A Virus Infection in the Brown Alga Sorocarpus uvaeformis (Lyngbye) Pringsheim (Phaeophyta, Ectocarpales)

Virus-like particles were obsemed in zoospores and less frequentlyin vegetative cells of the filaments of mature plants of thebrown alga Sorocarpus uvaeformls. The particles, measuring approximately170 nm in diameter, are isometric in profile and show threedistinct zones. An electron dense rim (coat), 10 nm in thickness,is separated from a dense core, 110 nm in diameter, by an electronlight space 20 nm in width. When closely packed the particlesare usually separated from each other by a regular halo-likespace. Besides the isometric particles long flexuous structuresof variable length and measuring 75 nm in width were also found.The infection could be induced experimentally in healthy cellsby using either medium prevenient from infected cultures orcrude extracts obtained from infected plants.     

RecA protein self-assembly. Multiple discrete aggregation states

Light scattering, sedimentation and electron microscopy have been used to investigate the aggregation states of highly purified RecA protein in solution. We show that RecA protein will self-assemble into a discrete series of quaternary structures depending upon protein concentration, ionic environment, and nucleotide cofactors. In a stock solution at moderate concentration (10 to 50 microM) RecA protein exists as small particles approximately 4 nm in diameter, larger particles approximately 12 nm in diameter (most probably rings of RecA protein), 10 nm diameter rods varying from 50 to 200 nm in length, and finally as much larger bundles of rods. The addition of monovalent salt shifts the distribution of RecA protein between its various oligomeric states. Increasing protein concentration favors more highly aggregated structures. At a given protein concentration, addition of mM levels of MgCl2 promotes the rapid formation of rods and slow formation of bundles. Under conditions typical of in vitro strand exchange reactions, RecA protein was found to exist as a mixture of rods and 12 nm particles with relatively few monomers.     

Nonoccluded baculovirus- and filamentous virus-like particles in the spotted cucumber beetle,diabrotica undecimpunctata (coleoptera: chrysomelid)

Nonoccluded baculovirus-and filamentous virus-like particles were found in nuclei of hemocytes or midgut cells of field-collected spotted cucumber beetles. Each type of particle was associated with a different type of virogenic stroma containing various viral components similar to those referred to as capsid, nucleocapsid, viroplasm, and viral envelope in other known baculovirus infections. Nucleocapsids of the virus which occured only in hemocytes were rod-shaped particles approximately 230 nm long and 52 nm wide and were enveloped singly by a trilaminar unit membrane. Enveloped and partly enveloped particles appeared to be released from the nucleus to the cytoplasm by budding through the nuclear envelope acquiring additional membranes. The nucleocapsids of the virus which occurred only in nuclei of midgut cells were filamentous particles with an average diameter of 25 nm and variable length up to 2 μm. Some extremely long particles were bent almost 360° near the middle, resulting in a hairpin-like configuration. The particles were always enveloped singly. No particles budding through the nuclear envelope were observed.     

Formation of amyloid fibrils via longitudinal growth of oligomers

Mature amyloid fibrils are believed to be formed by the lateral association of discrete structural units designated as protofibrils, but this lateral association of protofibrils has never been directly observed. We have recently characterized a thioesterase from Alcaligenes faecalis, which was shown to exist as homomeric oligomers with an average diameter of 21.6 nm consisting of 22 kDa subunits in predominantly beta-sheet structure. In this study, we have shown that upon incubation in a 75% ethanol solution, the oligomeric particles of protein were transformed into amyloid-like fibrils. TEM pictures obtained at various stages during fibril growth helped us to understand to a certain extent the early events in the fibrillization process. When incubated in 75% ethanol, oligomeric particles of protein grew to approximately 35-40 nm in diameter before fusion. Fusion of two oligomers of 35-40 nm resulted in the formation of a fibril. Fibril formation was accompanied by a reduction in the diameter of the particle to approximately 20-25 nm along with concomitant elongation to approximately 110 nm, indicating reorganization and strengthening of the structure. The elongation process continued by sequential addition of oligomeric units to give fibers 500-1000 nm in length with a further reduction in diameter to 17-20 nm. Further elongation resulted in the formation of fibers that were more than 4000 nm in length; the diameter, however, remained constant at 17-20 nm. These data clearly show that the mature fibrils have assembled via longitudinal growth of oligomers and not via lateral association of protofibrils.     

Basal lamina anionic sites in the embryonic submandibular salivary gland: resolution and distribution using ruthenium red and polyethyleneimine as cationic probes

The basal lamina of the embryonic submandibular epithelium is a dynamic compartment of the extracellular matrix required for branching morphogenesis. A transmission electron microscopy (TEM) structural analysis of the basal lamina, at a time of intense branching activity, was conducted, comparing standard glutaraldehyde-fixed preparations with ones that included tannic acid in the primary fixative, and comparing anionic site resolution and distribution with two cationic probes, ruthenium red (RR) and polyethyleneimine (PEI). Standard TEM revealed a conventional basal lamina structure, with a lamina densa, a lamina lucida interna and a lamina lucida externa. Fine filaments emanated from the lamina densa, traversing both lamina lucidae. Tannic acid revealed approximately 35 nm diameter electron-dense particles in the lamina densa with a spacing repeat of approximately 45 nm. Basal lamina anionic sites were resolved as approximately 26 nm diameter RR-particles and approximately 50 nm diameter PEI-particles, present in the lamina lucida interna and associated with the lamina lucida externa. RR-particle linear spacing was 70 nm in the externa and 50 nm in the interna, while the PEI-particle spacing repeat was 90 nm in both compartments. Binding of both probes was blocked by testicular hyaluronidase or chondroitinase treatment, a result suggesting that the anionic sites were chondroitin sulfate proteoglycan, hyaluronic acid, or both. The greater particle spacing observed with PEI was not simply a physical limitation resulting from the average PEI particle diameter being almost twice that of RR particles, since PEI-resolved anionic sites on interstitial collagen were much more closely spaced (approximately 60 nm) than RR-resolved sites (approximately 105 nm).(ABSTRACT TRUNCATED AT 250 WORDS)     

Preliminary Observations on the Fine Structure of the Pansporoblast of Thelohania bracteata (Strickland, 1913) (Microsporida, Nosematidae) as Revealed by Freeze-Etching Electron Microscopy

SYNOPSIS. The ultrastructure of a microsporidan pansporoblast was observed with freeze-etching electron microscopy. The cross-fractured face of ovoid mature spores, with the upper part of the spore coat fractured off, revealed the spore membrane; the convex face had many small depressions and the concave face bore fine particles. In cross-section the spore-coat was highly laminated and about 0.5 μ in diameter.
In the cytoplasm of the pansporoblast, fluid-filled and finger-print-life profiles of vesicles were observed. The vesicles were approximately 180 nm in diameter and laminated, each lamella being about 15–18 nm thick. In addition to these vesicles, a population of elevations, each with an average diameter of 40 nm, was evenly distributed in the pansporoblast among the spores. No other cytoplasmic organelles were observed within the pansporoblast. The pansporoblast wall was about 15–19 nm thick with particles 15–18 nm in diameter on its outer surface.     

Electron Microscopy of Colicin I-Producing Cells

Electron microscope examination of mitomycin C-induced strains of Escherichia coli colicinogenic for colicins Ia or Ib reveals particles of approximately 15 to 20 nm in diameter associated with the cell surface. Such structures are not present on uninduced colicinogenic strains nor on a mitomycin C-treated non-colicinogenic strain. In contrast to wild-type colicinogenic strains, induction of a colicinogenic strain known to be lacking the specific colicin I receptor was shown to result in the release of colicin activity into the cell medium. Examination of such induced mutants by electron microscopy showed their cell surfaces to be free of the particles observed on the wild-type strain. The cell medium of such induced mutant strains contained large numbers of particles of similar size and shape to those found on the surface of induced colicinogenic wild-type strains.     

Gradient gel electrophoresis-immunoblot analysis (GGEI): a sensitive method for apolipoprotein profile determinations

A method is described which will determine the distribution of individual apolipoproteins within the HDL subclasses. This method requires 1-2 microliters of plasma per determination and involves six steps: 1) electrophoresis of samples on non-denaturing 2-30% concave acrylamide gradient gels; 2) electrophoretic transfer of the lipoproteins to charge-modified nylon membranes; 3) fixation of the transferred lipoproteins with glutaraldehyde; 4) immunolocalization of the apolipoproteins with iodinated monospecific antibodies; 5) autoradiography followed by densitometry; and 6) reduction of the data to provide a plot of percent distribution versus particle size. When this method was applied to the analysis of rat apolipoproteins, differences were noted in the distribution of apoA-I, apoA-IV, and apoE. The majority of apoA-I was localized to HDL particles between 9 and 12 nm in diameter, with a median diameter of 10.0 nm, while apoE resided on substantially larger particles with a median diameter of 12.5 nm. ApoA-IV could be localized to three distinct areas: an HDL particle with a median diameter approximately 0.4 nm larger than apoA-I HDL, a particle smaller than albumin (lipoprotein-free apoA-IV), and a particle of 7.6 nm that does not appear to contain apoA-I or apoE.     

Visualization by freeze fracture, in vitro and in vivo, of the products of fat digestion

The technique of freeze fracture was used to visualize triglyceride (TG) hydrolysis and the production of lipolytic products (LPs) in vitro and in vivo in the presence of bile salts (BS). Three systems were investigated: pure lipolytic products (oleic acid and monoolein) in the presence of a pure bile salt (taurodeoxycholate (TDC)), lipolytic products produced from TG by pancreatic lipase in the presence of a variety of bile salts, and lipolytic products produced in the intestine of the killifish, Fundulus heteroclitus, after fat feeding. In vitro, lamellae (4-5 nm thick with 0-8-nm water spacings) appeared on the surface of TG droplets in all preparations with LP/BS molar ratios of 1.5 or greater and spherical vesicles (diameter range, 20-130 nm) were produced from these lamellae. With model killifish bile (taurocholate-cholate 1:1) at LP/BS ratios between 1.5 and 4, homogeneous vesicles or particles (mean diameter, 23.8 nm) were produced by lipase at pH 6.9. In vivo, lamellar product phases also occurred after fat feeding. The smallest visible LP/BS structures by freeze fracture electron microscopy were approximately 20 nm globular particles. Large disc-shaped micelles either were not present or were below the resolution limit of the replica (approximately 10 nm). The dominant aggregated lipolytic product phase was composed of multiple layers of rough-textured lamellae. No evidence of cubic structure was seen. These results show that lamellar and vesicular lipolytic product phases can be intermediates in intestinal fat digestion. However, no evidence for the direct endocytotic absorption of these product phases by the intestinal microvillus membrane was found.     

从一种地方性水牛病的组织中见到的病毒样颗粒

多年来在江苏淮河流域丘陵地区,流行一种以高热、败血症为特征的水牛病,病牛经多种药物治疗无效而死亡。自1984年以来,用电镜技术对病牛的病变组织和血液进行了超微结构研究,在病变组织和白细胞中发现了近似予逆转录病毒科中的RNA肿瘤病毒。这在国内外文献上尚未有报道。现将我们的观察结果报道如下,供商讨。 取8头刚宰杀的晚期病牛的淋巴结、肝脾组织;另取5头病牛的血液,用分层液提取白     

Gap junctions. Structural changes after uncoupling procedures

The freeze-fracture appearance of rat stomach and liver gap junctions changes after uncoupling procedures such as inhibition of the metabolism of perfusion with hypertonic sucrose. In control stomach, either fixed immediately or kept for 1 h in a well-oxygenated Tyrode's solution at 37 degrees C, most gap junctions between mucous cells contain particles irregularly packed at an average center-to-center spacing of 10.3-10.5 nm. After 1-h treatment with 2,4-dinitrophenol (DNP), at the same temperature and oxygenation, most particles aggregate hexagonally at an average spacing of approximately 8.5 nm. Similar changes are seen in hypoxic specimens. In control liver, fixed by perfusion, most junctional particles are irregularly packed at an average center-to-center spacing of approximately 10 mm. Small areas of fairly regular hexagonal packing are occasionally seen, where the average particle spacing is 9.2-9.5 nm. In hypoxic liver, the junctional particles form regular hexagonal packings in which the average center-to-center particle spacing is approximately 8.5 nm. In liver perfused with hypertonic sucrose-calcium solutions, following EDTA solutions, most junctions are pulled apart. The separated junctional membranes, expected to be highly impermeable, contain particles regularly and tightly packed as in hypoxic or DNP-treated junctions. Preliminary measurements indicate also a possible change in particle diameter, from approximately 8.6 nm (control) to approximately 7.7 nm (treated). The structural changes are similar to those previously reported in crayfish and may reflect conformational changes in particle subunits resulting in functional uncoupling.     

A freeze-fracture study of afferent and efferent synapses of hair cells in the sensory epithelium of the organ of Corti in the guinea pig

Summary Afferent and efferent synapses of hair cells in the organ of Corti of the guinea pig have been examined in freeze-fracture replicas.Afferent synapse In the inner hair cells, intramembranous particles 10 nm in diameter are aggregated on the ridge on the P-face of the presynaptic membrane directly beneath the synaptic rod. In the outer hair cells, in which the synaptic rod is located in the presynaptic cytoplasm underneath the presynaptic membrane, small aggregations of intramembranous particles 10 nm in diameter can be found on the P-face of the presynaptic membrane corresponding to the site of the presynaptic dense projection. Intramembranous particles 10 nm in diameter are also densely aggregated on the P-face of the postsynaptic membrane of the outer hair cells.Efferent synapse of the outer hair cells Large intramembranous particles 13 nm in diameter are distributed in clusters composed of four to ten particles on the P-face of the presynaptic membrane. In the P-face of the postsynaptic membrane, disc-like aggregations of intramembranous particles 9 nm in diameter are found. The subsynaptic cistern covers the cytoplasmic surface of the postsynaptic membrane of the efferent synapse; it may cover more than one postsynaptic membrane when several efferent synapses are in close proximity to one another.     

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     

Topography and functional information of plasma membrane

By using atomic force microscope (AFM), the topography and function of the plasmalemma surface of the isolated protoplasts from winter wheat mesophyll cells were observed, and compared with dead protoplasts induced by dehydrating stress. The observational results revealed that the plasma membrane of living protoplasts was in a state of polarization. Lipid layers of different cells and membrane areas exhibited distinct active states. The surfaces of plasma membranes were unequal, and were characterized of regionalisation. In addition, lattice structures were visualized in some regions of the membrane surface. These typical structures were assumed to be lipid molecular complexes, which were measured to be 15.8±0.09 nm in diameter and 1.9±0.3 nm in height. Both two-dimensional and three-dimensional imaging showed that the plasmalemma surfaces of winter wheat protoplasts were covered with numerous protruding particles. In order to determine the chemical nature of the protruding particles, living protoplasts were treated by proteolytic enzyme. Under the effect of enzyme, large particles became relatively looser, resulting that their width was increased and their height decreased. The results demonstrated that these particles were likely to be of protein nature. These protein particles at plasmalemma surface were different in size and unequal in distribution. The diameter of large protein particles ranged from 200 to 440 nm, with a central micropore, and the apparent height of them was found to vary from 12 to 40 nm. The diameter of mid-sized protein particles was between 40―60 nm, and a range of 1.8―5 nm was given for the apparent height of them. As for small protein particles, obtained values were 12―40 nm for their diameter and 0.7―2.2 nm for height. Some invaginated pits were also observed at the plasma membrane. They were formed by the endocytosis of protoplast. Distribution density of them at plasmalemma was about 16 pits per 15 μm2. According to their size, we classified the invaginated pits into two types―larger pits measuring 139 nm in diameter and 7.2 nm in depth, and smaller pits measuring 96 nm in diameter and 2.3 nm in depth. On dehydration-induced dead pro-toplasts, the degree of polarization of plasma membranes decreased. Lipid molecular layers appeared relatively smooth, and the quantity of integral proteins reduced a lot. Invaginated pits were still de-tectable at the membrane surface, but due to dehydration-induced protoplast contraction, the orifice diameter of pits reduced, and their depth increased. Larger pits averagely measuring 47.4 nm in di-ameter and 31.9 nm in depth, and smaller pits measuring 26.5 nm in diameter and 43 nm in depth at average. The measured thickness of plasma membranes of mesophyll cells from winter wheat examined by AFM was 6.6―9.8 nm, thicker in regions covered with proteins.     

Single nuclear pores visualized by confocal microscopy and image processing.

How nuclear pore complexes, mediating the transport of nucleic acids, proteins, and metabolites between cell nucleus and cytoplasm, are arranged in the nuclear envelope is essentially unknown. Here we describe a method combining high-resolution confocal imaging with image processing and pattern recognition to visualize single nuclear pore complexes (120 nm diameter), determine their relative positions with nanometer accuracy, and analyze their distribution in situ. The method was tested by means of a model system in which the very same sample areas could be imaged by confocal and electron microscopy. It was thus found that single fluorescent beads of 105 nm nominal diameter could be localized with a lateral accuracy of <20 nm and an axial accuracy of approximately 20 nm. The method was applied to digitonin-permeabilized 3T3 cells, whose nuclear pore complexes were fluorescently labeled with the anti-nucleoporin antibody mAb414. Stacks of optical sections were generated by confocal imaging at high resolution. Herein the nuclear pore complexes appeared as bright diffraction-limited spots whose centers were localized by fitting them by three-dimensional gaussians. The nearest-neighbor distribution function and the pair correlation function were calculated and found to agree well with those of randomly distributed hard cylinders of 138 +/- 17 nm diameter, but not with those of randomly distributed points or nonrandomly distributed cylinders. This was supported by a cluster analysis. Implications for the direct observation of the transport of single particles and molecules through individual nuclear pore complexes are discussed.     

Ultrastructure of free ribonucleoprotein complexes in spread mammalian nuclei

Mouse erythroleukemia cell nuclei obtained by three different methods were spread for electron microscopy under low ionic conditions. It was found that this procedure allows the observation of free large ribonucleoprotein (RNP) complexes released from the nuclei during the centrifugation. The morphology of these complexes was readily affected by the conditions of cell treatment and spreading. Two extreme forms of free nuclear RNP structures were obtained, both consisting of spherical particles with diameters of approximately 17-20 nm. The first type was of loosened complexes of irregularly assembled particles interconnected with RNA fibrils. The second represented tightly packed particles forming mostly branched structures. The latter structures appeared to be closer to the native form of the nuclear RNP particles, differing from polyribosomes by their characteristic branching and stability in EDTA solutions.     

Infantile enteritis viruses: morphogenesis and morphology.

A new virus was found to be associated with acute gastroenteritis in children. In duodenal biopsies, it was observed infecting only intestinal epithelial cells, and it resembled orbiviruses in its morphogenesis. For diagnsotic purposes the virus was readily demonstrated by negative staining of fecal extracts. Two forms of particles were seen: double-sheeled particles (70 to 75 nm in diameter) resembling those of reovirus with a sharper outline, and single-shelled particles (60 nm in diameter) with obvious capsomer structure and resembling those of orbiviruses. The morphological resemblance of this human virus to the viruses of "Nebraska" calf scours and epizootic diarrhoea of infant mice is emphasized.     

Dynamic light scattering from polydisperse suspensions of large spheres. Characterization of isolated secretory granules.

Dynamic light scattering is useful in determining the diameter of submicrometer particles in suspension. When both static scattering intensity P(K) and apparent diffusion coefficient D can be measured in a wide range of the length of the scattering vector K, it is possible to determine the number-average diameter dn and sharpness in size distribution of spheres. We derived approximate, but very simple, expressions for mean value of P(K) and mean value of D/D(dn) applicable to very large spheres for which the so-called Rayleigh-Debye condition is perturbed, where mean value of ... stands for the size average. These approximate expressions were compared with the numerical results based on the Mie scattering theory. Experimental results for isolated secretory granules, zymogen (dn approximately 800 nm) and chromaffin (dn approximately 400 nm) granules, were analyzed by use of the present formulation, and the dispersion in size distribution, sigma/dn = [(the mean of d2)/d2n - 1]1/2, was found to be about 0.2 for both types of granules.