Electron-microscopic analysis of ground elder (Aegopodium podagraria L.) lectin: evidence for a new type of supra-molecular protein structure

The lectin of ground elder (Aegopodium podagraria L.) was investigated electron-microscopically after negative staining with uranyl salts. Affinity-purified preparations of this glycoprotein were highly heteromorphous as they contained small particles approximately 4.6 nm in diameter and very large particles of different shapes. Among the latter, circular and helicoidal structures were the most regular in appearance. The circles were 9.3 nm in diameter, whereas the helices were 9 nm or 20 nm in diameter and up to 60 nm in length. After photographic enhancement, pictures of the molecules indicated that both the larger structures and the small particles could be obtained in pure forms by gel filtration of the lectin on Sepharose 4B. Since the former were the only constituents of the excluded fraction (M_r>5000000), whereas they were totally absent in the fraction eluting with an apparent molecular weight of about 500000, these supra-molecular structures revealed by the electron microscope cannot be artefacts generated during preparation of the lectin for electron-microscopic observation.Abbreviations APA Aegopodium podagraria agglutinin - EM electron microscopy - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Synthesis and spectral investigations of Cu(II) ion‐doped NaCaAlPO4F3 phosphor

Cu(II) ion‐doped NaCaAlPO₄F₃ phosphor has been synthesized using a solid state reaction method. The prepared sample is characterized by powder X‐ray diffraction, scanning electron microscope, optical absorption, electron paramagnetic resonance photoluminescence and Fourier transform infrared spectroscopy techniques. The crystallite size evaluated from x‐ray diffraction data is in nanometers. Scanning electron microscopy micrographs showed the presence of several irregular shaped particles. From optical absorption and electron paramagnetic resonance spectral data the doped Cu(II) ions are ascribed to distorted octahedral site symmetry. The synthesized phosphor exhibits emission bands in ultraviolet, blue and green regions under the excitation wavelength of 335 nm. The CIE chromaticity coordinates (x = 0.159, y = 0.204) also calculated for the prepared sample from the emission spectrum. The Fourier transform infrared spectroscopy spectrum revealed the characteristic vibrational bands of the prepared phosphor material. Copyright © 2014 John Wiley & Sons, Ltd.     

Effect of naloxone on renal cortical microcirculation in haemorrhagic shock

In order to assess the effect of opioid receptor antagonists, naloxone and noradrenaline, on renal cortical microcirculation, India ink infusion was made through the renal artery, one hour after treatment with each drug, in dogs subjected to haemorrhagic shock. Naloxone (1 mg/kg) treatment showed a dual beneficial effect of significant improvement (P < 0.001) in the mean arterial pressure without increasing the renal resistance as indicated by the presence of ink particles in about 75% of the cortical glomeruli. However, in the case of noradrenaline (2 Μ/kg/min)-treated animals, although mean arterial pressure increased significantly (P < 0.001) only very few glomeruli (25%) in the cortical region showed ink particles, demonstrating severe vasoconstriction. In the control group infused only with saline, although most of the glomeruli (92%) were filled with ink particles, there was a significant decline in the mean arterial pressure (P < 0.001).     

Synthesis,characterization and thermoluminescence studies of (ZnS)1‐x(MnTe)x nanophosphors

The present paper reports the thermoluminescence (TL) of (ZnS)_1‐x(MnTe)_x nanophosphors that were prepared by a wet chemical synthesis method. The structure investigated by X‐ray diffraction patterns confirms the formation of a sphalerite phase whose space group was found to be F 3m. From XRD, TEM and SEM analyses the average sizes of the particles were found to be 12 nm, 11 nm and 15 nm, respectively. Initially the TL intensity increased with increasing values of x because the number of luminescence centres increased; however, for higher values of x the TL intensity decreased because of the concentration quenching. Thus the TL, mechanoluminescence and photoluminescence intensities are optimum for a particular value of x, that is for x = 0.05. Thermoluminescence of the (ZnS)_1‐x (MnTe)_x nanophosphor has not been reported previously. There were two peaks seen in the thermoluminescence glow curves in which the first peak lay at 105–100 °C and the second peak lay at 183.5–178.5 °C. The activation energies for the first and second peaks were found to be 0.45 eV and 0.75 eV, respectively.     

In vivo effects of lipopolysaccharide on lymphoid and non-lymphoid cells in the mouse spleen

Summary In the present study the effects of lipopolysaccharide (LPS) on the cellular composition and phagocytosis of India ink in the inner parts of the periarteriolar lymphocyte sheaths (PALS) are described.Staining for B-, T-lymphocytes, and reticulin fibers in the spleen of normal and LPS-injected mice shows that the B-dependent follicular area is increased in size after LPS administration. However, the number of T-lymphocytes in the inner PALS is reduced markedly and a relatively high number of B-lymphocytes can be found in this area. The significance of this phenomenon is discussed.In untreated mouse spleen, carbon particles become localized in strongly acid-phosphatase (AP)-positive macrophages of the red pulp, marginal zone and white pulp 24 h after an intravenous injection of India ink. All these macrophages contain numerous carbon particles. After LPS pretreatment, the phagocytosis of carbon particles in the inner PALS is dramatically diminished, although many strongly AP-positive macrophages can be found in this area. The phagocytosis of carbon particles in the other compartments of the spleen did not change. It appears that injection of 2 g LPS or more is sufficient to induce this phenomenon which is most significant when LPS is injected 24 or 48 h before exposure to India ink.Abbreviations LPS lipopolysaccharide - PALS periarteriolar lymphocyte sheath - AP acid phosphatase - IDC interdigitating cells     

Bifunctional silica nanoparticles for the exploration of biofilms of Pseudomonas aeruginosa

Luminescent silica nanoparticles are frequently employed for biotechnology applications mainly because of their easy functionalization, photo-stability, and biocompatibility. Bifunctional silica nanoparticles (BSNPs) are described here as new efficient tools for investigating complex biological systems such as biofilms. Photoluminescence is brought about by the incorporation of a silylated ruthenium(II) complex. The surface properties of the silica particles were designed by reaction with amino-organosilanes, quaternary ammonium-organosilanes, carboxylate-organosilanes and hexamethyldisilazane. BSNPs were characterized extensively by DRIFT, ¹³C and ²⁹Si solid state NMR, XPS, and photoluminescence. Zeta potential and contact angle measurements exhibited various surface properties (hydrophilic/hydrophobic balance and electric charge) according to the functional groups. Confocal laser scanning microscopy (CLSM) measurements showed that the spatial distribution of these nanoparticles inside a biofilm of Pseudomonas aeruginosa PAO1 depends more on their hydrophilic/hydrophobic characteristics than on their size. CLSM observations using two nanosized particles (25 and 68?nm) suggest that narrow diffusion paths exist through the extracellular polymeric substances matrix.     

A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopy

A study is presented on the EPR characteristics of the paramagnetic groups in the respiratory chain present in membrane particles of Paracoccus denitrificans, the respiratory system of which is very similar to that in submitochondrial particles from beef heart. All paramagnetic prosthetic groups of the mitochondrial system are also found in the bacterial plasma membrane. Their properties suggest that the respiratory groups are embedded in very similar protein environments in the two systems.     

Nanoscale uniformity of pore architecture in diatomaceous silica: a combined small and wide angle x-ray scattering study

Combined small and wide angle X‐ray scattering (SAXS and WAXS) analysis was applied to purified biogenic silica of cultured diatom frustules and of natural populations sampled on marine tidal flats. The overall WAXS patterns did not reveal crystalline phases (WAXS domain between 0.07 to 0.5 nm) in this biogenic silica, which is in line with previous reports on the amorphous character of the SiO₂ matrix of diatom frustules. One exception was the silica of the pennate species Cylindrotheca fusiformis Reimann et Lewin, which revealed wide peaks in the WAXS spectra. These peaks either indicate the presence of a yet unknown crystalline phase with a repetitive distance (d‐value ≈0.06 nm) or are caused by the ordering of the fibrous silica fragments; numerous girdle bands. The SAXS spectra revealed the size range of pores (diameter d between 3.0 and 65 nm), the presence of distinct pores (slope transitions), and structure factors (oscillation of the spectra). All slopes varied in the range of ?4.0 to ?2.5, with two clear common regions among species: d < 10 nm (slopes –4, denoted as region I and also called the Porod region), and 10.0 < d < 40.0 nm (slopes ?2.9 to ?3.8, denoted as region II). The existence of these common regions suggests the presence of comparable form (region I) and structure (region II) factors, respectively the shape of the primary building units of the silica and the geometry of the pores. Contrast variation experiments using dibromomethane to fill pores in the SiO₂ matrix showed that scattering was caused by pores rather than silica particles. Electron microscopic analysis confirmed the presence of circular, elliptical, and rectangular pores ranging in size from 3 to 65 nm, determining the structure factor. The fine architecture (length/width ratio of pore diameters) and distribution of the pores, however, seemed to be influenced by environmental factors, such as the salinity of and additions of AlCl₃ to the growth medium. The results indicate that diatoms deposit silica with pores <50 nm in size and are highly homologous with respect to geometry. Consequently, it is suggested that in diatoms, whether pennate or centric, the formation of silica at a nanoscale level is a uniform process.     

Magnetizable antibody-like proteins

Use of paramagnetic particles to isolate molecules or cells from complex media is well established. Typically, particles are manufactured and coated with a biological molecule that confers specific biorecognition. Incubation of particles with sample and exposure to magnetic fields isolates the species of interest. We have designed, produced and assessed magnetized fusion proteins consisting of the antigen-binding portion of an antibody (single chain variable fraction; scFv) fused to the heavy chain of the iron-binding protein ferritin. The fusion protein subunits expressed in E. coli assemble to form a fusion protein consisting of a ferritin sphere with scFvs on the surface. The fusion proteins were chemically magnetized by introducing a paramagnetic iron core. The resultant fusion protein was shown to be magnetizable and capable of binding target antigens. These “organic” magnetizable particles possess a number of theoretical advantages over traditional inorganic particles.     

Correlation of photosystem-II complexes with exoplasmatic freeze-fracture particles of thylakoids of the cyanobacterium Synechococcus sp.

The supramolecular structure of the exoplasmic freeze-fracture particles of thylakoids of the thermophilic cyanobacterium Synechococcus sp. is compared with that of isolated photosystem-II complexes. The in-situ EF particles are scattered on the thylakoids or organized in rows of variable length; the latter aligned particles measure 10 nmx20 nm and are separated perpendicular to their long axis into two parts. We propose that they represent dimers composed of two monomeric 10-nm EF particles side by side. Isolated photosystem (PS)II particles correspond in size to the monomeric 10-nm EF particles as analysed by negative contrast and freeze-fracture electron microscopy. Dimeric PSII particles, very similar to the in-situ 10 nmx20 nm EF particles, are obtained after incorporation of purified PSII complexes into liposomes made from phospholipid and cholesterol. Each monomeric complex consists of the reaction center, the water-splitting system, the chlorophyll antennae and phycobilisome-binding polypeptides. We propose that the dimeric complexes bind one hemidiscoidal phycobilisome at their domains exposed to the external side of the thylakoids. The implications of this arrangement of the PSII-phycobilisome complexes within the thylakoids upon excitation-energy distribution are discussed.Abbreviations EF exoplasmic fracture face - LDS lithium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - PS photosystem - SDS sodium dodecyl sulfate - SPC-buffer 0.5 M sucrose, 0.5 M K₂HPO₄/KH₂PO₄, 0.3 M Nacitrate, pH 7.0 This study is dedicated to Professor W. Nultsch on the occasion of his 60th birthday.     

Membrane routing during exocytosis and endocytosis in neuroendocrine neurones and endocrine cells: use of colloidal gold particles and immunocytochemical discrimination of membrane compartments

Summary The hypothesis that the retrieval of membranes of neurohypophysial neurosecretory granules (NSG) and small electron-lucent microvesicles occurs by different routes was tested by incubating neurohypophysial neurosecretosomes with colloidal gold particles of various sizes. Neurosecretosomes derived from normal Long Evans rats and incubated in media of normal ionic composition endocytosed a few small (<25 nm) gold particles into 40–50 nm electron-lucent microvesicles. After depolarisation, more small gold particles were found in microvesicles, and small and large (>25 nm) gold particles in vacuoles. Oxytocin-containing neurosecretosomes derived from Brattleboro rats, which contain 160 nm-diameter NSG, endocytosed gold particles in a pattern indistinguishable from that of neurosecretosomes from Long Evans rats. However, neurosecretosomes derived from defective vasopressin neurones of Brattleboro rats, which contain microvesicles, small vacuoles, and a few 100 nm dense-cored vesicles, but not 160 nm NSG, endocytosed only small colloidal gold particles. Early after depolarisation the gold particles were present only in microvesicles, but later some could be found in vacuoles and lysosome-like structures. Immunogold cytochemistry using a polyclonal antiserum raised against microvesicle-rich neurosecretosomes derived from Brattleboro rats labelled microvesicles in the posterior pituitary strongly, NSG weakly, and vacuoles to a variable extent. These data together indicate that, after exocytosis, the membranes of NSG are recaptured as large vacuoles. Microvesicles are exocytosed and endocytosed separately.     

Atomic Force Microscopic Observation of Escherichia coli Ribosomes in Solution

Ribosomes of Escherichia coli were visualized in buffer solution by atomic force microscopy (AFM). A series of time-dependent AFM images showed that ribosomes spontaneously adsorb on mica. Although ribosomes observed in air are forced to flatten on the surface, the height of ribosomal particles obtained under a physiological buffer solution is 21.8±0.5 nm, which is consistent with the ideal diameter. We succeeded in observing single ribosomes in a near-native condition.     

Phototropism of maize coleoptiles Influences of light gradients

The lateral fluence-rate gradients in unilaterally irradiated maize (Zea mays L.) coleoptiles were calculated on the basis of the proportions of P _fr (far-red-absorbing form of phytochrome) measured spectroscopically in transverse slices of the coleoptiles (top 1 cm). The results showed the occurrence of significant gradients that are wavelength-dependent. The gradient at 449 nm was steeper than those measured at 516, 534 and 551 nm, which were steeper than that measured at 665 nm. The ratios between the sides proximal and distal to the light source were, for example, 1:0.12 (449 nm), 1:0.23 (534 nm), and 1:0.28 (665 nm). Fluence-response curves for coleoptile phototropism (first positive curvature produced by less than 100 s unilateral irradiation) were measured at 449, 516, 534 and 551 nm. Comparison of the threshold fluences indicated that the responsiveness to 551 nm is about 10^4.8 less than that to 449 nm. Increasing wavelengths led to a decrease in maximal curvature, which correlated with the decrease of the fluence-rate ratios between the proximal and distal sides. Phototropic fluence-response curves were also measured using bilateral irradiation (449 nm). In one set of experiments, the fluence ratio was kept constant (either 1:1/2, 1:1/4 or 1:1/16) and the total fluence was varied, and in the other set the fluence applied to one side was kept constant and the fluence ratio was varied. A simple model based on the assumption that only one photoreaction occurs, and that the response is a function of the difference between the proximal and distal sides in the local photoreceptor action was tested. A fluence-response curve for this local photoreceptor action was calculated based on the fluence-rate ratio and the phototropic fluence-response curve measured for 449 nm. This curve was used, in conjunction with the measured fluence-rate ratios, as a basis for calculating phototropic fluence-response curves for other wavelengths and those for 449 nm obtained with bilateral irradiation. The calculated fluence-response curves showed excellent agreement with the experimental data. It is concluded that the threshold for maize coleoptile phototropism reflects the apparent photoconversion cross-section of the blue-light receptor whereas the maximal curvature depends on the steepness of the light gradient across the coleoptile.Abbreviations and symbols I(x) fluence rate at the depth x - P _fr phytochrome (far-red absorbing) - P _r phytochrome (red absorbing) - P _tot total phytochrome (P _r+P _fr) - photoconversion cross-section     

The detection of single-stranded RNA in an isometric virus-like particle from Shiitake mushroom [Lentinus edodes (Berk.) Sing.]

Isometric virus-like particles (VLPs) with diameter of approximately 34 nm containing ss-RNA were purified from abnormal mycelium of Shiitake mushroom, Lentinus edodes (Berk.) Sing. SDS-polyacrylamide gel elec-trophoresis demonstrated that the virions contain a single capsid polypeptide with molecular weight of about 22 000 daltons. The nucleic acid extract from purified VLPs preparations showed only one band with a size of approximately 7.3 kilobases. The susceptibility to RNase 1 and S1 digestions, resistance to DNase and thermal denaturation behaviour of the viral genome indicated that it is a single-stranded RNA. To our knowledge, isometric single-stranded RNA VLPs isolated from Shiitake mushroom mycelia have not been reported before.     

Diffuse Reflectance Infrared Spectroscopic Identification of Dispersant/Particle Bonding Mechanisms in Functional Inks

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers. A typical inkjet ink is a colloidal dispersion containing approximately ten components including solvent, the nano to micron scale particles which will comprise the printed layer, polymeric dispersants to stabilize the particles, and polymers to tune layer strength, surface tension and viscosity. To rationally and efficiently formulate such an ink, it is crucial to know how the components interact. Specifically, which polymers bond to the particle surfaces and how are they attached? Answering this question requires an experimental procedure that discriminates between polymer adsorbed on the particles and free polymer. Further, the method must provide details about how the functional groups of the polymer interact with the particle. In this protocol, we show how to employ centrifugation to separate particles with adsorbed polymer from the rest of the ink, prepare the separated samples for spectroscopic measurement, and use Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) for accurate determination of dispersant/particle bonding mechanisms. A significant advantage of this methodology is that it provides high level mechanistic detail using only simple, commonly available laboratory equipment. This makes crucial data available to almost any formulation laboratory. The method is most useful for inks composed of metal, ceramic, and metal oxide particles in the range of 100 nm or greater. Because of the density and particle size of these inks, they are readily separable with centrifugation. Further, the spectroscopic signatures of such particles are easy to distinguish from absorbed polymer. The primary limitation of this technique is that the spectroscopy is performed ex-situ on the separated and dried particles as opposed to the particles in dispersion. However, results from attenuated total reflectance spectra of the wet separated particles provide evidence for the validity of the DRIFTS measurement.     

Expression of HBcAg mutant with long internal deletion in Saccharomyces cerevisiae and observation of its self-assembly particles by atomic force microscopy (AFM)

An internally truncated C gene of adr hepatitis B virus core antigen with long internal deletion (aa81–aa116) (ΔHBcAg with 36aa truncation) was expressed in Saccharomyces cerevisiae and the products (ΔrHBcAg) were purified from a crude lysate of the yeast by three steps: Sephrose CL-4B chromatography, sucrose step-gradient ultracentrifugation and CsCl-isopycnic ultracentrifugation. Results of ELISA test and density analysis of CsCl-isopycnic ultracentrifugation indicated that the purified products (ΔrHBcAg protein) with HBeAg antigenicity mainly located at the densities of 1.23 g ml⁻¹. Observation and analysis of the purified ΔrHBcAg products by AFM indicated that the ΔrHBcAg (core) protein produced in S. cerevisiae could self-assemble into three or more size classes of core particles which exhibited a polymorphous distribution of ΔrHBcAg (core) particles. These different size classes of core particles mainly centred on the range whose mean diameter was from 10 nm to 48 nm, especially on the position of 11 nm, 15.6 nm and the range from 27 nm to 41 nm, respectively. Furthermore, the most number of core particles mainly centred on the range whose mean diameter was from 27 nm to 41 nm. These results above indicated that the truncated internal long fragment (aa81–aa116) probably had no effect on self-assembly of the HBcAg core particles which implied the internal length fragment (aa81–aa116) was not the sole domain for self-assembly of HBcAg dimer or the truncated HBcAg protein subunit formed the fresh interactive domain with each other. These initial results above by AFM analysis were very important for further research on the self-assembly, ultrastructure, subunit interaction and core internal deletion mutant (CIDM) function of HBcAg core particles.     

Electron paramagnetic resonance study of honeybee Apis mellifera abdomens

Although ferromagnetic material has been detected in Apis mellfera abdomens and identified as suitable for magnetic reception, physical and magnetic properties of these particles are still lacking. Electron paramagnetic resonance is used to study different magnetic materials in these abdomens. At least four iron structures are identified: isolated Fe3+ ions, amorphous FeOOH, isolated magnetite nanoparticles of about 3 x 10(2) nm3 and 10(3) nm3 volumes, depending on the hydration degree of the sample, and aggregates of these particles. A low-temperature transition (52-91 K) was observed and the temperature dependence of the magnetic anisotropy constant of those particles was determined. These results imply that biomineralized magnetites are distinct from inorganic particles and the parameters presented are relevant for the refinement of magnetoreception models in honeybees.     

Localization of phytohemagglutinin in the embryonic axis of Phaseolus vulgaris with ultra-thin cryosections embedded in plastic after indirect immunolabeling

We have examined the properties and subcellular localization of phytohemagglutinin (PHA), the major lectin of the common bean (Phaseolus vulgaris.), in the axis cells of nearly mature and imbibed mature seeds. On a protein basis the axis contained about 15% as much PHA as the cotyledons. Localization of PHA was done with an indirect immunolabeling method (rabbit antibodies against PHA, followed by colloidal gold particles coated with goat antibodies against rabbit immunoglobulins) on ultra-thin cryosections which were embedded in plastic on the grids after the immunolabeling procedure. The embedding greatly improved the visualization of the subcellular structures. The small (4 nm) collodial gold particles, localized with the electron microscope, were found exclusively over small vacuoles or protein bodies in all the cell types examined (cortical parenchyma cells, vascular-bundle cells, epidermal cells). The matrix of these vacuoles-protein bodies appears considerably less dense than that of the protein bodies in the cotyledons, but the results confirm that in all parts of the embryo PHA is localized in similar structures.Abbreviations IgG immunoglobulin G - M_r relative molecular weight - PBS phosphate-buffered saline - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

EPR and optical changes of the photosystem II reaction center produced by low temperature illumination

Electron paramagnetic resonance (EPR) and absorption spectroscopy have been used to study the low temperature photochemical behavior of the Photosystem II D-1/D-2/ cytochrome b559 reaction center complex. The reaction center displays large triplet state EPR signals which are attenuated after actinic illumination at low temperatures in the presence of sodium dithionite. Concomitant with the triplet attenuation is the buildup of a structured radical signal with an effective g value of 2.0046 and a peak-to-peak width of 11.9 G. The structure in the signal is suggestive of it being comprised in part of the anion radical of pheophytin a. This assignment is corroborated by low temperature optical absorbance measurements carried out after actinic illumination at the low temperatures which show absorption bleachings at 681 nm, 544 nm and 422 nm and an absorbance buildup at 446 nm indicating the formation of reduced pheophytin.Abbreviations EPR electron paramagnetic resonance