Amelogenin "nanorods" formation during proteolysis by Mmp-20

Amelogenin is cleaved by enamelysin (Mmp-20) soon after its secretion, and the cleavage products accumulate in specific locations during enamel formation, suggesting that parent amelogenin proteolysis is necessary for activating its functions. To investigate the precise roles of Mmp-20 and its influence on the assembly of amelogenin, an in vitro enzymatic digestion process mimicking the initial stages of amelogenin proteolysis was investigated at near-physiological conditions using recombinant porcine amelogenin (rP172) and enamelysin. Hierarchically organized nanorod structures formed during different digestion stages were detected by TEM. At the earliest stage, uniformly dispersed parent amelogenin spherical particles, mixed with some darker stained smaller spheres, and accompanying elongated chain-like nanostructures were observed. Cylindrical nanorods, which appeared to be the result of tight assembly of thin subunit cylindrical discs with thicknesses ranging from ∼2.5 to ∼6.0 nm, were formed after an hour of proteolysis. These subunit building blocks stacked to form nanorods with maximum length of ∼100 nm. With the production of more cleavage products, additional morphologies spontaneously evolved from the cylindrical nanorods. Larger ball-like aggregates ultimately formed at the end of proteolysis. The uniform spherical particles, nanorods, morphological patterns evolved from nanorods, and globular aggregated microstructures were successively formed by means of co-assembly of amelogenin and its cleavage products during a comparatively slow proteolysis process. We propose that, following the C-terminal cleavage of amelogenin, co-assembly with its fragments leads to formation of nanorod structures whose properties eventually dictate the super-structural organization of enamel matrix, controlling the elongated growth of enamel apatite crystals.     

Colonizing macroinvertebrates in the Upper Mississippi River with a comparison of basket and multiplate samplers *

SUMMARY. Colonizing aquatic macroinvertebrates were collected from two kinds of artificial substrate placed on wing dams in Pool 13 of the Upper Mississippi River in September 1978. Thirty-one taxa were collected from basket samplers containing cement spheres and twenty-one taxa from multiplate samplers constructed from tempered hardboard. Hydro-psychidae (Trichoptera), especially Cheumatopsyche sp., Potamyla flava and Hydropsyche sp., were the dominant macroinvertebrates which colonized both samplers. Basket samplers had a significantly greater macroinvertebrate density, biomass and number of taxa compared with multiplate samplers. Precision of the arithmetic mean for density, biomass and number of taxa was 19.9, 18.3 and 8.1% for basket samplers and 18.8, 18.7 and 8.5% for multiplate samplers. The number of sampling units required for a precision of 20% for macroinvertebrate density, biomass and number of taxa was 13, 11 and 2 for basket samplers and 11, 11 and 2 for multiplate samplers. Basket samplers with 7.5-cm cement spheres are recommended for use instead of multiplate samplers.     

Flow resistance and drag forces due to multiple adherent leukocytes in postcapillary vessels.

Computational fluid dynamics was used to model flow past multiple adherent leukocytes in postcapillary size vessels. A finite-element package was used to solve the Navier-Stokes equations for low Reynolds number flow of a Newtonian fluid past spheres adhering to the wall of a cylindrical vessel. We determined the effects of sphere number, relative geometry, and spacing on the flow resistance in the vessel and the fluid flow drag force acting to sweep the sphere off the vessel wall. The computations show that when adherent leukocytes are aligned on the same side of the vessel, the drag force on each of the interacting leukocytes is less than the drag force on an isolated adherent leukocyte and can decrease by up to 50%. The magnitude of the reduction depends on the ratio of leukocyte to blood vessel diameter and distance between adherent leukocytes. However, there is an increase in the drag force when leukocytes adhere to opposite sides of the vessel wall. The increase in resistance generated by adherent leukocytes in vessels of various sizes is calculated from the computational results. The resistance increases with decreasing vessel size and is most pronounced when leukocytes adhere to opposite sides of the vessel.     

Spectroscopy-Based Modelling of the 3D Structure of the β Subunit of the High Affinity IgE Receptor

Abstract

The high affinity IgE receptor, possesses a tetrameric structure. The 243 residue β subunit is a polytopic protein with four hydrophobic membrane-spanning segments, whereas the individual α and γ subunits are bitopic proteins each containing one transmembrane domain in their monomeric form. In the proposed topographical model (Blank et al., 1989), the four trans-membrane α helices of the β subunit are connected by three loop sequences.

To study the individual subunits and intact receptor, this membrane protein was divided into domains such as its loop peptides, cytoplasmic peptides and transmembrane helices according to Blank et al., 1989. The 3D structure of the synthesized loop peptides and cytoplasmic peptides were calculated; CD and/or NMR data were used as appropriate to generate the resultant structures which were then used as data basis for the higher level calculations.

The four individual transmembrane helices of the β subunit were characterised, first of all, by mapping the relative lipophilicity of their surfaces using lipophilic probes. A second procedure, docking of the individual helices in pairs, was used to predict helix–helix interactions.

The data on the relative lipophilicity of the surfaces as well as the surfaces that favoured helix–helix interactions were used in combination with the spectroscopy-based structures of the loops and cytoplasmic domains to calculate via molecular dynamics, the helix arrangement and 3D structure of the β subunit of the high affinity IgE receptor. In the final analysis, the molecular simulations yielded two structures of the β subunit, which should form a basis for the modelling of the whole high affinity IgE receptor.     

Simulated and experimental estimates of hydrodynamic drag from bio-logging tags

Drag force acting on swimming marine mammals is difficult to measure directly. Researchers often use simple modeling and kinematic measurements from animals, or computational fluid dynamics (CFD) simulations to estimate drag. However, studies that compare these methods are lacking. Here, computational simulation and physical experiments were used to estimate drag forces on gliding bottlenose dolphins (Tursiops truncatus). To facilitate comparison, variable drag loading (no-tag, tag, tag + 4, tag + 8) was used to increase force in both simulations and experiments. During the experiments, two dolphins were trained to perform controlled glides with variable loading. CFD simulations of dolphin/tag geometry in steady flow (1–6 m/s) were used to model drag forces. We expect both techniques will capture relative changes created by experimental conditions, but absolute forces predicted by the methods will differ. CFD estimates were within a calculated 90% confidence interval of the experimental results for all but the tag condition. Relative drag increase predicted by the simulation vs. experiment, respectively, differed by between 21% and 31%: tag, 4% vs. 33%; tag + 4, 47% vs. 68%; and tag + 8, 108% vs. 77%. The results from this work provide a direct comparison of computational and experimental estimates of drag, and provide a framework to quantify uncertainty.     

The association of mannitol oxidase with a distinct organelle in the digestive gland of the terrestrial slugArion ater

Summary Post nuclear supernatants prepared from homogenates of the digestive gland of the slugArion ater were analysed by rate-dependent and density-dependent density gradient zonal centrifugation. Mannitol oxidase carrying structures exhibited distinct centrifugal behaviour sedimenting more slowly than mitochondria, lysosomes and peroxisomes but faster than microsomes, and banding sharply at a density of 1.15 g/ml in sucrose gradients. By combining rate and isopycnic centrifugations mannitol oxidase carrying structures were largely separated from contaminating organelles. Electron microscopic examination of such mannitol oxidase-enriched fractions revealed the predominant presence of distinct structures of tubular form. SDS PAGE analysis indicated that the major polypeptide present had a mass of 68 kDa corresponding to the major subunit previously reported for partially purified mannitol oxidase ofHelix aspersa.Abbreviations ER endoplasmic reticulum - SDS PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis     

Dendrimers in gene transfection

Dendrimers are a new class of nanocomposite materials. They are branching polymers whose structure is formed by monomeric subunit branches diverging to all sides from a central nucleus. The type of nucleus, attached monomers, and functional groups can be chosen during synthesis, which produces dendrimers of definite size, shape, density, polarity, branch mobility, and solubility. This review deals with problems of dendrimer molecular structures and capability of in vitro, in vivo, ex vivo, and in situ transfection of genetic material. Advantages and shortcomings of different types of dendrimers in this respect are discussed.     

Transport properties of rigid, symmetrical oligomeric structures composed of prolate, ellipsoidal subunits

We have calculated translational and rotational diffusion coefficients and intrinsic viscosities of oligomeric structures composed of n identical subunits having a prolate ellipsoidal shape with axial ratio p. Results are presented for p = 1-6 for a variety of structures with n = 1-6. We compare our results with those obtained by a different modeling procedure, proposed by other workers, in which the monomeric subunit is represented as a string of touching, colinear spheres. If n and an estimate of p are known, the structure of the oligomer can be. in most cases, unambiguously determined by comparison of the experimental oligomer-to-monomer ratios of a given property with the numerical results of this work. As examples of the applicability of our results, we examine the relationship between structure and properties for neurophysin. bovine serum albumin, hemoglobin and phycocyanin.     

Determination of amino acid sequences of two subunits in sarcosine oxidase from Corynebacterium sp. U-96

The primary structures of the C and D subunits of sarcosine oxidase from Corynebacterium sp. U-96 were determined by sequencing the peptide fragments derived from their enzymatic digestions. The C and D subunits were shown to be composed of 199 and 92 residues, respectively. Each amino acid sequence showed a high homology with the sequence of the corresponding subunit from Corynebacterium sp. P-1. However, there were some differences between these two species, that is, four N-terminal residues were truncated in the C subunit, but six C-terminal residues were truncated in the D subunit. The D subunit contained three cysteine residues, but no disulfide bonds are in the subunit. Overall sequences of both subunit showed no homology with any other protein in the data base.     

Variable Subunit Contact and Cooperativity of Hemoglobins

Tertiary structures of proteins are conserved better than their primary structures during evolution. Quaternary structures or subunit organizations, however, are not always conserved. A typical case is found in hemoglobin family. Although human, Scapharca, and Urechis have tetrameric hemoglobins, their subunit contacts are completely different from each other. We report here that only one or two amino acid replacements are enough to create a new contact between subunits. Such a small number of chance replacements is expected during the evolution of hemoglobins. This result explains why different modes of subunit interaction evolved in animal hemoglobins. In contrast, certain interactions between subunits are necessary for cooperative oxygen binding. Cooperative oxygen binding is observed often in dimeric and tetrameric hemoglobins. Conformational change of a subunit induced by the first oxygen binding to the heme group is transmitted through the subunit contacts and increases the affinity of the second oxygen. The tetrameric hemoglobins from humans and Scapharca have cooperativity in spite of their different modes of subunit contact, but the one from Urechis does not. The relationship between cooperativity and the mode of subunit contacts is not clear. We compared the atomic interactions at the subunit contact surface of cooperative and non-cooperative tetrameric hemoglobins. We show that heme-contact modules M3–M6 play a key role in the subunit contacts responsible for cooperativity. A module was defined as a contiguous peptide segment having compact conformation and its average length is about 15 amino acid residues. We show that the cooperative hemoglobins have interactins involving at least two pairs of modules among the four heme-contact modules at subunit contact. Received: 12 January 2001 / Accepted: 3 April 2001     

Amphibian oocytes and sphere organelles: are the U snRNA genes amplified?

The sphere organelles (spheres) ofXenopus and other amphibian oocytes are known to contain small nuclear ribonucleoprotein particles (snRNPs) and have been suggested to play a role in snRNP complex assembly. Coupled with the similarities that exist between spheres and nucleoli and the quantitative and kinetic aspects of snRNA synthesis in theXenopus oocyte, we have investigated whether or not the U snRNA encoding genes are amplified inXenopus oogenesis, the spheres being possible sites for the location of such extrachromosomal gene copies. By applying a number of quantitative nucleic acid hybridization procedures to both total and fractionated oocyte and somatic DNA, employing both homologous and heterologous U snRNA gene probes and suitable amplification and non-amplification control probes, we show that the U snRNA genes do not undergo any major amplification inXenopus oogenesis. Therefore, the analogy between the sphere organelles and nucleoli appears to be limited. The role of the spheres and their relationship to other snRNP containing structures, specifically B snurposomes, and the sphere organizer loci remains obscure.by A. Spradling     

Biological characterization of the skin of shortfin mako shark Isurus oxyrinchus and preliminary study of the hydrodynamic behaviour through computational fluid dynamics

This study characterized the morphology, density and orientation of the dermal denticles along the body of a shortfin mako shark Isurus oxyrinchus and identified the hydrodynamic parameters of its body through a computational fluid‐dynamics model. The study showed a great variability in the morphology, size, shape, orientation and density of dermal denticles along the body of I. oxyrinchus. There was a significant higher density in dorsal and ventral areas of the body and their highest angular deviations were found in the lower part of the mouth and in the areas between the pre‐caudal pit and the second dorsal and pelvic fins. A detailed three‐dimensional geometry from a scanned body of a shark was carried out to evaluate the hydrodynamic properties such as drag coefficient, lift coefficient and superficial (skin) friction coefficient of the skin together with flow velocity field, according to different roughness coefficients simulating the effect of the dermal denticles. This preliminary approach contributed to detailed information of the denticle interactions. As the height of the denticles was increased, flow velocity and the effect of lift decreased whereas drag increased. The highest peaks of skin friction coefficient were observed around the pectoral fins.     

Structural characterization of components of protein assemblies by comparative modeling and electron cryo-microscopy

We explore structural characterization of protein assemblies by a combination of electron cryo-microscopy (cryoEM) and comparative protein structure modeling. Specifically, our method finds an optimal atomic model of a given assembly subunit and its position within an assembly by fitting alternative comparative models into a cryoEM map. The alternative models are calculated by MODELLER [J. Mol. Biol. 234 (1993) 313] from different sequence alignments between the modeled protein and its template structures. The fitting of these models into a cryoEM density map is performed either by FOLDHUNTER [J. Mol. Biol. 308 (2001) 1033] or by a new density fitting module of MODELLER (Mod-EM). Identification of the most accurate model is based on the correlation between the model accuracy and the quality of fit into the cryoEM density map. To quantify this correlation, we created a benchmark consisting of eight proteins of different structural folds with corresponding density maps simulated at five resolutions from 5 to 15 angstroms, with three noise levels each. Each of the proteins in the set was modeled based on 300 different alignments to their remotely related templates (12-32% sequence identity), spanning the range from entirely inaccurate to essentially accurate alignments. The benchmark revealed that one of the most accurate models can usually be identified by the quality of its fit into the cryoEM density map, even for noisy maps at 15 angstroms resolution. Therefore, a cryoEM density map can be helpful in improving the accuracy of a comparative model. Moreover, a pseudo-atomic model of a component in an assembly may be built better with comparative models of the native subunit sequences than with experimentally determined structures of their homologs.     

Air-permeable hole-pattern and nose-droop control improve aerodynamic performance of primary feathers

Primary feathers of soaring land birds have evolved into highly specialized flight feathers characterized by morphological improvements affecting aerodynamic performance. The foremost feathers in the cascade have to bear high lift-loading with a strong bending during soaring flight. A challenge to the study of feather aerodynamics is to understand how the observed low drag and high lift values in the Reynolds (Re) regime from 1.0 to 2.0E4 can be achieved. Computed micro-tomography images show that the feather responds to high lift-loading with an increasing nose-droop and profile-camber. Wind-tunnel tests conducted with the foremost primary feather of a White Stork (Ciconia ciconia) at Re = 1.8E4 indicated a surprisingly high maximum lift coefficient of 1.5 and a glide ratio of nearly 10. We present evidence that this is due to morphologic characteristics formed by the cristae dorsales as well as air-permeable arrays along the rhachis. Measurements of lift and drag forces with open and closed pores confirmed the efficiency of this mechanism. Porous structures facilitate a blow out, comparable to technical blow-hole turbulators for sailplanes and low speed turbine-blades. From our findings, we conclude that the mechanism has evolved in order to affect the boundary layer and to reduce aerodynamic drag of the feather.     

Microhydrodynamics simulation of protein crystallization. I. Static calculations.

A computer simulation method is proposed to study the effects of hydrodynamic interactions on protein crystallization. It is a combination of Stokesian dynamics and continuum hydrodynamics and is referred to as "microhydrodynamics." The method is checked against analytical expressions for Stokes drag and diffusion coefficients for unit spheres. For a number of protein molecules the diffusion coefficients have been calculated and compared with experimental values. It is shown that the method works well for stationary calculations. Using dynamical calculations interacting protein molecules will be simulated to study the events in the early stages of protein crystallization.     

Biological activity of invitro synthesised protein: Binding of Semliki Forest virus capsid protein to the large ribosomal subunit

Cell-free translation of the Semliki Forest virus-specific 26S RNA yielded primarily capsid protein. After treatment of the protein synthesising reaction with 25 mM EDTA, the capsid protein cosedimented with the large ribosomal subunit in sucrose gradients, and banded with the subunit at a density of 1.54 gm/cm³ in CsCl. Exposure to 0.5 M KCl released the protein from the subunit. Similar binding of the virus capsid protein to the large ribosomal subunit has been observed in infected HeLa cells, although its function is not clear. The nonstructural proteins, which are the major products translated from the virion 42S RNA, did not associate with sedimenting structures.     

The complete primary structure of ribosomal protein L1 fromThermus thermophilus

The primary structure of the 23S rRNA binding ribosomal protein L1 from the 50S ribosomal subunit ofThermus thermophilus ribosomes has been elucidated by direct protein sequencing of selected peptides prepared by enzymatic and chemical cleavage of the intact purified protein. The polypeptide chain contains 228 amino acids and has a calculated molecular mass of 24,694 D. A comparison with the primary structures of the corresponding proteins fromEscherichia coli andBacillus stearothermophilus reveals a sequence homology of 49% and 58%, respectively. With respect to both proteins, L1 fromT. thermophilus contains particularly less Ala, Lys, Gln, and Val, whereas its content of Glu, Gly, His, Ile, and Arg is higher. In addition, two fragments obtained by limited proteolysis of the intact, unmodified protein were characterized.     

Yolk sphere formation is initiated in oocytes before development of patency in follicles of the moth,Plodia interpunctella

We describe a provitellogenic stage, a previously unrecognized stage of follicle development in moths, and show that oocytes begin yolk sphere formation prior to the development of patency by the follicular epithelium. The vitellogenic activities of follicles from pharate adult femalePlodia interpunctella (Hübner) were determined by visualizing the subunits of vitellin (YP1 and YP3) and the follicular epithelium yolk protein (YP2 and YP4) using monospecific antisera to each subunit to immunolabel whole-mounted ovaries or ultrathin sections. At 92 h after pupation, yolk spheres that contained only YP2 began to proliferate in the oocytes. The inter-follicular epithelial cell spaces were closed at 92 h making vitellogenin inaccessible to the oocyte, and consequently, the vitellin subunits were not observed in the yolk spheres. YP2 uptake most likely occurred across the brush border from the follicular epithelial cells to the oocyte at this time. At 105 h, the inter-follicular epithelial cell spaces appeared closed yet trace amounts of labeling for vitellin were observed in the spaces and also in the yolk spheres along with YP2. Equivalent labeling for all four YPs in yolk spheres was finally observed at 112 h after pupation when the follicular epithelium had become patent. These data indicate that the provitellogenic stage is an extended transition period between the previtellogenic and vitellogenic stages that lasts for approximately 13 h, and it is marked at the beginning by YP2 yolk sphere formation in the oocyte and at the end by patency in the follicular epithelium.     

Terahertz Response of ZnS/Ge and ZnO/Ge Nanostructured Composites

We present a new set of nanostructured composites which can exhibit a phenomenon known as surface plasmon resonance in a broad frequency range from the deep infrared to the terahertz region. The structures are composites of two different kinds of non-overlapping spheres. These spheres are made from a high refractive index nonplasmonic material and a Drude-like plasmonic material. Our results are explained in the context of the extended Maxwell–Garnett theory. The effective permittivity and refractive index of zinc sulfide/Ge and zinc oxide/Ge composites have been calculated over terahertz frequencies.     

Low-resolution structural studies of mitochondrial ubiquinol:cytochrome c reductase in detergent solutions by neutron scattering

Mitochondrial ubiquinol:cytochrome c reductase (Mr approximately 600,000) was cleaved into a complex (Mr approximately 280,000) of the subunits III (cytochrome b), IV (cytochrome c1) and VI to IX, a complex (Mr approximately 300,000) of the subunits I and II, and the single subunit V (iron-sulphur subunit, Mr approximately 25,000). Neutron scattering was applied to the whole enzyme, the cytochrome bc1 complex, both in hydrogenated and deuterated alkyl (phenyl) polyoxyethylene detergents, and the complex of subunits I and II in detergent-free solution. The neutron parameters were compared with the structures of the enzyme and the cytochrome bc1 complex previously determined by electron microscopy. Using the method of hard spheres, comparison of the calculated and experimental radius of gyration implies that the length of the enzyme across the bilayer or the detergent micelle is between 150 and 175 A and of the cytochrome bc1 complex between 90 and 115 A. The subunit topography was confirmed. The cleavage plane between the cytochrome bc1 complex and the complex of subunits I and II lies at the centre of the enzyme and runs parallel to the membrane just outside the bilayer. The detergent uniformly surrounds the protein as a belt, which is displaced by 30 to 40 A from the protein centre of the enzyme and by about 20 A from the protein centre of the cytochrome bc1 complex. The low protein matchpoint of the whole enzyme as compared to the subunit complexes is accounted for in terms of the non-exchange of about 30 to 60% of the exchangeable protons within the intact enzyme. Polar residues are, on average, at the protein surface and non-polar residues and polar residues with non-exchanged protons are buried within the enzyme.