Shallow moving structures promote marine invader dominance

Global increases in urban development have resulted in severe habitat modification in many estuaries. Most are now dominated by artificial structures, which might have a myriad of effects on native species. The provision of extra hard substrata presents additional free space, and recent research suggests non-indigenous epifauna may be able to exploit these artificial structures (particularly pontoons) more effectively than native species. The early development of fouling assemblages was compared on settlement plates attached to fixed or moving experimental structures at depths of 0.5 m and 2 m. Invertebrate invaders as a group were disproportionately more numerous on shallow, moving plates (essentially floating surfaces) than on deeper plates, whereas native epifauna were less numerous than invaders in all treatments. Importantly, however, individual invasive species showed differing effects of movement and depth. Future management strategies should take into account the potential for shallow, moving structures to enhance invader dominance and strongly consider using fixed structures to reduce opportunities for invaders.     

Functional imaging of pericellular proteolysis in cancer cell invasion

Proteolytic interactions between cells and extracellular matrix (ECM) are involved in many physiological and pathological processes, such as embryogenesis, wound healing, immune response, and cancer. The visualization of cell-mediated proteolysis towards ECM is thus required to understand basic mechanisms of tissue formation and repair, such as the breakdown and structural remodelling of ECM, inflammatory changes of tissue integrity, and the formation of proteolytic trails by moving cells. A panel of synergistic techniques for the visualization of pericellular proteolysis in live and fixed samples allow monitoring the of proteolytic tumor cell invasion in three-dimensional (3D) fibrillar collagen matrices in vitro. These include the quantification of collagenolysis by measuring the release of collagen fragments, the detection of protease expression and local activity by dequenching of fluorogenic substrate, and the staining of cleavage-associated neoepitopes together with changes in matrix structure. In combination, these approaches allow the high-resolution mapping of pericellular proteolysis towards ECM substrata including individual focal cleavage sites and the interplay between cell dynamics and alterations in the tissue architecture.     

Cortactin (CTTN), N-WASP (WASL), and clathrin (CLTC) are present at podosome-like tubulobulbar complexes in the rat testis

Tubulobulbar complexes are actin filament-rich plasma membrane protrusions that form at intercellular junctions in the seminiferous epithelium of the mammalian testis. They are proposed to internalize intact junctions during sperm release and during the translocation of spermatocytes through basal junction complexes between neighboring Sertoli cells. Tubulobulbar complexes morphologically resemble podosomes found at cell/substrate attachments in other systems. In this study we probe apical tubulobulbar complexes in fixed epithelial fragments and fixed frozen sections of rat testis for two key actin-related components found at podosomes, and for the endocytosis-related protein clathrin. N-WASP and cortactin, two regulators of actin network assembly known to be components of podosomes, are concentrated at tubulobulbar complexes. Clathrin-positive structures occur in Sertoli cell regions containing tubulobulbar complexes when analyzed by immunofluorescence microscopy and occur at the ends of the complexes when evaluated by immunoelectron microscopy. Our results are consistent with the conclusion that tubulobulbar complexes are podosome-like structures. We propose that the formation of tubulobulbar complexes may be clathrin initiated and that their growth is due to the dendritic assembly of a membrane-related actin network.     

Actin cytoskeleton controls movement of intracellular organelles in pancreatic duct epithelial cells

In most eukaryotic cells, microtubules and filamentous actin (F-actin) provide tracks on which intracellular organelles move using molecular motors. Here we report that cytoplasmic movement of both mitochondria and lysosomes is slowed by F-actin meshwork formation in pancreatic duct epithelial cells (PDEC). Mitochondria and lysosomes were labeled with fluorescent Mitotracker Red CMXRos and Lysotracker Red DND-99, respectively, and their movements were monitored using epi-fluorescence and confocal microscopy. Mitochondria and lysosomes moving actively at rest stopped rapidly within several seconds after an intracellular Ca(2+) rise induced by activation of P2Y(2) purinergic receptors. The 'freezing' of the organelles was inhibited by blocking the Ca(2+) rise or by pretreatment with latrunculin B, an inhibitor of F-actin formation. Indeed, this freezing effect on the organelles was accompanied by the formation of F-actin in the whole cytoplasm as stained with Alexa 488-phalloidin in fixed PDEC. For real-time monitoring of F-actin formation in live cells, we expressed sGFP-fimbrin actin binding domain2 (fABD2) in PDEC. Rapid recruitment of the fluorescent probe near the nucleus and lysosomes suggested dense F-actin formation around intracellular structures. The development of F-actin paralleled that of organelle freezing. We conclude that rapid Ca(2+)-dependent F-actin formation physically restrains intracellular organelles and reduces their mobility non-selectively in PDEC.     

Analysis of catalytic properties of hen egg white lysozyme during renaturation from denatured and reduced material.

Catalytic properties of hen egg white lysozyme were analyzed during the renaturation of the enzyme from completely reduced and denatured material. The formation of intermediate folding products and the generation of native lysozyme was monitored by acetic acid/urea electrophoresis. The results showed that during the beginning of renaturation almost all reduced and denatured lysozyme is converted to forms possessing lower compactness than native lysozyme, probably as a result of formation of only one or two disulfide bonds. Kinetic analysis of lysozyme during renaturation showed that the generation of lysozyme with four disulfide bonds was not necessarily equivalent to the formation lysozyme with native-like catalytic properties. It appeared that the formation rate of the structures of the structures of the substrate binding site and of the catalytic site were limited by the generation of four disulfide bonds containing lysozyme. The catalytic properties of intermediate folding products made it evident that the final structures of the substrate binding site and of the catalytic site were formed after the generation of all disulfide bonds.     

A model for the formation of ring-shaped structures in colonies of mycelial fungi

Mathematical model of the development of the pattern of colonies is considered. The model represents the systems of differential equations of the first order. It includes non-dimensional parameters characterizing the following features: concentration of substrate, concentration of metabolic products--growth inhibitor, mycelium and spores, radial and specific rate of mycelium growth, rate of substrate consumption and production of metabolic products, coefficients of diffusion of substrate and metabolic products, initial concentration of mycelium and substrate, time of delay of mycelium reaction on metabolic products and spore formation, threshold concentration of metabolic products. The model is adequate to the experiments with cultivation of Penicillium chrysogenum. It was shown that necessary condition for the formation of the circle periodical structures (zoning) in the colonies is an ability for the production of growth inhibitors (antibiotics, etc.). It was proved that formation of colonies of "continuous lawn" type is caused by restrictions on growth because of mycelium satiation or exhaustion of substrate. Such growth scenario is realized in experiments either on reach substrate or on hungry agar. For the appearance of regulating of "zone structure" type limitation on critical level of metabolic product concentration is very important. The number of periodical zone structures and their widths are determined by the above parameters.     

Kinematics of surface growth

 In this paper a general mathematical framework is developed to describe cases of fixed and moving growth surfaces. This formulation has the mathematical structure suggested by Skalak (1981), but is extended herein to include discussion of possible singularities, incompatibilities, residual stresses and moving growth surfaces. Further, the general theoretical equations necessary for the computation of the final form of a structure from the distribution of growth velocities on a growth surface are presented and applied in a number of examples. It is shown that although assuming growth is always in a direction normal to the current growth surface is generally sufficient, growth at an angle to the growth surface may represent the biological reality more fully in some respects. From a theoretical viewpoint, growth at an angle to a growth surface is necessary in some situations to avoid postulating singularities in the growth velocity field. Examples of growth on fixed and moving surfaces are developed to simulate the generation of horns, seashells, antlers, teeth and similar biological structures. Received 20 February 1996; received in revised form 15 October 1996     

Functional analysis of hyperthermophilic endocellulase from Pyrococcus horikoshii by crystallographic snapshots

A hyperthermophilic membrane-related β-1,4-endoglucanase (family 5, cellulase) of the archaeon Pyrococcus horikoshii was found to be capable of hydrolysing cellulose at high temperatures. The hyperthermophilic cellulase has promise for applications in biomass utilization. To clarify its detailed function, we determined the crystal structures of mutants of the enzyme in complex with either the substrate or product ligands. We were able to resolve different kinds of complex structures at 1.65-2.01?? (1??=0.1?nm). The structural analysis of various mutant enzymes yielded a sequence of crystallographic snapshots, which could be used to explain the catalytic process of the enzyme. The substrate position is fixed by the alignment of one cellobiose unit between the two aromatic amino acid residues at subsites +1 and +2. During the enzyme reaction, the glucose structure of cellulose substrates is distorted at subsite -1, and the β-1,4-glucoside bond between glucose moieties is twisted between subsites -1 and +1. Subsite -2 specifically recognizes the glucose residue, but recognition by subsites +1 and +2 is loose during the enzyme reaction. This type of recognition is important for creation of the distorted boat form of the substrate at subsite -1. A rare enzyme-substrate complex was observed within the low-activity mutant Y299F, which suggested the existence of a trapped ligand structure before the formation by covalent bonding of the proposed intermediate structure. Analysis of the enzyme-substrate structure suggested that an incoming water molecule, essential for hydrolysis during the retention process, might be introduced to the cleavage position after the cellobiose product at subsites +1 and +2 was released from the active site.     

Characterization of covalent Ene adduct intermediates in "hydride equivalent" transfers in a dihydropyridine model for NADH reduction reactions

A study of the reactions of an NADH model, 1,4-di(trimethylsilyl)-1,4-dihydropyridine, 7, with a series of α,β-unsaturated cyano and carbonyl compounds has produced the first direct evidence for an obligatory covalent adduct between a dihydropyridine and substrate in a reduction reaction. The reactions were monitored by NMR spectroscopy. In all reactions studied, the covalent adduct was the first new species detected and its decomposition to form products could be observed. Concentrations of adducts were sufficiently high at steady-state that their structures could be determined directly from NMR spectra of the reaction mixtures; adduct structures are those expected from an Ene reaction between 7 and the substrate. This first reaction step results in transfer of the C(4) hydrogen nucleus of 7 to the substrate and formation of a covalent bond between C(2) of the dihydropyridine ring and the substrate α-atom. Discovery of these Ene-adduct intermediates completes the spectrum of mechanisms observed in NADH model reactions to span those with free radical intermediates, no detectable intermediates and now covalent intermediates. The geometry of the transition state for formation of the Ene adduct is compared with those of theoretical transition state models and crystal structures of enzyme-substrate/inhibitor complexes to suggest a relative orientation for the dihydropyridine ring and the substrate in an initial cyclic transition state that is flexible enough to accommodate all observed mechanistic outcomes.     

Ab initio study of the reaction mechanism of ribonuclease A with cytidyl-3',5'-adenosine. I. Geometry optimization of cytidyl-3', 5'-adenosine.

As a first part of the ab initio study of the reaction mechanism of ribonuclease A with cytidyl-3',5'-adenosine, the geometry of the cytidyl-3',5'-adenosine substrate has been optimized using the Hartree-Fock method. Eleven different starting structures of cytidyl-3',5'-adenosine have been studied. To guarantee a proper alignment with the active site of the ribonuclease A enzyme, a part of the substrate was fixed during the geometry optimization. The geometry and intramolecular interactions of the refined conformations have been evaluated and two possible prototype structures have been proposed. One of these prototypes is more in accordance with the results of a molecular dynamics simulation and is therefore presented as a model for the geometry of cytidyl-3', 5'-adenosine in the initial step of the reaction with ribonuclease A.     

Turing structures in an enzyme-induction system with gap junction-mediated non-linear diffusion

Two cells, each containing a reaction system modeling genetic induction, are coupled by diffusion. The substrate is moving through gap junctions, the number of which is regulated by the adjacent cells. This leads to a non-linear substrate diffusion term in the rate equations. Stability analysis reveals the conditions for the emergence of stable asymmetric solutions (dissipative structures). Due to non-linear diffusion rigid restrictions on the ratio of the two diffusion constants no longer exist. We demonstrate that substances operating as regulators of intercellular communication and participating in cellular metabolism may exhibit morphogenetic functions.     

The angular orientation of the movement detectors acting on the flight lift response in flies

The lift response of houseflies Musca domestica in fixed flight to periodic gratings movins in 12 different orientations has been measured. Two projectors were arranged symmetrically to the flies stimulating successively 18 circular patches of 50° (25°) diameter (9 for each eye) in their visual field. The shapes of the lift responses measured as a function of the orientation of the moving grating varied when different patches in the visual field were stimulated. A qualitative comparison of these response curves leads to the conclusion that the orientation of the movement detecting substrate acting on the flight lift response varies as a function of the stimulated area in the visual field. A straightforward correlation between the geometry of the ommatidial pattern and the orientation of the movement detecting substrate valid for all stimulated areas of the compound eyes does not seem very likely.     

Large conformational changes in the catalytic cycle of glutathione synthase

Glutathione synthase catalyzes the final ATP-dependent step in glutathione biosynthesis, the formation of glutathione from gamma-glutamylcysteine and glycine. We have determined structures of yeast glutathione synthase in two forms: unbound (2.3 A resolution) and bound to its substrate gamma-glutamylcysteine, the ATP analog AMP-PNP, and two magnesium ions (1.8 A resolution). These structures reveal that upon substrate binding, large domain motions convert the enzyme from an open unliganded form to a closed conformation in which protein domains completely surround the substrate in the active site.     

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors

The structures of 5-aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4-oxosebacic acid and 4,7-dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P-side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A- and P-side substrates). The most intriguing result is the novel finding that 4,7-dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P-side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A-side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A- and P-sites is proposed.     

Dipole models of alpha-rhythm generators

Different hypothesis of the alpha rhythm origin were tested by dipole simulation of the alpha rhythm sources. EEG was recorded during driving photic stimulation in order to increase the signal-to-noise ratio. Models with fixed and moving dipoles were analyzed. Dipole sources were compared with magnetic resonance imaging (MRI) scans to find the exact location of oscillations in brain anatomical structures. A two-level multiple dipole model was found to fit the EEG most adequately. The first level was represented by two oscillators localized in the thalamic reticular nuclei, and the second level is associated with two modality-specific oscillators localized in the respective cortical areas.     

Crystal structure of RS21-C6, involved in nucleoside triphosphate pyrophosphohydrolysis

RS21-C6, which is highly expressed in all vertebrate genomes and green plants, is proposed to have nucleoside triphosphate pyrophosphohydrolase activity. Here, we report the crystal structures of the core fragment of RS21-C6, named RSCUT, and the complex with the substrate 5-methyl dCTP. The refined structure of RSCUT consists mainly of alpha-helices and shows formation of a tightly associated tetramer. On the basis of the structure of the RSCUT-m5dCTP complex and the results of pyrophosphatase activity assays, several key residues involved in the substrate binding of RS21-C6 have been identified. Tetramer formation is shown to be required for substrate binding.     

Acyl-CoA: glycine N-acyltransferase: in vitro studies on the glycine conjugation of straight- and branched-chained acyl-CoA esters in human liver

Apparent kinetic constants (Km and Vmax values) were determined for human liver acyl-CoA: glycine acyltransferase (glycine-N-acylase) towards isobutyryl-CoA, 2-methyl butyryl-CoA, isovaleryl-CoA, butyryl-CoA, hexanoyl-CoA, octanoyl-CoA, and decanoyl-CoA. These acyl-CoA esters were selected because of their relevance to the human diseases with cellular accumulation of these esters, i.e., especially to metabolic defects in the acyl-CoA dehydrogenation steps of the branched-chain amino acids, lysine, 5-hydroxy lysine, tryptophan, and fatty acid oxidation pathways. With the acyl-CoA ester as the fixed substrate, the Km value for glycine ranged from 0.5 to 2.9 mole/liter, and with glycine as fixed substrate, the Km values for the acyl-CoA esters varied from 0.3 to 5.6 mmole/liter. It is concluded that the substrate concentration is decisive for the glycine conjugate formation and that the occurrence in urine of acylglycines reflects an intramitochondrial accumulation of the corresponding acyl-CoA ester.     

Dihydropteroate synthase from Streptococcus pneumoniae: structure, ligand recognition and mechanism of sulfonamide resistance

DHPS (dihydropteroate synthase) catalyses an essential step in the biosynthesis of folic acid and is the target for the sulfonamide group of antimicrobial drugs. In the present paper we report two crystal structures of DHPS from the respiratory pathogen Streptococcus pneumoniae: the apoenzyme at 1.8 A (1 A=0.1 nm) resolution and a complex with DHPP (6-hydroxymethyl-7,8-dihydropterin monophosphate) at 2.4 A resolution. The enzyme forms a alpha/beta barrel structure, with a highly conserved binding pocket for recognition of the pterin substrate, DHPPP (6-hydroxymethyl-7,8-dihydropterin pyrophosphate). There is a fixed order of substrate binding: DHPPP binds first, followed by the second substrate, pABA (p-aminobenzoic acid). Binding of PP(i) also allows the enzyme to recognize pABA or sulfonamide drugs, which act as pABA analogues. Using equilibrium and pre-steady state kinetic fluorescence measurements, we show that the on-rate for DHPPP binding to the enzyme is relatively low (2.6x10(5) M(-1) x s(-1)) and propose that binding of this substrate induces a large scale movement of the second loop in the enzyme structure to participate in the formation of the pABA-binding site. Two mutations which confer resistance to sulfonamide drugs do not affect DHPPP binding, but have a substantial effect on pABA and sulfonamide recognition. The results show that binding of DHPPP and pABA are separate distinguishable events in the reaction cycle, and that mutations which confer resistance to sulfonamide drugs act exclusively on the second step in the binding process.     

Method of preparing suspended cells for scanning electron microscopy]

To preserve the lifetime morphology of the surface of suspended cells, these must be fixed in suspensions. The subsequent stages of cell preparation for scanning electron microscopy (dehydratation, critical point drying, coating) are considerably facilitated if fixed cells are preliminary attached to some substrate surface. An effective substrate should provide a firm rather than selective attachment of the overwhelming majority of fixed cells; the substrate should be also available for a wide application. The trial of different types of substrates showed a sufficient effectivity of plates made of commercial aluminium foil. In tests with murine embryonal and transformed fibroblasts as well as with human blood leukocytes, in average 90 per cent of cells fixed with glutaraldehyde in suspensions got attached to foil substrate surfaces; the fixed cells both settled from suspension and attached were seen distributed evenly on the substrate surface. The use of aluminium foil substrates made it possible to study the surface topography of some types of suspended cells.     

Biosynthesis of the Class III Lantipeptide Catenulipeptin

Lantipeptides are ribosomally synthesized and posttranslationally modified peptides containing lanthionine and/or labionin structures. In this study, a novel class III lantipeptide termed catenulipeptin was discovered from Catenulispora acidiphila DSM 44928, and its biosynthesis was reconstituted in vitro. The multifunctional enzyme AciKC catalyzes both dehydration and cyclization of its peptide substrate AciA and installs two labionin structures in catenulipeptin. AciKC shows promiscuity with respect to cosubstrate and accepts all four NTPs. The C-terminal domain of AciKC is responsible for the labionin formation in catenulipeptin. The cyclase activity of AciKC requires the leader peptide of AciA substrate but does not require ATP or Zn(2+). Mutagenesis studies suggest that the labionin cyclization may proceed in a C-to-N-terminal direction. Catenulipeptin partially restores aerial hyphae growth when applied to surfactin-treated Streptomyces coelicolor.