pharmaceuticals or pesticides. (C) 2001 Academic Press.

Engineering the structures of nanoporous clays with micelles of alkyl polyether surfactants

Composite clay nanostructures (CCNs) were observed in intercalating Laponite clay with alumina in the presence of alkyl polyether surfactants which contain hydrophobic alkyl chains and ether groups. Such nanostructured clays are highly porous solids consisting of randomly orientated clay platelets intercalated with alumina nanoparticles. The pores in the product solids are larger than the dimension of the surfactant molecules, ranging from 2 to 10 nm. This suggests that the micelles of the surfactant molecules, rather than the molecules, act as templates in the synthesis. Interestingly, it is found that the size of the framework pores was directly proportional to the amount of the surfactants in terms of moles, but shows no evident dependence on the size of the surfactant molecules. Broad pore size distributions were observed for the product CCNs. This study demonstrates that introducing surfactants in the pillaring process of clays is a powerful strategy for tailoring the pore structures of nanoporous clays. With this new technique, it is possible to design and engineer such composite clay nanostructures with desired pore and surface properties by the proper choice of surfactant amounts and preparation conditions.

Significance of serum early pregnancy factor concentrations during pregnancy and embryonic development in Sminthopsis macroura (Spencer) (Marsupialia : Dasyuridae)

Marsupial pregnancy differs from that in eutherians in duration, placentation and hormonal profile so much so that maternal recognition of pregnancy may not occur in polyovular marsupials. However, a comparison of gravid and non-gravid uteri reveals differences indicative of histological and physiological adaptations to pregnancy. In the present study, the hypothesis that embryo-maternal signalling occurs in polyovular marsupials was tested by examining serum from non-pregnant and pregnant Sminthopsis macroura for the presence of early pregnancy factor (EPF), a serum protein secreted by the ovary in response to the presence of a newly fertilized egg in the oviduct. EPF is detectable in the serum of pregnant, but not in non-pregnant, females in all eutherians studied to date. In the present study, EPF was detected in S. macroura serum by the rosette inhibition test during the first 9 days of the 10.7 day gestation period in this marsupial. However, EPF was not detected on day 10, just before parturition, or in non-pregnant or preovulatory animals. Immunohistochemical analysis of ovaries from gravid and non-gravid animals demonstrates that EPF is found in the capillaries, interstitial spaces and secretory cells of the corpus luteum. It is concluded that the spatiotemporal pattern of EPF activity described strongly indicates that maternal recognition of pregnancy in marsupials is mediated, at least in part, by EPF. Because the endocrinological milieu is the same in pregnant and non-pregnant marsupials, the possibility of using marsupials as an experimental system for studying EPF function unconfounded by hormonal effects is presented.

Homeotic genes influence the axonal pathway of a Drosophila embryonic sensory neuron

Each abdominal hemisegment of the Drosophila embryo has two sensory neurons intimately associated with a tracheal branch. During embryogenesis, the axons of these sensory neurons, termed the v'td2 neurons, enter the CNS and grow toward the brain with a distinctive pathway change in the third thoracic neuromere. We show that the axons use guidance cues that are under control of the bithorax gene complex (BX-C). Pathway defects in mutants suggest that a drop in Ultrabithorax expression permits the pathway change in the T3 neuromere, while combined Ultrabithorax and abdominal-A expression represses it in the abdominal neuromeres. We propose that the axons do not respond to a particular segmental identity in forming the pathway change; rather they respond to pathfinding cues that come about as a result of a drop in BX-C expression along the antero-posterior axis of the CNS.

Crystal structures of free, IMP-, and GMP-bound Escherichia coli hypoxanthine phosphoribosyltransferase

Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 Angstrom resolution) and for the enzyme in complex with the reaction products, inosine 5'-monophosphate (IMP) and guanosine 5-monophosphate (GMP) (2.8 Angstrom resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Calpha atoms of 1.0 Angstrom. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for divalent metal ion, Usually Mg2+ in vivo. In the free structure, a Mg2+, is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp 163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg2+ complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.

Linear ketenimines. Variable structures of C,C-dicyanoketenimines and C,C-bis-sulfonylketenimines

C,C-Dicyanoketenimines 10a-c were generated by flash vacuum thermolysis of ketene NS-acetals 9a-c or by thermal or photochemical decomposition of alpha-azido-,beta-cyanocinnamonitrile 11. In the latter reaction, 3,3-dicyano-2-phenyl-1-azirine 12 is also formed. IR spectroscopy of the keteniminines isolated in Ar matrixes or as neat films, NMR spectroscopy of 10c, and theoretical calculations (B3LYP/6-31G*) demonstrate that these ketenimines have variable geometry, being essentially linear along the CCN-R framework in polar media (neat films and solution), but in the gas phase or Ar matrix they are bent, as is usual for ketenimines. Experiments and calculations agree that a single CN substituent as in 13 is not enough to enforce linearity, and sulfonyl groups are less effective that cyano groups in causing linearity. C,C-Bis(methylsulfonyl)ketenimines 4-5 and a C-cyano-C-(methylsulfonyl)ketenimine 15 are not linear. The compound p-O2NC6H4N=C= C(COOMe)2 previously reported in the literature is probably somewhat linearized along the CCNR moiety. A computational survey (B3LYP/6-31G*) of the inversion barrier at nitrogen indicates that electronegative C-substituents dramatically lower the barrier; this is also true of N-acyl substituents. Increasing polarity causes lower barriers. Although N-alkylbis(methylsulfonyl)ketenimines are not calculated to be linear, the barriers are so low that crystal lattice forces can induce planarity in N-methylbis(methylsulfonyl)ketenimine 3.

Expert system application on preliminary design of water retaining structures

Design of liquid retaining structures involves many decisions to be made by the designer based on rules of thumb, heuristics, judgment, code of practice and previous experience. Various design parameters to be chosen include configuration, material, loading, etc. A novice engineer may face many difficulties in the design process. Recent developments in artificial intelligence and emerging field of knowledge-based system (KBS) have made widespread applications in different fields. However, no attempt has been made to apply this intelligent system to the design of liquid retaining structures. The objective of this study is, thus, to develop a KBS that has the ability to assist engineers in the preliminary design of liquid retaining structures. Moreover, it can provide expert advice to the user in selection of design criteria, design parameters and optimum configuration based on minimum cost. The development of a prototype KBS for the design of liquid retaining structures (LIQUID), using blackboard architecture with hybrid knowledge representation techniques including production rule system and object-oriented approach, is presented in this paper. An expert system shell, Visual Rule Studio, is employed to facilitate the development of this prototype system. (C) 2002 Elsevier Science Ltd. All rights reserved.

Coordinated expression of scleraxis and Sox9 genes during embryonic development of tendons and cartilage

Embryonic development of tendons is in close association with that of cartilage and bone. Although these tissues are derived from mesenchymal progenitor cells which also give rise to muscle and fat, their fates clearly diverse in early embryonic stages, Transcription factors may play pivotal roles in the process of determination and differentiation of tendon cells as well as other cells in the skeletal system. Scleraxis, a basic helix-loop-helix (bHLH) type transcription factor. is expressed in mesenchymal progenitors that later form connective tissues including tendons. Sox9 is an HMG-box containing transcription factor, which is expressed at high levels in chondrocytes. We hypothesized that the two transcription factors regulate the fate of cells that interact with each other at the interface between the two tissues during divergence of their differentiation pathways, To address this point, we investigated scleraxis and Sox9 rnRNA expression during mouse embyogenesis focusing on the coordinated development of tendons and skeletons, In the early stage of mesenchymal tissue development at 10.5 d.p.c., scleraxis and Sox9 transcripts were expressed in the mesenchymal progenitor cells in the appendicular and axial mesenchyme. At 11.5 d.p.c.. scleraxis transcripts were observed in the mesenchymal tissue surrounding skeletal primordia which express Sox9. From this stage, scleraxis expression was closely associated with, but distinct from, formation of skeletal primordia, At 13.5 d.p.c., scleraxis was expressed broadly in the interface between muscle and skeletal primordia while Sox9 expression is confined within the early skeletal primordia. Then. at 15.5 d.p.c., scleraxis transcripts were more restricted to tendons. These observations revealed the presence of temporal and spatial association of scleraxis expression during embryonic development of tendon precursor cells in close association with that of So,0 expression in chondrogenic cells in skeletal tissues. (C) 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.

Discovery and structures of the cyclotides: novel macrocyclic peptides from plants

Circular disulfide-rich polypeptides were unknown a decade ago but over recent years a large family of such molecules has been discovered, which we now refer to as the cyclotides. They are typically about 30 amino acids in size, contain an N- to C-cyclised backbone and incorporate three disulfide bonds arranged in a cystine knot motif. In this motif, an embedded ring in the structure formed by two disulfide bonds and their connecting backbone segments is penetrated by the third disulfide bond. The combination of this knotted and strongly braced structure with a circular backbone renders the cyclotides impervious to enzymatic breakdown and makes them exceptionally stable. This article describes the discovery of the cyclotides in plants from the Rubiaceae and Violaceae families, their chemical synthesis, folding, structural characterisation, and biosynthetic origin. The cyclotides have a diverse range of biological applications, ranging from uterotonic action, to anti-HIV and neurotensin antagonism. Certain plants from which they are derived have a history of uses in native medicine, with activity being observed after oral ingestion of a tea made from the plants. This suggests the possibility that the cyclotides may be orally bioavailable. They therefore have a range of potential applications as a stable peptide framework.

Expression and role of roundabout-1 in embryonic Xenopus forebrain

The receptor Roundabout-1 (Robo1) and its ligand Slit are known to influence axon guidance and central nervous system (CNS) patterning in both vertebrate and nonvertebrate systems. Although Robo-Slit interactions mediate axon guidance in the Drosophila CNS, their role in establishing the early axon scaffold in the embryonic vertebrate brain remains unclear. We report here the identification and expression of a Xenopus Robo1 orthologue that is highly homologous to mammalian Robo1. By using overexpression studies and immunohistochemical and in situ hybridization techniques, we have investigated the role of Robo1 in the development of a subset of neurons and axon tracts in the Xenopus forebrain. Robo1 is expressed in forebrain nuclei and in neuroepithelial cells underlying the main axon tracts. Misexpression of Robo1 led to aberrant development of axon tracts as well as the ectopic differentiation of forebrain neurons. These results implicate Robo1 in both neuronal differentiation and axon guidance in embryonic vertebrate forebrain. (C) 2002 Wiley-Liss, Inc.

Characteristic zirconium-rich coring structures in Mg-Zr alloys

The most characteristic feature of the microstructure of a magnesium alloy that contains more than a few tenths per cent soluble zirconium is the zirconium-rich cores that exist in most grains. The morphology, distribution and composition of cores observed in a Mg-0.56%Zr alloy and the small particles present in them were investigated. (C) 2002 Acta Materialia Inc. Published by Elsevier Science Ltd. All rights reserved.

Exploring privileged structures: the combinatorial synthesis of cyclic peptides

Head-to-tail cyclic peptides have been reported to bind to multiple, unrelated classes of receptor with high affinity. They may therefore be considered to be privileged structures. This review outlines the strategies by which both macrocyclic cyclic peptides and cyclic dipeptides or diketopiperazines have been synthesised in combinatorial libraries. It also briefly outlines some of the biological applications of these molecules, thereby justifying their inclusion as privileged structures.