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PubMed1976

Effects of cations upon chloroplast membrane subunit. Interactions and excitation energy distribution.

When isolated chloroplasts from mature pea (Pisum sativum) leaves were treated with digitonin under "low salt" conditions, the membranes were extensively solubilized into small subunits (as evidenced by analysis with small pore ultrafilters). From this solubilized preparation, a photochemically inactive chlorophyll - protein complex (chlorophyll alpha/beta ratio, 1.3) was isolated. We suggest that the detergent-derived membrane fragment from mature membranes is a structural complex within the membrane which contains the light-harvesting chlorophyll alpha/beta protein and which acts as a light-harvesting antenna primarily for Photosystem II. Cations dramatically alter the structural interaction of the light-harvesting complex with the photochemically active system II complex. This interaction has been measured by determining the amount of protein-bound chlorophyll beta and Photosystem II activity which can be released into dispersed subunits by digitonin treatment of chloroplast lamellae. When cations are present to cause interaction between the Photosystem II complex and the light-harvesting pigment - protein, the combined complexes pellet as a "heavy" membranous fraction during differential centrifugation of detergent treated lamellae. In the absence of cations, the two complexes dissociate and can be isolated in a "light" submembrane preparation from which the light-harvesting complex can be purified by sucrose gradient centrifugation. Cation effects on excitation energy distribution between Photosystems I and II have been monitored by following Photosystem II fluorescence changes under chloroplast incubation conditions identical to those used for detergent treatment (with the exception of chlorophyll concentration differences and omission of detergents). The cation dependency of the pigment - protein complex and Photosystem II reaction center interactions measured by detergent fractionation, and regulation of excitation energy distribution as measured by fluorescence changes, were identical. We conclude that changes in substructural organization of intact membranes, involving cation induced changes in the interaction of intramembranous subunits, are the primary factors regulating the distribution of excitation energy between Photosystems II and I.

Cell Fractionation↗
PubMed1990

Five children with del (2)(q31q33) and one individual with dup (2)(q31q33) from a single family: review of brain, cardiac, and limb malformations.

Five matings to a dir ins (6;2)(q16;q31q33) carrier have produced a high frequency (42%) of offspring with unbalanced karyotypes. Five children have the derivative chromosome 2 resulting in del (2)(q31q33) and one individual received the derivative chromosome 6 leading to dup (2)(q31q33). The findings associated with the deletion include pre- and postnatal growth retardation, developmental delay, minor facial anomalies, seizures, complex structural heart defects, and limb deficiency. Autopsy of one individual showed complex brain malformations including hydrocephalus secondary to obstruction of the foramina of Monro, extensive heterotopias and polymicrogyria, and an unusual form of total anomalous pulmonary venous return. We compare the findings in these children to those of previously reported cases and construct an overview of the range of anomalies. Apparently, no other individual with dup (2)(q31q33) has been described. We compare the physical peculiarities of our patient with those of individuals with duplications of overlapping regions of 2q.

Abnormalities, Multiple↗
PubMed1995

Structures of partridge egg-white lysozyme with and without tri-N-acetylchitotriose inhibitor at 1.9 A resolution.

The three-dimensional structures of native partridge egg-white lysozyme (PEWL) and PEWL complexed with tri-N-acetylchitotriose inhibitor have been determined crystallographically and refined at 1.9 A resolution. Crystals of native and complexed protein are isomorphous and have space group and cell dimensions that are identical to those of hen egg-white lysozyme (HEWL) under similar crystallization conditions. Full occupancy of the trisaccharide in the inhibitor complex has allowed definitive modeling and refinement of all three sugar residues, located at subsites A, B, and C in the PEWL active site. A comparison has been made with HEWL/inhibitor complexes in which coordinates were either not refined (Blake CCF, et al., 1967, Proc R Soc B 167:378-388) or were refined at partial occupancy (Cheetham JC, Artymiuk PJ, Phillips DC, 1992, J Mol Biol 224:613-628). Although the loop comprising residues 70-75 is located on the surface of the protein and not near the active site, it appears to be affected indirectly by trisaccharide binding such that the loop shifts toward the active site and becomes relatively immobilized. The source of this loop movement appears to be the anchoring of Trp62, located in the active site cleft, as it forms a hydrogen bond with O6 of the N-acetylglucosamine at site C. Good electron density for the trisaccharide in the PEWL complex structure shows that Asp 101 is involved in hydrogen bonding interactions with the terminal sugar residue.

Animals↗
PubMed2006

Crystal structures of human glycerol 3-phosphate dehydrogenase 1 (GPD1).

Homo sapiens L-alpha-glycerol-3-phosphate dehydrogenase 1 (GPD1) catalyzes the reversible biological conversion of dihydroxyacetone (DHAP) to glycerol-3-phosphate. The GPD1 protein was expressed in Escherichia coli, and purified as a fusion protein with glutathione S-transferase. Here we report the apoenzyme structure of GPD1 determined by multiwavelength anomalous diffraction phasing, and other complex structures with small molecules (NAD+ and DHAP) by the molecular replacement method. This enzyme structure is organized into two distinct domains, the N-terminal eight-stranded beta-sheet sandwich domain and the C-terminal helical substrate-binding domain. An electrophilic catalytic mechanism by the epsilon-NH3+ group of Lys204 is proposed on the basis of the structural analyses. In addition, the inhibitory effects of zinc and sulfate on GPDHs are assayed and discussed.

Catalysis↗
PubMed2004

A small fraction of dermatan sulfate with significantly increased anticoagulant activity was selected by interaction with the first complement protein.

Dermatan sulfate (DS) is a member of the family of structurally complex, sulfated, linear heteropolysaccharides called glycosaminoglycans (GAGs). It has a similar structure to heparin and heparan sulfate (HS), but with acetylgalactosamine replacing glucosamine, and the uronic acid moiety, mainly iduronic, joined 1-->3 to the hexosamine. We are studying the relationships between structure and activities of dermatan sulfate, in particular those associated with the thrombin inhibition mediated by heparin cofactor II (HCII). As we have demonstrated with heparin, a small fraction of dermatan sulfate was isolated by precipitation with the first component of the complement system, under very specific conditions of low ionic strength, and the presence of calcium ions. The sulfate content and the anticoagulant activity of the dermatan sulfate fraction isolated in the precipitate were three and four times greater respectively than the starting material. Our in vivo studies showed that this fraction has threefold higher thrombolytic activity than the DS. All these results suggest that this fraction could be used as a therapeutic agent for thrombi dissolution.

Acetylgalactosamine↗
PubMed1998

Perceiving topological structure of 2-D patterns.

Four experiments investigated observers' sensitivity to the topological structure of visual stimuli. Three factors were taken to capture the topological structure of 2-D patterns: The number of disconnected components, the number of holes (connections), and inclusion relationships. If studied in isolation, any given topological property is typically confounded with the presence of particular features such as line terminations and contour length, or with Gestalt principles of perceptual organization. We went beyond existing studies and attempted to systematically remove potential confounds from the stimulus displays. Results showed that processing speeds for two-dimensional patterns are a function of their topological properties. The more patterns differ in their topological structure the easier they can be discriminated. Not only do all three topological factors contribute to pattern discriminability, they also can be combined to provide an overall measure of structural complexity. Forced choice comparison techniques agreed well with similarity judgments. Topological structure thus contributed to discriminability above and beyond many confounding variables. Claims suggesting a general topological analyzer in visual processing are discussed.

Adult↗
PubMed1998

The molecular characterisation of chicken pituitary N-terminal pro-opiomelanocortin (POMC).

Monoclonal antibodies (Mabs) specifically recognizing the chicken pituitary corticotropes were used to isolate a population of closely related peptides from crude chicken pituitary extracts. A homogeneous N-terminal sequence homologous to the extreme N-terminus of mammalian and amphibian pro-opiomelanocortin (POMC) was revealed. Further physicochemical analysis proved the existence of a series of C-terminally truncated peptides including 3 major molecular species corresponding to Ser1-Gly64, Ser1-Arg73 and Ser1-Gly105 respectively. The two latter molecules were shown to be N-glycosylated at position Asn67, with mass spectrometric data indicating a carbohydrate structure of the oligomannose 5 type, in addition to two more complex structures. No evidence was found in favour of O-glycosylation on Ser47. Degenerated PCR primers were deduced from the above protein sequence and from the known chicken adrenocorticotropic hormone (ACTH) sequence. The nucleotide sequence obtained by reversed transcription PCR (RT-PCR) completely confirmed the new amino acid sequence data including pro-gamma-MSH, the joining peptide and ACTH.

Adrenocorticotropic Hormone↗
PubMed1999

RNA as a small-molecule drug target: doubling the value of genomics.

Recent advances in the determination of RNA structure and function have led to new opportunities that will have a significant impact on the pharmaceutical industry. RNA, which, among other functions, serves as a messenger between DNA and proteins, was thought to be an entirely flexible molecule without significant structural complexity. However, recent studies have revealed a surprising intricacy in RNA structure. This observation unlocks opportunities for the pharmaceutical industry to target RNA with small molecules. Because both proteins and their specific mRNAs are potential drug-binding sites, the number of targets revealed from genome sequencing efforts is effectively doubled. Perhaps more importantly, drugs that bind to RNA might produce effects that cannot be achieved by drugs that bind to proteins.

Journal Article↗
PubMed2006

Isolation and characterization of glycosylphosphatidylinositol-anchored peptides by hydrophilic interaction chromatography and MALDI tandem mass spectrometry.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are posttranslationally processed proteins that become tethered to the extracellular leaflet of the plasma membrane via a C-terminal glycan-like moiety. Since the first GPI-AP was described in the 1970s, more than 500 GPI-APs have been reported in a range of species, including plants, microbes, and mammals. GPI-APs are probably involved in cell signaling, cell recognition, and cell remodeling processes, and they may potentially serve as cell surface antigens or vaccine targets in pathogenic microorganisms or transformed mammalian cells. Due to the structural complexity and physicochemical properties of GPI-APs, their identification and structural characterization is a demanding analytical task. Here, we report a simple, fast and sensitive method for isolation and structural analysis of GPI-anchors using a combination of hydrophilic interaction liquid chromatography and matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight tandem mass spectrometry. This method allowed analysis of GPI peptides derived from low picomole levels of the porcine kidney membrane dipeptidase. Furthermore, it allowed unambiguous assignment of the omega site via amino acid sequencing of the modified peptides. GPI-anchor-specific diagnostic ions were observed by MALDI-MS/MS at m/z 162, 286, 422, and 447, corresponding to glucosamine, mannose ethanolamine phosphate, glucosamine inositol phosphate, and mannose ethanolamine phosphate glucosamine, respectively. Thus, the methodology described herein may enable sensitive and specific detection of GPI-anchored peptides in large-scale proteomic studies of plasma membrane proteins.

Amino Acid Sequence↗
PubMed2003

Traveling stripes on the skin of a mutant mouse.

In the course of animal development, complex structures form autonomously from the apparently shapeless egg. How cells can produce spatial patterns that are much larger than each cell is one of the key issues in developmental biology. It has been suggested that spatial patterns in animals form through the same principles by which dispatched structures are formed in the nonbiological system. However, because of the complexity of biological systems, molecular details of such phenomena have been rarely clarified. In this article, we introduce an example of a pattern-forming phenomenon that occurs in the skin of mutant mice. The mutant mouse has a defect in splicing of the Foxn1 (Whn or nude) gene, which terminates hair follicle development just after pigment begins to accumulate in the follicle. The immature follicles are rapidly discharged, and a new hair cycle resumes. Eventually, the skin color of the mouse appears to oscillate. The color oscillation is synchronous in juvenile mice, but the phase gradually shifts among skin regions to eventually form traveling, evenly spaced stripes. Although the time scale is quite different, the pattern change in the mutant mouse shares characteristics with the nonlinear waves generated on excitable media, such as the Belousov-Zhabotinskii reaction, suggesting that a common principle underlies the wave pattern formation. Molecular details that underlie the phenomenon can be conjectured from recent molecular studies.

Age Factors↗
PubMed2001

Modelling the dose-volume response of the spinal cord, based on the idea of damage to contiguous functional subunits.

PURPOSE: To investigate the response of the spinal cord of experimental animals to homogeneous irradiation, the main purpose being to propose a new version of the Critical Volume Normal Tissue Complication Probability (NTCP) model, incorporating spatial correlation between damaged functional subunits (FSU). METHOD: The standard Critical Volume NTCP model and its modified version, the Contiguous Damage model promoted here, are described in mathematical terms. Also, a fiber-like structure of the spinal cord is considered, which is a more complex structure than the standard Critical Volume NTCP model assumes. It is demonstrated that the Contiguous Damage model predicts different responses to two-segment irradiation and to single-segment irradiation to the same combined length as observed in experiments on rats, a result that cannot be described by the standard Critical Volume NTCP model. RESULTS AND CONCLUSIONS: Both the Critical Volume model and the Contiguous Damage model, are fitted to two sets of canine spinal cord radiation data corresponding to two different fractionation regimes of irradiation. Whole-organ irradiation as well as partial irradiation to different lengths are considered, allowing the investigation of dose-volume effects. Formal goodness-of-fit investigation shows that both models fit the canine spinal cord data equally well.

Animals↗
PubMed1992

Structural features in TAR RNA of human and simian immunodeficiency viruses: a phylogenetic analysis.

A comparative analysis of TAR RNA structures in human and simian immunodeficiency viruses reveals the conservation of certain structural features despite the divergence in sequence. Both the TAR elements of HIV-1 and SIV-chimpanzee can be folded into relatively simple one-stem hairpin structures. Chemical and RNAase probes were used to analyze the more complex structure of HIV-2 TAR RNA, which folds into a branched hairpin structure. A surprisingly similar RNA conformation can be proposed for SIV-mandrill, despite considerable divergence in nucleotide sequence. A third structural presentation of TAR sequences is seen for SIV-african green monkey. These results are generally consistent with the classification of HIV-SIV viruses in four subgroups based on sequence analyses (both nucleotide- and amino acid-sequences). However, some conserved TAR structures were detected for members of different virus subgroups. It is therefore proposed that RNA structure analysis might provide an additional tool for determining phylogenetic relationships among the HIV-SIV viruses.

Base Composition↗
PubMed1997

Jaw muscle development as evidence for embryonic repatterning in direct-developing frogs.

The Puerto Rican direct-developing frog Eleutherodactylus coqui (Leptodactylidae) displays a novel mode of jaw muscle development for anuran amphibians. Unlike metamorphosing species, several larval-specific features never form in E. coqui; embryonic muscle primordia initially assume an abbreviated, mid-metamorphic configuration that is soon remodelled to form the adult morphology before hatching. Also lacking are both the distinct population of larval myofibres and the conspicuous, larval-to-adult myofibre turnover that are characteristic of muscle development in metamorphosing species. These modifications are part of a comprehensive alteration in embryonic cranial patterning that has accompanied life history evolution in this highly speciose lineage. Embryonic 'repatterning' in Eleutherodactylus may reflect underlying developmental mechanisms that mediate the integrated evolution of complex structures. Such mechanisms may also facilitate, in organisms with a primitively complex life cycle, the evolutionary dissociation of embryonic, larval, and adult features.

Animals↗
PubMed2006

Mapping complex myoarchitecture in the bovine tongue with diffusion-spectrum magnetic resonance imaging.

The ability to resolve complex fiber populations in muscular tissues is important for relating tissue structure with mechanical function. To address this issue in the case of tongue, we employed diffusion spectrum imaging (DSI), an MRI method for determining three-dimensional myoarchitecture where myofiber populations are variably aligned. By specifically varying gradient field strength, molecular displacement in a tissue can be determined by Fourier-transforming the echo intensity against gradient strength at fixed gradient pulse spacing. The displacement profiles are visualized by graphing three-dimensional isocontour icons for each voxel, with the isocontour shape and size representing the magnitude and direction of the constituting fiber populations. To validate this method, we simulated a DSI experiment within the constraints of arbitrary crossing fibers, and determined that DSI accurately depicts the angular relationships between these fibers. Considering the fiber relationships in the whole bovine tongue, we compared the images obtained by DSI with those obtained by diffusion tensor imaging in an anterior slice of the lingual core, a region known to possess extensive fiber crossing. In contrast to diffusion tensor imaging, which depicts the anterior core solely as a region with low anisotropy due to the presence of mixed-orientation fiber populations, DSI shows two distinct fiber populations, with an explicit orthogonal relationship to each other. In imaging the whole lingual tissue, we discerned arrays of crossing and noncrossing fibers involving the intrinsic and extrinsic muscles, which merged at regions of interface. We conclude that DSI has the capacity to determine three-dimensional fiber orientation in structurally complex muscular tissues.

Animals↗
PubMed1976

[Electronmicroscopical and morphometrical study of rat hippocampal synapses].

The neuronal structure and synaptology of the hippocampus of the rat are studied using neurohistological, electron microscopical and morphometrical methods. After Golgi-impregnations the neurons of the hippocampus were classified by reason of special morphological features (pericaryon, dendritic, ramification, course and termination of the axons). The neurons of the hippocampus show from the region CA4 towards the region CA1 an increasing "pyramidalization" which is explained respecting the neuronal structure and synaptic architectonics as the result of a specific presynaptic representation of afferents at the neurons. The high degree of differentiation of the CA1-pyramids speaks for the progressive developmental tendency in the phylogenesis of these pyramids compared to the neocortical neurons. The ultrastructural investigations of the hippocampus show that each individual layer of the regions CA1, CA3 and CA4 has a characteristic synaptic architectonics. In all hippocampal layers the axo-spino-dendritic contacts are the most frequent type of synapses with the structural characteristics of the excitatory synapses; the synaptic contact appears as "Bouton en passage" respectively as "Bouton terminal". The axosomatic synapses of the stratum pyramidale (CA1 to CA3) predominantly are developed as symmetrical contacts with inhibitory function. As the terminals of the basket cells they represent the morphological correlate for the recurrent inhibitation of the pyramids by the inhibitory effecting basket cells. In the supra- and infrapyramidal layers of the regions CA3 and CA4 special contacts are developed for the connection withe the mossy fiber terminals--at the pericarya and the main dendrites as so-called somatic and dendritic protrusions and as micro-dendrites. As has been established for the neocortex, the dendritic spines are the decisive receptive postsynaptic structural characteristic of the hippocampus as well. Pre- and postsynaptic elements show a broad scope of variation in forming their structures. This also applies to the intrahippocampal systems with their highly complex structure which is the morphological expression of an extremely high degree of differentiation. The morphometrical investigations of the ultrastructure of synapses enclose the calculation of the number of synapses per mm3 of the volume of layers, the average individual volume of synapses, the presynaptic portion of volume of the relative volume of cortex of the hippocampal layers as well as the average number and size of the synaptic vesicles. The average number of synapses per mm3 brain tissue runs in CA1 to 5,9 X 10(9), in CA3 to 7,9 X 10(9) andin CA4 to 8,7 X 10(9) synapses per mm3. The average individual volume of synapses amounts to 0,2-0,3 mm3 whereas the average portion of volume of the presynaptic structures per relative cortical volume comes to 14% in CA1, 16-19% in CA3 and 15% in CA4.

Animals↗
PubMed1987

The high-resolution X-ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech Hirudo medicinalis. Structural analysis, subtilisin structure and interface geometry.

Triclinic crystals of the complex formed by eglin with subtilisin Carlsberg were analyzed by X-ray diffraction. The crystal and molecular structure of this complex was determined with data that extended to 0.12-nm resolution by a combination of Patterson search methods and isomorphous replacement techniques. Its structure was refined to a crystallographic R value of 0.178 (1.0-0.12 nm) using an energy-restraint least-squares procedure. The complete subtilisin molecule could be traced without ambiguity in the refined electron density. The eglin component, from which an amino-terminal segment is cleaved off, is only defined from Lys8I (i.e. the lysine residue 8 of the inhibitor) onwards. Per unit cell, 436 fixed solvent molecules and 2 calcium ions were located. In spite of 84 amino acid replacements and one deletion, subtilisin Carlsberg exhibits a very similar polypeptide fold to subtilisin BPN'. The root-mean-square deviations of all alpha-carbon atoms (excluding those at the deletion site) from models of subtilisin BPN' [Alden, R. A., Birktoft, J. J., Kraut, J., Robertus, J. D. & Wright, C. S. (1971) Biochem. Biophys. Res. Commun. 45, 337-344] and subtilisin Novo [Drenth, J., Hol, W. G. J., Jansonius, J. N. & Kockoek, R. (1972) Eur. J. Biochem. 25, 177-181] are 0.077 nm and 0.103 nm. Most of these deviations result from global shifts rather than changes of the local geometry. The single-residue deletion at position 56 affects only the surrounding conformation. Two sites of high electron density and close distances to surrounding oxygen ligands have been found in the Carlsberg enzyme which are probably occupied by calcium ions. Eglin consists of a twisted four-stranded beta-sheet flanked by an alpha-helix and by an exposed proteinase binding loop on opposite sides. Around the reactive site, Leu45I-Asp46I, this loop is mainly stabilized by electrostatic/hydrogen bond interactions with the side chains of two arginine residues which project from the hydrophobic core [Bode, W., Papamokos, E., Musil, D., Seemüller, W. & Fritz, H. (1986) EMBO J. 5, 813-818]. The reactive site loop conformation resembles that found in other 'small' proteinase inhibitors. The scissile peptide bond is not cleaved but its carbonyl group is slightly distorted from planar geometry. Most of the intermolecular contacts are contributed by the nine residues of the reactive-site loop Gly40I-Arg48I.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

The structure of endothiapepsin complexed with the gem-diol inhibitor PD-135,040 at 1.37 A.

The crystal structure of endothiapepsin complexed with the gem-diol inhibitor PD-135,040 has been anisotropically refined to a resolution of 1.37 A. The structure of this inhibitor complex is in agreement with previous structures of endothiapepsin gem-diol inhibitor complexes that have been used to develop proposed catalytic mechanisms. However, the increase in resolution over previous structures confirms the presence of a number of short hydrogen bonds within the active site that are likely to play an important role in the catalytic mechanism. The presence of low-barrier hydrogen bonds was indicated in a previous one-dimensional H NMR spectrum.

Aspartic Acid Endopeptidases↗
PubMed2000

How does the brain learn language? Insights from the study of children with and without language impairment.

Neurobiological studies have generated new ways of thinking about development of brain structure and function. Development involves more than just growth from simple to complex structures. The initial over-abundance of neurons and synaptic connections is subsequently pruned of those that are non-functional. In addition, as behavioural and cognitive functions emerge and become automatized, the underlying brain representations are reorganized. In this paper, I shall argue that these different modes of neurodevelopmental change provide a useful metaphor for examining language acquisition. It will be argued that language acquisition can involve learning to ignore and inhibit irrelevant information, as well as forming new ways of representing complex information economically. Modular organization is not present from the outset, but develops gradually. This analysis suggests a new way of assessing specific language impairment (SLI). There has been much debate as to whether children with SLI lack specific modular components of a language processing system. I propose instead that these children persist in using inefficient ways of representing language. Finally, I consider what we know about the neurobiological basis of such a deficit. There is mounting evidence that children with SLI have subtle structural anomalies affecting the language areas of the brain, which are largely genetically determined. We should not, however, conclude that the language difficulties are immutable.

Brain↗