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Many proteins of widely differing functionality and structure are capable of binding heparin. Structural characterisations of the many types of such complexes are being reported in ever-increasing number and at improved resolution. Several crystal structures of complexes formed through the interaction of heparin-derived oligosaccharides with one or more protein partners have been described.
Inhibition of CDK2/CA (cyclin-dependent kinase 2/cyclin A complex) activity through blocking of the substrate recognition site in the cyclin A subunit has been demonstrated to be an effective method for inducing apoptosis in tumor cells. We have used the cyclin binding motif (CBM) present in the tumor suppressor proteins p21(WAF1) and p27(KIP1) as a template to optimize the minimal sequence necessary for CDK2/CA inhibition. A series of peptides were prepared, containing nonnatural amino acids, which possess nano- to micromolar CDK2-inhibitory activity. Here we present X-ray structures of the protein complex CDK2/CA, together with the cyclin groove-bound peptides H-Ala-Ala-Abu-Arg-Ser-Leu-Ile-(p-F-Phe)-NH(2) (peptide 1), H-Arg-Arg-Leu-Ile-Phe-NH(2) (peptide 2), Ac-Arg-Arg-Leu-Asn-(m-Cl-Phe)-NH(2) (peptide 3), H-Arg-Arg-Leu-Asn-(p-F-Phe)-NH(2) (peptide 4), and H-Cit-Cit-Leu-Ile-(p-F-Phe)-NH(2) (peptide 5). Some of the peptide complexes presented here were obtained through the novel technique of ligand exchange within protein crystals. This method may find general application for obtaining complex structures of proteins with surface-bound ligands.
The monooxo rhenium(V) complexes of cysteine (complex 1) and cysteine methyl ester (complex 2) were synthesised via a ligand exchange reaction starting from gluconatooxorhenium(V). Unexpectedly, the obtained oxorhenium(V) complex with cysteine methyl ester (2) was partially saponified. Both complexes were characterised by common analytical techniques in their solid state. Thus, an octahedral complex structure with 2(NH2,S) co-ordination in the equatorial plane and one carboxyl group bound trans to the oxo group is proven for complex 2 by X-ray diffraction. Furthermore, the existence of a dioxo species at higher pH was proven for the first time with this type of ligand by determining the nearest co-ordination sphere of the rhenium centre in solution at a pH of 12 using extended X-ray absorption fine structure spectroscopy.
"This article analyses the social factors that promote the formation of complex family structures--with several [nuclear families]...living in the same household, in Spanish society. Although it could be thought that these complex family structures are associated [with] the household structure typical of traditional peasant communities, it is true that they also exist in...modern urban households." Data are primarily taken from the 1990 Active Population Survey. (SUMMARY IN ENG)
We have employed fast atom bombardment mass spectrometry (FAB-MS) to screen the N-linked oligosaccharides of Bowes melanoma tissue plasminogen activator (mt-PA), and recombinant t-PAs produced by Chinese hamster ovary cells (rt-PA) and by a gene-enriched melanoma cell line (rmt-PA). These studies have confirmed the published structures for rt-PA, but are not in agreement with some of the structures reported for mt-PA. In the latter glycoprotein we have identified a novel structure as the major oligosaccharide attached to Asn-184 and Asn-448. This is a biantennary oligosaccharide consisting of a fucosylated trimannosyl core to which are attached two GalNAc(1----4)GlcNAc antennae, one of which carries a sialic acid linked at the 6-position of the GalNAc. Minor constituents are sialylated on both or neither antennae. The sialylated GalNAc moiety is unique in N-linked glycoproteins. The majority of complex structures in rmt-PA contain N-acetyllactosamine moieties at both the Asn-184 and Asn-448 sites with the novel oligosaccharide occurring as a minor component at the Asn-184 site. This study demonstrates the power of mass spectrometric strategies based on high-field two-sector FAB-MS for structure elucidations of natural and recombinant glycoproteins.
[Structure: see text] A recyclable, air- and moisture-stable chiral indium complex in [hmim][PF6-] ionic liquid has been developed. The cycloaddition of a variety of cyclic and open-chained dienes to 2-methacrolein and 2-bromoacrolein resulted in good yields and excellent enantioselectivities (up to 98% ee). Moreover, the chiral In(III) complex can be reused for seven successive cycles with comparable enantioselectivities and yields without loss of catalytic activity.
Ultraviolet spectra of docosahexaenoic acid (DHA) ester and its complex with silver ion were determined, and shifts of ultraviolet absorption peak before and after the complexing reaction were observed. Meanwhile, complexing bonds of DHA ester-silver ion were explained by molecular orbital theory. Thus, complex structure and complexing mechanism were discovered. The results showed that ultraviolet characteristic absorption peak of DHA ester-silver ion complex moved to lower wavelength. It pointed out that distance between energy levels for outer electron transition of complex is increased, and the energy for outer electron transition of complex is bigger than that of DHA ester.
Pex6p belongs to the AAA family of ATPases. Its CHO mutant, ZP92, lacks normal peroxisomes but contains peroxisomal membrane remnants, so called peroxisomal ghosts, which are detected with anti-70-kDa peroxisomal membrane protein (PMP70) antibody. No peroxisomal matrix proteins were detected inside the ghosts, but exogenously expressed green fluorescent protein (GFP) fused to peroxisome targeting signal-1 (PTS-1) accumulated in the areas adjacent to the ghosts. Electron microscopic examination revealed that PMP70-positive ghosts in ZP92 were complex membrane structures, rather than peroxisomes with reduced matrix protein import ability. In a typical case, a set of one central spherical body and two layers of double-membraned loops were observed, with endoplasmic reticulum present alongside the outer loop. In the early stage of complementation by PEX6 cDNA, catalase and acyl-CoA oxidase accumulated in the lumen of the double-membraned loops. Biochemical analysis revealed that almost all the peroxisomal ghosts were converted into peroxisomes upon complementation. Our results indicate that 1) Peroxisomal ghosts are complex membrane structures; and 2) The complex membrane structures become import competent and are converted into peroxisomes upon complementation with PEX6.
An inhibitor complex structure of glycinamide ribonucleotide transformylase (GAR-Tfase; EC 2.1.2.2) from Escherichia coli has been determined with a multisubstrate adduct BW1476U89 to an R-value of 19.1% at 1.96 A resolution. The structure was determined by a combination of molecular and single isomorphous replacement using data from two different monoclinic crystal lattices and collecting data from crystals soaked in 20% (w/v) methyl-pentanediol as cryoprotectant for shock-freezing at -150 degrees C. The multisubstrate adduct is bound in an extended crevice at the interface between the two functional domains of the enzyme. This inhibitor is positioned in the binding site by three sets of tight interactions with its phosphate, glutamate and pyrimidone ring moieties, while its interventing linker atoms are more flexible and adopt two distinct sets of conformations. The highly conserved Arg103, His108 and Gln170 residues that are key in ligand binding and catalysis (His108), have compensatory conformational variation that gives some clues as to their role in substrate specificity and in the formyl transfer. The molecular design of 1476U89 as a multisubstrate adduct inhibitor (Ki approximately 100 pM at pH 8.5), is confirmed as it closely mimics the shape, molecular interaction and combined binding constants of the natural 10-formyltetrahydrofolate (10-CHO-H4F; Km approximately 77.4 microM at pH 8.5) and glycinamide-ribonucleotide (GAR; Km approximately 8.1 microM at pH 8.5) substrates. The stereochemistry of this ligand complex suggests that His108 may act as an electrophile stabilizing the oxyanion of the tetrahedral intermediate that is formed as a result of the direct attack on the 10-CHO-H4F by the amino group of GAR. Structural comparison of the folate binding modes among GAR-Tfase, dihydrofolate reductase and thymidylate synthase reveals that folate derivates bound to GAR-Tfase differentially adopt the trans conformation for the dihedral angle between atoms C-6 and C-9 providing a handle for targeting specific folate-dependent enzymes. The structural information derived from two different discrete conformations of the ligand in the binding site also suggests several leads for the de novo design of inhibitors of GAR-Tfase that may develop into useful chemotherapeutic agents.
We have used two fluorescent probes, NBD and dansyl, attached site-specifically to the serpin plasminogen activator inhibitor-1 (PAI-1) to address the question of whether a common mechanism of proteinase translocation and full insertion of the reactive center loop is used by PAI-1 when it forms covalent SDS-stable complexes with four arginine-specific proteinases, which differ markedly in size and domain composition. Single-cysteine residues were incorporated at position 119 or 302 as sites for specific reporter labeling. These are positions approximately 30 A apart that allow discrimination between different types of complex structure. Fluorescent derivatives were prepared for each of these variants using both NBD and dansyl as reporters of local perturbations. Spectra of native and cleaved forms also allowed discrimination between direct proteinase-induced changes and effects solely due to conformational change within the serpin. Covalent complexes of these derivatized PAI-1 species were made with the proteinases trypsin, LMW u-PA, HMW u-PA, and t-PA. Whereas only minor perturbations of either NBD and dansyl were found for almost all complexes when label was at position 119, major perturbations in both wavelength maximum (blue shifts) and quantum yield (both increases and decreases) were found for all complexes for both NBD and dansyl at position 302. This is consistent with all four complexes having similar location of the proteinase catalytic domain and hence with all four using the same mechanism of full-loop insertion with consequent distortion of the proteinase wedged in at the bottom of the serpin.
Increasing complexity and increased restraints affect the task of patient management in High Dependency Environments, which has become intricate and difficult. Medical knowledge alone is not enough any longer for proper patient care. Management ability and facilities are required. Current medical knowledge should be expanded by management methods and techniques. By looking at management models in the industry, we found striking similarities between the industrial management situation and clinical patient management. Both systems share complexity in structure, complexity in interaction and evolutionary character. Clinical patient management can be compared with a navigation process. The patient is steered by a control system, and course information is given by control dimensions. Clinical patient management becomes a succession of steering activities influenced by the surrounding systems. This system can be structured in three interacting layers: an operational level, in which information is collected and actions executed; a strategic level in which strategies based on goal-oriented mental anticipation of a probabilistic system are formulated; and a normative level at which principles and norms are defined. It is possible then, to define the tools which have to be developed and implemented to improve clinical management capabilities. At the operational level these tools are addressed to improve clinical decision making by providing information in an ergonomical way. They include artifact elimination, data reduction, increase in meaningful information and unwanted data filtering. At the strategic level, tools to check the feasibility of the applied strategies have to be developed, such as: ideal patient course plots and increased training in strategic thinking.(ABSTRACT TRUNCATED AT 250 WORDS)
Most of the techniques used to visualize structures of the synaptonemal complex (SC) are based on specific staining properties or immunocytochemical detection of proteinaceous SC components. The SC is therefore considered to be mostly protein. We have now accomplished visualization of the DNA within the SC by use of the BrdU antibody technique, following BrdU substitution during the last premeiotic S phase. Preparations of mouse meiotic chromosomes were obtained by spreading on a water surface. The DNA content in the SCs, which appeared as light threads, was clearly lower than the DNA content in the surrounding chromatin. At higher magnification, dark, longitudinal structures appeared in these threads. These structures are made up of DNA, which forms the inner part of the lateral elements of the SCs. Within the SCs, DNA is confined mostly to these threads. Thus, DNA staining reveals the same structures already known from protein staining of SCs. The DNA mass surrounding the SCs is often nonhomogeneously distributed along the chromosome axis. The more dense parts appear to be chromomeres. The DNA staining technique described in this paper may therefore be a useful complement to standard protein staining techniques for pachytene chromosomes.
The characterization of autoantibody specificities in rheumatic diseases is important in both diagnostic and basic research areas. Identification of the epitopes recognized by autoantibodies and their clinical and biological significance is not a trivial task. Epitopes may range in complexity from simple linear sequences of amino acids to complex quaternary structures. In addition to this structural complexity the frequency with which an autoantigen and its epitopes are recognized in a patient population may be useful in diagnosis, defining disease subgroups, and may offer information on disease prognosis. In this review recent advances in the epitope mapping of autoantigens in connective tissue diseases are discussed, with particular emphasis placed on the methodologies used to identify epitopes and the classification of the structural features of epitopes. To illustrate the identification of epitope structure, clinically relevant autoantigens, including CENP-A, PM/Scl-100, fibrillarin, filaggrin, Ro-52, and dsDNA, are discussed as examples of each type of epitope.
We have investigated the structure of complexes formed between a series of poly(A)n (n = 30 to 480) and HD40 (helix-destabilizing protein, molecular weight of 40,000), the major protein component of 30 S heterogeneous nuclear ribonucleoprotein particles (hnRNP) from the brine shrimp Artemia salina. Protein HD40 is similar to corresponding hnRNP proteins from higher eukaryotes and the complexes it forms with single-stranded nucleic acids are strikingly similar to the native "beads-on-a-string" structure of hnRNP. Using analytical ultracentrifugation and electron microscopy we find: (1) complexes formed between HD40 and long ribohomopolymers also have a beads-on-a-string structure, showing that the ability to form this structure is an inherent property of HD40, and is not dependent on any structural features of natural RNA; (2) complexes between HD40 and poly(A)160 form disks that are about 3 nm high by 18 nm in diameter and contain 20 HD40 molecules; (3) complexes of HD40 with poly(A)n with fewer than 160 nucleotides form sectors of a disk: 40 nucleotides give rise to a quarter of a disk, 80 nucleotides, half a disk, etc. The molecular weights increase with the size of poly(A)n at the rate of 5300 per nucleotide, a stoichiometry of eight nucleotides per HD40; (4) as the size of the poly(A)n increases beyond 160 nucleotides, the additional nucleoprotein elements may either initiate the formation of a second disk adjacent to the first or stack on top of the first disk to form a 6 nm high helix with a diameter of 18 nm. Based on these results, we propose that the existence of lateral protein-protein interactions that produce the basic 3 nm X 18 nm disk, combined with the marginal stability of the helix result in (a) interruptions of the helix that give rise to the beads-on-a-string appearance of the complexes, and (b) inherent heterogeneity of individual "beads" which may contain one or more turns of the helix. From measurements of HD40 complexes with coliphage MS2 RNA, phi X174 viral DNA as well as with the homopolymers, a bead is estimated to contain an average of approximately 300 nucleotides; approximately 1 X 8 turns of the helix.
Individuals at five grade levels (kindergarten, Grade 2, Grade 4, Grade 6, and college) made pair-comparison judgments of visual complexity. The influence of the presence or absence of six types of visual structure (double symmetry, vertical symmetry, horizontal symmetry, diagonal symmetry, checkerboard organization, and rotational organization) and of amount of contour were examined. Two general developmental trends were revealed: First, the age at which visual structure initially affected complexity judgments varied with the type of structure, independent of amount of contour, within the range of contour values used. Second, there was a uniform increase in the effect of structure on complexity judgments between the fourth and sixth grades. These results are discussed in relation to possible mechanisms of visual pattern encoding and complexity judgment.
Megalin (gp 330) is a large cell surface receptor expressed on the apical surfaces of epithelial tissues, that mediates the binding and internalization of a number of structurally and functionally distinct ligands. In this paper we report the first detailed structural characterization of megalin-derived oligosaccharides. Using strategies based on mass spectrometric analysis, we have defined the structures of the N-glycans of megalin. The results reveal that megalin glycoprotein is heterogeneously glycosylated. The major N-glycans identified belong to the following two classes: high mannose structures and complex type structures, with complex structures being more abundant than high mannose structures. The major nonreducing epitopes in the complex-type glycans are: GlcNAc, Galbeta1-4GlcNAc (LacNAc), NeuAcalpha2-6Galbeta1-4GlcNAc (sialylated LacNAc), GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4GlcNAc (Sd(a)) and Galalpha1-3Galbeta1-4GlcNAc. Most complex structures are characterized by the presence of (alpha1,6)-core fucosylation and the presence of a bisecting GlcNAc residue.
The evidence of supporting phonological deficit as a cause of developmental dyslexia has been accumulating rapidly over the past 2 decades, yet the exact mechanisms underlying this deficit remain controversial. Some authors assume that a temporal processing deficit is the source of the phonological disorder observed in dyslexic children. Others maintain that the phonological deficit in dyslexia is basically linguistic, not acoustic, in nature. Three experiments were conducted and tested the impact of the temporal alteration and the impact of complex syllabic structure on consonant order judgments. Thirteen phonological dyslexics (age 10-13) and 10 controls matched for chronologial age were compared on a Temporal Order Judgment (TOJ) task using the succession of two consonants (/p/ /s/) within a cluster. In order to test the possible relevance of the temporal deficit hypothesis, the task also included two additional conditions where either the two stimuli were artificially slowed or two phonological structures were opposed (CCV and CVCV). As expected, the TOJ performance was significantly poorer in dyslexics than in controls. Moreover, in the "slowed speech" condition dyslexics' performance improved to reach the normal controls' level, whereas manipulating the phonological structure complexity provided no significant improvement. Finally dyslexics' performances, especially on the slowed condition, were found correlated with several tests of phonological processing. These results lend support to the general temporal deficit theory of dyslexia.
The bacterial flagellar motor harnesses ion flow to drive rotary motion, at speeds reaching 100000 rpm and with apparently tight coupling. The functional properties of the motor are quite well understood, but its molecular mechanism remains unknown. Studies of motor physiology, together with mutational and biochemical studies of the components, place significant constraints on the mechanism. Rotation is probably driven by conformational changes in membrane-protein complexes that form the stator. These conformational changes occur as protons move on and off a critical aspartate residue in the stator protein MotB, and the resulting forces are applied to the rotor protein FliG. The bacterial flagellum is a complex structure built from about two dozen proteins. Its construction requires an apparatus at the base that exports many flagellar components to their sites of installation by way of an axial channel through the structure. The sequence of events in assembly is understood in general terms, but not yet at the molecular level. A fuller understanding of motor rotation and flagellar assembly will require more data on the structures and organization of the constituent proteins.