Sensillar types of the ovipositor of Dasineura brassicae: structure and relation to oviposition behaviour

ABSTRACT. The distal part of the ovipositor of Dasineura brassicae Winn. (Diptera; Cecidomyiidae) possesses forty to forty-five sensilla of three morphological types. Most are provided with a cuticular bristle, which projects from the surface of the ovipositor; fifteen have a taste/tactile function based on fine structural characteristics; about twenty-five are innervated by a single sensory cell, specialized for mechanoreception. Scolopidial sensory receptors are anchored to the cuticle inside the distal part of the ovipositor, they probably respond to changes in length of the ovipositor. Different sensory systems are involved in the choice of oviposition site; compound eyes and antennae are probably active in the earlier stages, whereas the receptors of the ovipositor appear well suited to govern the last steps in this behaviour.     

Localisation of thiamine pyrophosphatase within the cytoplasmic fine structure of trophozoites of Entamoeba histolytica and E. invadens

McCaul T.F. and Bird R.G. 1978. Localisation of thiamine pyrophosphatase within the cytoplasmic fine structure of trophozoites of Entamoeba histolytica and E. invadens. International Journal for Parasitology8: 501–506. The distribution of thiamine pyrophosphatase (TPPase) activity was studied in both formaldehyde and glutaraldehyde fixed trophozoites of Entamoeba histolytica and E. invadens. The activity was localised within certain vacuoles. No dense deposits for TPPase activity were seen in the small vesicles, elongated smooth-walled lacunae equated with endoplasmic reticulum, or the nucleus. The demonstration of small vesicles surrounding the larger vacuoles indicated that the Golgi-like vacuoles might be involved in the production of cell coat materials and primary lysosomes.     

Multi-resolution contour-based fitting of macromolecular structures

A novel contour-based matching criterion is presented for the quantitative docking of high-resolution structures of components into low-resolution maps of macromolecular complexes. The proposed Laplacian filter is combined with a six-dimensional search using fast Fourier transforms to rapidly scan the rigid-body degrees of freedom of a probe molecule relative to a fixed target density map. A comparison of the docking performance with the standard cross-correlation criterion demonstrates that contour matching with the Laplacian filter significantly extends the viable resolution range of correlation-based fitting to resolutions as low as 30 A. The gain in docking precision at medium to low resolution (15-30 A) is critical for image reconstructions from electron microscopy (EM). The new algorithm enables for the first time the reliable docking of smaller molecular components into EM densities of large biomolecular assemblies at such low resolutions. As an example of the practical effectiveness of contour-based fitting, a new pseudo-atomic model of a microtubule was constructed from a 20 A resolution EM map and from atomic structures of alpha and beta tubulin subunits.     

Amyloid fibril formation from full-length and fragments of amylin

Amyloiddeposits of fibrillar human amylin (hA) in the pancreas may be a causative factor in type-2 diabetes. A detailed comparison of in vitro fibril formation by full-length hA(1-37) versus fragments of this peptide-hA(8-37) and hA(20-29)-is presented. Circular dichroism spectroscopy revealed that fibril formation was accompanied by a conformational change: random coil to beta-sheet/alpha-helical structure. Fibril morphologies were visualized by electron microscopy and displayed a remarkable diversity. hA(20-29) formed flat ribbons consisting of numerous 3. 6-nm-wide protofibrils. In contrast, hA(1-37) and hA(8-37) formed polymorphic higher order fibrils by lateral association and/or coiling together of 5.0-nm-wide protofibril subunits. For full-length hA(1-37), the predominant fibril type contained three protofibrils and for hA(8-37), the predominant type contained two protofibrils. Polymerization was also monitored with the thioflavin-T binding assay, which revealed different kinetics of assembly for hA(1-37) and hA(8-37) fibrils. hA(20-29) fibrils did not bind thioflavin-T. Together the results demonstrate that the N-terminal region of the hA peptide influences the relative frequencies of the various higher order fibril types and thereby the overall kinetics of fibril formation. Furthermore, while residues 20-29 contribute to the fibrils' beta-sheet core, the flanking C- and N-terminal regions of the hA peptide determine the interactions involved in the formation of higher order coiled polymorphic superstructures.     

Quaternary organization of the Staphylothermus marinus phosphoenolpyruvate synthase: angular reconstitution from cryoelectron micrographs with molecular modeling

Digital electron images of frozen-hydrated preparations of the 2.25-MDa Staphylothermus marinus phosphoenolpyruvate synthase (EC 2.7.9.2) have been analyzed by single-particle classification and averaging and iterative quaternion-based angular reconstitution. Contrast transfer function correction of micrographs obtained at different defocus values was used to improve the informational quality of the projection averages. Three-dimensional reconstructions were obtained to roughly 3-nm spatial resolution, in which the 24 identical subunits were arranged to form an octahedral complex, although the amino-terminal nucleotide-binding domain was not resolved. An atomic model of the subunit was generated by homology modeling using as the reference the known X-ray crystallographic structure of the related enzyme pyruvate orthophosphate dikinase (EC 2.7.9.1) from Clostridium symbiosum (Protein Data Bank entry 1DIK). The S. marinus protein could be arranged into an assembly of 12 homodimers to match the three-dimensional reconstruction in terms of shape and size of the homodimers, as well as overall shape and size of the complex. The quaternary model indicated that active sites of three monomers were localized around cavities (or putative channels) centered at the threefold axes of rotational symmetry and that carboxyl-terminal alpha-helical segments of four monomers were localized at the fourfold axes of rotational symmetry where they could facilitate interdimer interaction. The quaternary arrangement also indicated numerous potential hydrophobic and electrostatic interactions at the interdimer interfaces that could contribute further to structural stability.     

The ITS2 Database

The internal transcribed spacer 2 (ITS2) has been used as a phylogenetic marker for more than two decades. As ITS2 research mainly focused on the very variable ITS2 sequence, it confined this marker to low-level phylogenetics only. However, the combination of the ITS2 sequence and its highly conserved secondary structure improves the phylogenetic resolution¹ and allows phylogenetic inference at multiple taxonomic ranks, including species delimitation^2-8.The ITS2 Database⁹ presents an exhaustive dataset of internal transcribed spacer 2 sequences from NCBI GenBank¹¹ accurately reannotated¹⁰. Following an annotation by profile Hidden Markov Models (HMMs), the secondary structure of each sequence is predicted. First, it is tested whether a minimum energy based fold¹² (direct fold) results in a correct, four helix conformation. If this is not the case, the structure is predicted by homology modeling¹³. In homology modeling, an already known secondary structure is transferred to another ITS2 sequence, whose secondary structure was not able to fold correctly in a direct fold.The ITS2 Database is not only a database for storage and retrieval of ITS2 sequence-structures. It also provides several tools to process your own ITS2 sequences, including annotation, structural prediction, motif detection and BLAST¹⁴ search on the combined sequence-structure information. Moreover, it integrates trimmed versions of 4SALE^15,16 and ProfDistS¹⁷ for multiple sequence-structure alignment calculation and Neighbor Joining¹⁸ tree reconstruction. Together they form a coherent analysis pipeline from an initial set of sequences to a phylogeny based on sequence and secondary structure.In a nutshell, this workbench simplifies first phylogenetic analyses to only a few mouse-clicks, while additionally providing tools and data for comprehensive large-scale analyses.     

Etude Ultrastructurale du Genre Enteromonas da Fonseca (Zoomastigophorea) et Révision de l'Ordre des Diplomonadida Wenyon

RESUME. Deux espèces d'Enteromonas sont observées, provenant, l'une de l'intestin de Triton, l'autre des crottes du Lapin domestique. La cellule piriforme porte un noyau antérieur et 4 flagelles insérts près du pôle ventral du noyau. Le flagelle récurrent (R) est logé dans une dépression ventrale ou cytostome. Les cinétosomes, disposés en une paire antérieure (#1, #2) et une paire postérieure (#3, R), sont liés entre eux par des microfibrilles. Une fibre microtubulaire située au-dessus du noyau est reliée au cinétosome #1. Une autre fibre microtubulaire sous-nucléaire est homologue de la fibre microtubulaire croisée qui existe chez les cellules de Diplozoaires. Le cytostome est bordé par 2 lèvres: la gauche proéminente et armée par plusieurs rangées de microtubules, la droite contenant seulement une mince fibre microtubulaire associée à des microfibrilles. Le cytostome occupe les 2/3 de la face ventrale. Le flagelle récurrent pénètre dans le cytostome puis dépasse l'extrémite de la cellule. Les Bactéries sont phagocytées au fond du cytostome, entre les 2 lèvres distendues. Elles sont digérées dans les nombreuses vacuoles et les corps résiduels sont évacués par rupture de la membrane cellulaire. L'ergastoplasme est concentré près de la périphérie de la cellule. Il n'y a pas de mitochondrie ni d'appareil de Golgi. Dans les kystes observés la cellule plurinucléée est enfermée dans une enveloppe kystique microfibrillaire, les axonèmes sont libres dans le cytoplasme. Les formes diplomonades sont nombreuses et ressemblent aux cellules d'Hexamita, excepté par le cytostome qui est différent. Dans ces formes, les 2 monades sont souvent disposées selon une symétrie axiale binaire mais quelquefois elles sont associées de façon plus anarchique. La cinétide d'Enteromonas est organisée comme celle d'un zoïde de Diplozoaire. Il est possible que le genre Enteromonas soit à l'origine des Diplomonadida et que l'état diplomonadien transitoire chez Enteromonas se soit stabilisé ensuite chez les Diplomonadida. Enteromonas apparaît plus primitif que les autres genres de Diplomonadida aussi nous proposons de créer 2 sous-ordres: celui des Enteromonadina avec le genre Enteromonas et celui des Diplomonadina avec les genres Trepomonas, Trigonomonas, Hexamita, Spironucleus, Octomitus, Giardia. La disposition des cinétosomes et l'existence du cytostome sont les principaux caractères communs entre Enteromonas et les Retortamonadida, cependant les fibres annexes ne sont pas homologues. Une étude plus complète de la division nucléaire et cellulaire de ces 2 ordres de Zooflagellés est nécessaire pour donner un meilleur schéma évolutif. SYNOPSIS. Fine structure of 2 species of Enteromonas, one from the intestine of the salamander, Triturus vulgaris, and another from the feces of domestic rabbit, Oryctolagus cuniculi, is described. The pyriform cell has an anteriorly located nucleus. The 4 flagella originate from an area near the anterior end of the nucleus. The recurrent flagellum (R) is lodged in a ventral depression or cytostome. The kinetosomes, arranged into 2 pairs, anterior (#1, #2) and posterior (#3, R), are interconnected by microfibrils. One microtubular fiber, connected to kinetosome #1, is situated near the anterior surface of the nucleus. Another, subnuclear, microtubular fiber is homologous to the “crossed'’fiber found in Diplozoa. The cytostome is bordered by 2 lips: the preeminent left lip is equipped with several rows of microtubules, while the right lip contains only a thin microtubular fiber associated with microfibrils. The cytostome occupies 2/3 of the ventral surface. The recurrent flagellum passes over the anterior surface of the cell and then comes to lie in the cytostome. The bacteria are phagocytosed in the bottom part of the cytostome between the 2 distended lips. They are digested in numerous vacuoles. The undigested residual bodies are evacuated by a rupture of the cell membrane. The ergastoplasm is concentrated near the cell periphery. Mitochondria and the Golgi apparatus are absent. In the cyst stage, the multinucleate cell is enclosed in a microfibrillar membrane; the axonemes lie free in the cytoplasm. Diplomonad forms of Enteromonas resembling Hexamita are numerous, except that the cytostome is different in these 2 genera. In such forms, the arrangement of the 2 individuals often has binary axial symmetry, but on occasion they are associated in a more anarchic fashion. The mastigont of Enteromonas is organized like that of a single zooid of a diplozoon. It is possible that the genus Enteromonas is ancestral to Diplomonadida and that the diplomonad state, transitory in Enteromonas, became permanently established in Diplomonadida. Enteromonas appears to be more primitive than the other genera of Diplomonadida. Thus we propose 2 suborders: Enteromonadina, subord. nov. with the genus Enteromonas, and Diplomonadina Wenyon, emend., with the genera Trepomonas, Trigonomonas, Hexamita, Spironucleus, Octomitus, Giardia. The arrangement of the kinetosomes and the existence of a cytostome are the principal characters common to Enteromonas and Retortamonadida, while their “accessory'’fibers are not homologous. A more complete study of division of the 2 zooflagellate orders is necessary for the presentation of a more detailed evolutionary scheme of these groups.     

Integrative Structural Investigation on the Architecture of Human Importin4_Histone H3/H4_Asf1a Complex and Its Histone H3 Tail Binding

Importin4 transports histone H3/H4 in complex with Asf1a to the nucleus for chromatin assembly. Importin4 recognizes the nuclear localization sequence located at the N-terminal tail of histones. Here, we analyzed the structures and interactions of human Importin4, histones and Asf1a by cross-linking mass spectrometry, X-ray crystallography, negative-stain electron microscopy, small-angle X-ray scattering and integrative modeling. The cross-linking mass spectrometry data showed that the C-terminal region of Importin4 was extensively cross-linked with the histone H3 tail. We determined the crystal structure of the C-terminal region of Importin4 bound to the histone H3 peptide, thus revealing that the acidic patch in Importin4 accommodates the histone H3 tail, and that histone H3 Lys14 contributes to the interaction with Importin4. In addition, we show that Asf1a modulates the binding of histone H3/H4 to Importin4. Furthermore, the molecular architecture of the Importin4_histone H3/H4_Asf1a complex was produced through an integrative modeling approach. Overall, this work provides structural insights into how Importin4 recognizes histones and their chaperone complex.     

1H NMR-visible mobile lipid domains correlate with cytoplasmic lipid bodies in apoptotic T-lymphoblastoid cells

The presence of nuclear magnetic resonance (NMR)-visible mobile lipid (ML) domains in apoptotic lymphoblasts suggests alterations in neutral lipid metabolism and compartmentation during programmed cell death. The detection of similar ML signals in activated lymphocytes raises questions about common mechanisms of ML formation during apoptosis and upon lymphoblast stimulation. Structure and subcellular localization of ML domains were therefore investigated by NMR, fluorescence and electron microscopy in Jurkat T-lymphoblasts either induced to apoptosis (by anthracyclines or dexamethasone or by serum deprivation) or activated by phorbol myristate acetate (PMA) plus ionomycin. ML contents in drug-treated cells correlated linearly with apoptosis, irrespective of the specific inducer and cell cycle arrest phase (r=0.993, P<0.001). Similar ML levels were measured in drug-induced apoptotic cells (A≈30–40%) and in non-apoptotic PMA/ionomycin-treated lymphoblasts (72 h). Lower ML contents were instead formed in serum-deprived apoptotic cells, with respect to controls. Increases in ML signals were associated, in either apoptotic or activated cells, with the accumulation of cytoplasmic, osmophilic lipid bodies (diameter≤1.0 μm), surrounded by own membrane, possessing intramembrane particles. The results support the hypothesis that ML are formed in the cytoplasm of drug-induced apoptotic cells during an early, ‘biochemically active’ phase of programmed cell death.