Abstract: Forisomes are protein polymers found in leguminous plants that have the remarkable ability to undergo reversible "muscle-like" contractions in the presence of divalent cations and in extreme pH environments. To gain insight into the molecular basis of forisome structure and assembly, we used confocal laser scanning microscopy to monitor the assembly of fluorescence-labeled artificial forisomes in real time, revealing two distinct assembly processes involving either fiber elongation or fiber alignment. We also used scanning and transmission electron microscopy and X-ray diffraction to investigate the ultrastructure of forisomes, finding that individual fibers are arranged into compact fibril bundles that disentangle with minimal residual order in the presence of calcium ions. To demonstrate the potential applications of artificial forisomes, we created hybrid protein bodies from forisome subunits fused to the B-domain of staphylococcal protein A. This allowed the functionalization of the artificial forisomes with antibodies that were then used to target forisomes to specific regions on a substrate, providing a straightforward approach to develop forisome-based technical devices with precise configurations. The functional contractile properties of forisomes are also better preserved when they are immobilized via affinity reagents rather than by direct contact to the substrate. Artificial forisomes produced in plants and yeast therefore provide an ideal model for the investigation of forisome structure and assembly and for the design and testing of tailored artificial forisomes for technical applications.
Abstract: We report on the application of laser-induced breakdown spectroscopy (LIBS) to the determination of elements distinctive in terms of their biological significance (such as potassium, magnesium, calcium, and sodium) and to the monitoring of accumulation of potentially toxic heavy metal ions in living microorganisms (algae); in order to trace e.g. the influence of environmental exposure and other cultivation and biological factors having an impact on them. Algae cells were suspended in liquid media or piesented in a form of adherent cell mass on a surface (biofilm) and, consequently, characterized using their spectra. In our feasibility study we used three different experimental arrangements employing double-pulse LIBS technique in order to improve on analytical selectivity and sensitivity for potential industrial biotechnology applications, e.g. for monitoring of mass production of commercial biofuels, utilization in the food industry and control of the removal of heavy metal ions from industrial waste waters. (C) 2012 Elsevier B.V. All rights reserved.
Abstract: The sieve element occlusion (SEO) gene family originally was delimited to genes encoding structural components of forisomes, which are specialized crystalloid phloem proteins found solely in the Fabaceae. More recently, SEO genes discovered in various non-Fabaceae plants were proposed to encode the common phloem proteins (P-proteins) that plug sieve plates after wounding. We carried out a comprehensive characterization of two tobacco (Nicotiana tabacum) SEO genes (NtSEO). Reporter genes controlled by the NtSEO promoters were expressed specifically in immature sieve elements, and GFP-SEO fusion proteins formed parietal agglomerates in intact sieve elements as well as sieve plate plugs after wounding. NtSEO proteins with and without fluorescent protein tags formed agglomerates similar in structure to native P-protein bodies when transiently coexpressed in Nicotiana benthamiana, and the analysis of these protein complexes by electron microscopy revealed ultrastructural features resembling those of native P-proteins. NtSEO-RNA interference lines were essentially devoid of P-protein structures and lost photoassimilates more rapidly after injury than control plants, thus confirming the role of P-proteins in sieve tube sealing. We therefore provide direct evidence that SEO genes in tobacco encode P-protein subunits that affect translocation. We also found that peptides recently identified in fascicular phloem P-protein plugs from squash (Cucurbita maxima) represent cucurbit members of the SEO family. Our results therefore suggest a common evolutionary origin for P-proteins found in the sieve elements of all dicotyledonous plants and demonstrate the exceptional status of extrafascicular P-proteins in cucurbits.
Abstract: The biosynthesis of rubber is thought to take place on the surface of rubber particles in laticifers, highly specialized cells that are present in more than 40 plant families. The small rubber particle protein (SRPP) has been supposed to be involved in rubber biosynthesis, and recently five SRPPs (TbSRPP1-5) were identified in the rubber-producing dandelion species Taraxacum brevicorniculatum. Here, we demonstrate by immunogold labeling that TbSRPPs are localized to rubber particles, and that rubber particles mainly consist of TbSRPP3, 4 and 5 as shown by high-resolution two-dimensional gel electrophoresis and mass spectrometric analysis. We also carried out an RNA-interference approach in transgenic plants to address the function of TbSRPPs in rubber biosynthesis as well as rubber particle morphology and stability. TbSRPP-RNAi transgenic T. brevicorniculatum plants showed a 40-50% reduction in the dry rubber content, but neither the rubber weight average molecular mass nor the polydispersity of the rubber were affected. Although no phenotypical differences to wild-type particles could be observed in vivo, rubber particles from the TbSRPP-RNAi transgenic lines were less stable and tend to rapidly aggregate in expelling latex after wounding of laticifers. Our results prove that TbSRPPs are very crucial for rubber production in T. brevicorniculatum, probably by contributing to a most favourable and stable rubber particle architecture for efficient rubber biosynthesis and eventually storage.
Abstract: Chlorosomes, the antenna complexes of green bacteria, are unique antenna systems in which pigments are organized in aggregates. Studies on isolated chlorosomes from Chlorobaculum tepidum based on SDS-PAGE, immunoblotting and molecular biology have revealed that they contain ten chlorosomal proteins, but no comprehensive information is available about the protein composition of the entire organelle. To extend these studies, chlorosomes were isolated from C. tepidum using three related and one independent isolation protocol and characterized by absorption spectroscopy, tricine SDS-PAGE, dynamic light scattering (DLS) and electron microscopy. Tricine SDS-PAGE showed the presence of more than 20 proteins with molecular weights ranging between 6 and 70 kDa. The chlorosomes varied in size. Their hydrodynamic radius (R(h) ) ranged from 51 to 75 nm and electron microscopy indicated that they were on average 140 nm wide and 170 nm long. Furthermore, the mass of 184 whole chlorosome organelles determined by scanning transmission electron microscopy ranged from 27 to 237 MDa being on average 88 (±28) MDa. In contrast their mass-per-area was independent of their size, indicating that there is a strict limit to chlorosome thickness. The average protein composition of the C. tepidum chlorosome organelles was obtained by MS/MS-driven proteomics and for the first time a detailed protein catalogue of the isolated chlorosomal proteome is presented. Based on the proteomics results for chlorosomes isolated by different protocols, four proteins that are involved in the electron or ion transport are proposed to be tightly associated with or incorporated into C. tepidum chlorosomes as well as the ten Csm proteins known to date.
Abstract: Golgi-localized, γ-ear-containing, ADP ribosylation factor-binding (GGA) proteins are monomeric adaptors implicated in clathrin-mediated vesicular transport between the trans Golgi network and endosomes, characterized mainly from cell culture analysis of lysosomal sorting. To provide the first demonstration of GGA's role in vivo, we used Drosophila which has a single GGA and a single lysosomal sorting receptor, lysosomal enzyme receptor protein (LERP). Using RNAi knockdowns, we show that the Drosophila GGA is required for lysosomal sorting. We further identified authentic components of the Drosophila lysosomal sorting system--the sorting receptor LERP, the sorting adaptor GGA and the lysosomal cargo cathepsins B1, D and L--to show that GGA depletion results in lysosomal dysfunction. Abnormal lysosomal morphology, missorting of lysosomal cathepsins and impaired lysosomal proteolysis show disturbed LERP trafficking after GGA depletion. GGA is highly expressed in the mushroom bodies and the pigment cells of the retina, and increasing or decreasing the levels of GGA in the eyes leads to retinal defects. Reduced GGA levels also enhance an eye defect caused by overexpression of the autophagy-associated protein Blue cheese (Bchs), implicating GGA in autophagic processes. This shows that Drosophila provides an excellent whole-animal model to gain new insights into the function of GGA in the physiological environment of a multicellular organism.
Abstract: Hollow polymeric capsules prepared from Layer-by-Layer coating of colloidal templates with polyelectrolyte multilayers are promising materials for drug delivery and release applications. Details of the wall structure arising from the core dissolution process are investigated by high resolution scanning electron microscopy (SEM), using both secondary (SE) and backscattered electron (BSE) imaging modes. Atomic force microscopy (AFM) of the capsules in liquid was used as an independent technique. BSE images of the coated templates may be used for a rough estimation of the wall thickness. A freeze-drying procedure allows for the first time the investigation of dried multilayer capsules with an intact shape by SEM. Details of the nanostructure of the capsule walls are obtained, the topography shows structures on the scale of several 10 nm, corresponding to single chains. These structures are confirmed by AFM in liquid. In addition, after core dissolution single holes with sizes above 10 nm can be identified in the capsule wall. These holes are the structural property controlling the permeation and release and are here visualized for the first time. The number of holes per capsule as well as their distribution and size are analysed and discussed in their relevance for release applications.
Abstract: High-angle annular dark-field scanning transmission electron microscopy (HAADF STEM) at low energies (≤30 keV) was used to study quantitatively electron scattering in amorphous carbon and carbon-based materials. Experimental HAADF STEM intensities from samples with well-known composition and thickness are compared with results of Monte Carlo simulations and semiempirical equations describing multiple electron scattering. A well-defined relationship is found between the maximum HAADF STEM intensity and sample thickness which is exploited (a) to derive a quantitative description for the mean quadratic scattering angle and (b) to calculate the transmitted HAADF STEM intensity as a function of the relevant materials parameters and electron energy. The formalism can be also applied to determine TEM sample thicknesses by minimizing the contrast of the sample as a function of the electron energy.
Abstract: Human rhinoviruses (HRVs) are the major cause of the common cold and account for 30-50% of all acute respiratory illnesses. Although HRV infections are usually harmless and invade only the upper respiratory tract, several studies demonstrate that HRV is involved in the exacerbation of asthma. VP1 is one of the surface-exposed proteins of the viral capsid that is important for the binding of rhinoviruses to the corresponding receptors on human cells. Here we investigated its potential usefulness for vaccination against the common cold. We expressed VP1 proteins from two distantly related HRV strains, HRV89 and HRV14, in Escherichia coli. Mice and rabbits were immunised with the purified recombinant proteins. The induced antibodies reacted with natural VP1 and with whole virus particles as shown by immunoblotting and immunogold electron microscopy. They exhibited strong cross-neutralising activity for different HRV strains. Therefore, recombinant VP1 may be considered a candidate HRV vaccine to prevent HRV-induced asthma exacerbations.
Abstract: Electron microscopy has been used to measure the mass of biological nanoparticles since the early 60s, and is the only way to obtain the mass of large structures or parameters such as the mass-per-length of filaments. The ability of this method to sort heterogeneous samples both in terms of mass and shape promises to make it a key tool for proteomics down to the single cell level. A new multiplatform software package, MASDET, that can be run under MATLAB or as a standalone program is described. Based on a user-friendly graphical interface MASDET streamlines mass evaluation and greatly increases the speed of required optimisation procedures. Importantly, the immediate application of Monte-Carlo simulations to describe multiple scattering is possible, allowing the mass analysis of thicker samples and the generation of mass thickness maps.
Abstract: Pollen from timothy grass (Phleum pratense L.) was subjected to various aqueous and non-aqueous fixation and preparation protocols for transmission electron microscopy. Only in the cytoplasm of anhydrously prepared pollen grains were conspicuous inclusions observed that range in size from less than 1 mum up to 8 or 10 mum. These bodies have so far not been described in the literature. Higher magnifications show that these inclusions consist of bundles of hexagonally arranged small tubules. In order to obtain details of the ultrastructure of this novel pollen component, TEM micrographs of ultrathin sections of hexagonally arranged tubules were analyzed using Fourier transform techniques of image analysis. It was found that the tubules form groups with quasi-periodic hexagonal arrangement, with an average centre-to-centre spacing between the neighbouring tubules of approximately 42 nm. Individual tubules are formed by 12 or 13 particles. The outer diameter of the tubules ranges between 22 and 24 nm. From our experiments, we conclude that the quasi-periodic hexagonally arranged tubules forming conspicuous cytoplasmic inclusions in dry timothy grass pollen grains are structurally similar to microtubules.
Abstract: S-layers are surface layers of bacterial cell walls. They are formed by two-dimensional, monomolecular crystalline arrays of identical units of protein or glycoprotein macromolecules (subunits). In general, each S-layer exhibits one of four possible 2-D lattice types: oblique (p1 or p2 symmetry), triangle (p3 symmetry), square (p4 symmetry) or hexagonal (p6 symmetry). The S-layer protein compasses up to 15% of the total protein of the bacterial cell and thus represents its major protein. Since 1972, S-layers have also been found in cyanobacteria. So far, they have been observed in 60 strains (isolates) of 23 species, belonging to 12 genera of unicellular Chroococcales and in just five strains or isolates (four species, four genera-only with p1 and p4 lattice symmetry) of filamentous Oscillatoriales; in further families of filamentous cyanobacteria (Nostocales, Stigonematales) they have not been detected, although filamentous cyanobacteria have been frequently studied in the electron microscope. In Chroococcales, relatively large cells of planktonic genera harbouring gas vesicles, S-layers are often present, while picoplanktonic species without gas vesicles usually do not have them. The p6 lattice symmetry appears to be the most common in cyanobacteria, having been found in 41 out of the 60 S-layers observed. All cells of a given strain, all strains capable of forming S-layers and all S-layer forming species of a given genus (as far as it is known) form S-layers of the same lattice type. Hence, the ability to form an S-layer appears to be useful as a supportive morphological marker for species classification. In 41 S-layer formers, the center-to-center spacing of their lattice unit arrays has been measured; the lattice constants range from 5 to 22nm, measured directly on surface of fixed cells. Coarse S-layers of p6 symmetry are the most frequent (with spacing of 15.0-22.0nm); p1 and p2 S-layers are the finest ones (with spacing of 5.0-10.0nm). Medium-spaced lattices (11.0-14.0nm) may be both of the p4 or p6 symmetry types. When measured on isolated S-layers, the spacings show a 10-60% higher value. All the hexagonal unit lattices have the same molecular architecture. Each S-layer unit resembles a truncated cone with an axial pore and with six protein subunits symmetrically placed around its opening. Adjoining units are interspaced by relatively fine channels. The fine detail of every S-layer of every individual strain is unique. Only the S-layer protein subunits of Synechococcus sp. strain GL24 have been analysed by electrophoresis. When incorporated into the S-layer units they confer a net neutral charge to the cell surface. This cyanobacterium induces mineralization of fine-grain gypsum and calcite in a saturated lake fresh water solution. This process is involved in the formation of stromatolites.
Abstract: Methods for the analysis of continuously distorted quasiperiodic images are improved. Especially techniques of 2-D reconstruction are studied. The locally normalized correlation is used in the correlation-averaging method. To eliminate Image distortion the function of the distortion is approximated step by step by the linear fractional function or by the Lagrange interpolation polynomials. The modification of the methods is illustrated in figures of cyanobacterial S layers, namely, on negatively strained S layers of Synechocystis aquatilis Kovacik 1990/8 and Microcystis aeruginosa Hindak 1971/1 and the metal-shadowed S layer of Microcystis of. wesenbergii Bitov 1994. (C) 2000 Society of Photo-Optical Instrumentation Engineers. [S0091-3286(00)02504-6].
Abstract: Trace mineral analysis of the body is invaluable in biology, medicine and dentistry when considering the role of mineral nutrition and metabolism in the context of maintaining human health. The presence of key elements in the body, such as boron, calcium, chromium, copper, iron, silicon and zinc are known to be of vital importance, but are often found to be present in inadequate quantity. In sharp contrast, the accumulation of other elements, such as e.g. aluminium, cadmium, lead and mercury is less favourable since frequently these metals are already toxic at extremely low concentration levels, interfering with essential chemical processing of the vitamins and minerals. Here we report on the application of Laser Induced Breakdown Spectroscopy (LIBS) and Laser Induced Fluorescence Spectroscopy (LIFS) to the analysis of important minerals and of toxic elements within the body. Samples from different parts of the body have been studied, including specimen of skin tissue, finger nails and teeth. It is particularly noteworthy that for none of these laser spectroscopic measurements specific sample preparation was needed but that specimens could be used as taken from source.
Abstract: Methods for analysis, especially 2D reconstruction, of cyanobacterial S-layers were improved. The locally normalized correlation was used in the course of correlation averaging. For elimination of image distortion, the function of the distortion was approximated step by step by the linear fractional function or by the Lagrange interpolation polynomials. The modification of the methods is illustrated on figures of S-layers of Synechocystis aquatilis Kovacik 1990/8 and Microcystis aeruginosa Hindak 1971/1 (negatively stained), and Microcystis cf. wesenbergii Bitov 1994 (metal shadow).
Abstract: Two-dimensional reconstruction of continuously distorted quasi-periodical images is discussed. The use of classical methods, the method of modification of spatial-frequency spectrum and the method of correlation averaging, give accurate results only for images with very small distortion. In the paper the methods are modified by the use of a simple technique for elimination of the distortion and formula for correlation.
Abstract: The application of Laser Induced Breakdown Spectroscopy (LIBS) to real-time, in-sih and remote analysis of trace amounts in liquid samples is described, to be used for example in harsh or difficult-to-reach environments within industry for the analysis of pollutants in water. Numerous elements, including a range of toxic heavy metals, have been measured over a wide range of concentrations. Detection limits usually are in the range of a few parts per million; for several elements even lower limits could be realised. The analysis system is based on a fibre delivery assembly which is capable to both deliver the laser light to, and to collect the micro-plasma light from the target area, up to 30m. Alternatively, a telescopic arrangement for in a "line-of-sight" arrangement was employed. In order to provide quantitative data in the evaluation of laser generated plasmas, parameters such as electron densities, plasma temperature, line shape functions, and others need to be known; their measurement and determination are outlined. For internal standardisation and the generation of concentration calibration curves, reference lines of carefully chosen elements were used.
Abstract: S-layers are now considered a common cell wall structure in Bacteria and Archaea as well as in some algae. Morphological and chemical studies have revealed that S-layers consist of crystalline arrays of protein or glycoprotein subunits forming oblique, square or hexagonal lattices on the cell surface, Electron microscopy and computer image enhancement techniques have been applied to obtain structural information down to the nanometer range. This chapter deals with the wide distribution of S-layers among cyanobacteria, and their morphological and chemical characterization, and the potential of high resolution electron microscopic studies applied to the cell envelope of Pyrodictium. The occurrence of S-layers in cyanobacteria was investigated by cryomethods and ultrathin sectioning. These investigations indicate that the ultrastructure of S-layers may be exploited as an auxiliary taxonomic criterion in the classification of cyanobacteria. Pyrodictium is the first organism which has shown an optimum growth temperature above 100 degrees C. The highly irregularly shaped, flagellated cells are interconnected by extracellular tubules, The three-dimensional structure of this network was visualized with high resolution scanning electron microscopy while the fine structure of the cell wall architecture was studied in detail with Various electron microscopic techniques, Both contributions demonstrate that the investigation of the fine structure of S-layers is fundamental for establishing structure-function relationships for these two-dimensional crystalline arrays.
Abstract: The application of Laser Induced Breakdown Spectroscopy (LIBS) to real-time, in-situ and remote analysis of trace amounts in liquid samples is described, to be used for example in harsh or difficult-to-reach environments within industry for the analysis of pollutants in water. Numerous elements, including a range of toxic heavy metals, have been measured over a wide range of concentrations. Detection limits usually are in the range of a few parts per million; for several elements even lower limits could be realized. The analysis system is based on a fibre delivery assembly which is capable to both deliver the laser light to, and to collect the micro-plasma light from the target area, up to 30 m. Alternatively, a telescopic arrangement for in a `line-of-sight' arrangement was employed. In order to provide quantitative data in the evaluation of laser generated plasmas, parameters such as electron densities, plasma temperature, line shape functions, and others need to be known; their measurement and determination are outlined. For internal standardization and the generation of concentration calibration curves, reference lines of carefully chosen elements were used.
