Replication of double-stranded RNA in particles of Penicillium stoloniferum virus S.

RNA polymerase activity was assayed in different particle classes of Penicillium stoloniferum virus S. RNA polymerase activity was found to be associated with H particles, which contain double-stranded RNA and single-stranded RNA, but not with L particles, which contain only double-stranded RNA and not with M particles, which contain only single-stranded RNA. In H particles the reaction occurred with the formation of one new molecule of double-stranded RNA (or two complementary single strands of RNA) per virus particle and the production of product particles (P particles), which contained two molecules of double-stranded RNA (or its equivalent). This RNA polymerase is therefore a replicase, which catalyses the synthesis of the two complementary strands of double-stranded RNA in a single virus particle. This is the first report of this type of RNA polymerase system.     

The biophysics of pit construction by antlion larvae (Myrmeleon, Neuroptera)

An antlion pit is lined with fine particles during construction. This feature appears to increase the effectiveness of the pit in prey capture. Pit structure is influenced by physical properties of sand and the building behaviour of the antlion. Two physical properties of sand govern pit structure: the angle of repose and Stoke's Law drag force. These two properties complement each other as follows: (a) Since larger particles have a lower angle of repose than smaller particles, fine sand grains tend to stay on the pit walls, whereas larger particles fall to the pit's centre. (b) Large particles have a lower drag to momentum ratio than do small particles. Thus, larger particles are more likely to be thrown out of the pit than are smaller particles. Several behavioural modifications were demonstrated that increase the number of fine particles on the pit walls while reducing construction costs for the antlion. (a) A trajectory angle of 45° is used when the antlion throws particles out of the pit. This angle will maximize the distance to which larger particles are thrown. A trajectory angle of 60° is used at the end of pit construction when the antlion is throwing fine particles on the sides of the pit. This angle reduces the number of these fine particles leaving the pit. (b) Antlions can alter the velocity with which they throw particles. When discarding prey carcasses and debris that have accumulated during prey capture, they use a velocity that is approximately 39% higher than the velocity used during pit construction. (c) By vibrating their forelegs, antlions appear to sift out the finer particles before each throw. This increases the percentage of larger particles discarded from the pit.     

Freeze-fracture and electrophysiological studies of newly developed acetylcholine receptors in Xenopus embryonic muscle cells

The development of acetylcholine receptors on Xenopus embryonic muscle cells both in culture and in situ was studied using electrophysiology and freeze-fracture electron microscopy. Acetylcholine sensitivity first appeared at developmental stage 20 and gradually increased up to about stage 31. Freeze-fracture of muscle cells that were nonsensitive to acetylcholine revealed diffusely distributed small P-face intramembraneous particles. When cells acquired sensitivity to acetylcholine, a different group of diffusely distributed large P-face particles began to appear. This group of particles was analyzed by subtracting the size distribution found on nonsensitive cells from that found on sensitive cells. We call this group of particles difference particles. The sizes of difference particles were large (peak diameter 11 nm). The density of difference particles gradually increased with development. The density of small particles (less than 9 nm) did not change with development. At later stages (32-36) aggregates of large particles appeared, which probably represent acetylcholine receptor clusters. The size distribution of difference particles was close to that of the aggregated particles, suggesting that at least part of difference particles represent diffusely distributed acetylcholine receptors. Difference particles exist mostly in solitary form (occasionally double), indicating that an acetylcholine receptor can be functional in solitary form. This result also shows that diffuse acetylcholine receptors that have previously been observed with 125I- alpha-bungarotoxin autoradiography do indeed exist in solitary forms not as microaggregates.     

Particle size influence in an impaction bone grafting model. Comparison of fresh-frozen and freeze-dried allografts

BackgroundImpaction bone grafting with large particles is considered as mechanically superior to smaller morsels. Interest of freeze-dried irradiated bone for impaction bone grafting has been observed with small particles. Influence of bone process on other particle sizes still needed to be assessed.Material and methodsTwenty-four osteoarthrotic femoral heads were used to prepare fresh-frozen and freeze-dried irradiated cancellous bone. Each group was divided into four batches of different particle sizes, each batch containing 18 samples. The different particle sizes were obtained with a Retsch Cross Beater Mill SK 100, Noviomagus rotating bone mills with two sizes of rasps and a Luer bone rongeur. Bone grafts were impacted in a contained cylinder. Stiffness was monitored during impaction.ResultsFreeze-dried irradiated grafts showed higher stiffness than fresh-frozen bone whatever the size of the particles. Large particles obtained with the rongeur and the large rasp from the Noviomagus bone mill were mechanically superior than small particles up to 30 impactions.InterpretationLarge particles offer better mechanical performance as a greater magnitude of force would be required to deform and break the particles. Freeze-dried irradiated bone brittleness reduces this advantage after 30 impactions. Large particles embrittlement leads to similar mechanical results as small particles at higher impaction rate. This may account for partial collapse of the graft layer in clinical situation when impaction rate is lower. This model supports the use of small particles obtained with thin rasp bone mill when freeze-dried irradiated bone for impaction bone grafting and large particles obtained with the Rongeur when fresh-frozen bone is available.     

Effect of DNA length and H4 acetylation on the thermal stability of reconstituted nucleosome particles

To examine the factors involved with nucleosome stability, we reconstituted nonacetylated particles containing various lengths (192, 162, and 152 base pairs) of DNA onto the Lytechinus variegatus nucleosome positioning sequence in the absence of linker histone. We characterized the particles and examined their thermal stability. DNA of less than chromatosome length (168 base pairs) produces particles with altered denaturation profiles, possibly caused by histone rearrangement in those core-like particles. We also examined the effects of tetra-acetylation of histone H4 on the thermal stability of reconstituted nucleosome particles. Tetra-acetylation of H4 reduces the nucleosome thermal stability by 0.8 degrees C as compared with nonacetylated particles. This difference is close to values published comparing bulk nonacetylated nucleosomes and core particles to ones enriched for core histone acetylation, suggesting that H4 acetylation has a dominant effect on nucleosome particle energetics.     

Molecular composition and surface properties of storage lipid particles in wax bean (Phaseolus vulgaris)

Lipid particles have been isolated from seeds of wax bean (Phaseolus vulgaris), a species in which starch and protein rather than lipid are the major seed storage reserves. These lipid particles resemble oil bodies present in oil-rich seeds in that > 90% of their lipid is triacylglycerol. Moreover, this triacylglycerol is rapidly metabolized during seed germination indicating that it is a storage reserve. The phospholipid surfaces of oil bodies are known to be completely coated with oleosin which prevents their coalescence, particularly during desiccation of the developing seed. This would appear to be necessary since lipid is the major storage reserve in oil seeds, and there are very few alternate types of storage particles in the cytoplasm of oil seed endosperm to provide a buffer against coalescence of oil bodies by isolating them from one another. The present study indicates that the surfaces of lipid particles from wax bean are not completely coated with oleosin and feature regions of naked phospholipid. This finding has been interpreted as reflecting the fact that lipid particles in wax been seeds are less prone to coalescence than oil bodies of oil-rich seeds. This arises because the individual lipid particles are interspersed in situ among highly abundant protein bodies and starch grains and hence less likely to come in contact with one another, even during desiccation of the developing seed.     

A rapid method for staining large chylomicrons

In this report, we present a rapid method for producing high-quality micrographs suitable for determining the size distributions of particles in concentrated samples of postprandial chylomicrons and chylomicron remnants. The procedure consists of mixing particles with osmium tetroxide in water to stabilize the lipids of the particles. These fixed and positively stained particles are then negatively stained with phosphotungstate in the presence of dilute sucrose. This dual staining procedure prevents the fusion and clustering of chylomicrons during processing for electron microscopy and is effective with particles of different lipid compositions. In addition, this procedure is simple and rapid, adding only one mixing step and 5 min to the preparation time required for conventional negative stains.     

Movement of vault particles visualized by GFP-tagged major vault protein

Vaults are abundant large ribonucleoprotein particles. They frequently colocalize with microtubules and accumulate in filamentous actin-rich lamellipodia. To examine the movement of vaults in living cells, a chimera between the green fluorescent protein and the major vault protein was created. This fusion protein assembled into vault particles as assayed by biochemical fractionation and direct observation of living or fixed cells. By fluorescence recovery after photobleaching, we analyzed the bulk transport of vault particles into neuritic tips of PC12 cells treated with nerve growth factor. Confocal laser scanning microscopy demonstrated co-localization of the major vault protein and microtubules. Video microscopy indicated that, whereas the majority of vault particles were stationary, some individual vault particles moved rapidly, consistent with the action of a microtubule-based or actin-based molecular motor. This work was supported by the United States Public Health Service, National Institutes of Health (grant GM38097 to L.H.R.) and by the North Atlantic Treaty Organization (grant CRG972834 to W.V.).     

Viruslike particles in Gyratrix hermaphroditus (Turbellaria: Rhabdocoela)

During an ultrastructural examination, viruslike particles were observed in the turbellarian Gyratrix hermaphroditus. This is the first time viruslike particles have been found in a noncultivated platyhelminth species. The particles are 70 nm in diameter and have a capsidlike outer layer and an inner core measuring 40–50 nm in diameter. They occur in a crystalline arrangement in the nucleus as well as in the cytoplasm. Numerous cytoplasmic abnormalities were seen in connection with the particles. The occurrence of the particles in different tissues and their significance for the host are discussed.     

Use of constant composition polyvinylpyrrolidone columns to study the interaction of fat particles with plasma

Fat particles (lipoproteins of S(f) > 400) have been obtained from dog and human lymph and from human post-alimentary plasma. They were flocculated by a solution of 5% polyvinylpyrrolidone (PVP) in 10% NaCl and layered at the bottom of tubes of PVP of various concentrations between 2% and 5% (w/v). As the concentration of PVP increased, a greater proportion of the particles accumulated at the top of the tube ("primary" particles). When the concentration of PVP in the tube was held constant at 3% PVP, dilution of the sample with plasma was found to produce an increased proportion of bottom particles ("secondary" particles). This observation suggests that bottom particles result from an interaction of top particles with plasma.     

Murine Intracisternal A Type Particles Fail to Separate from the Membrane of the Endoplasmic Reticulum

Analysis of serial sections of murine cells containing intracisternal A particles revealed that over 99% of all A particles remain in a budding configuration. This indicates that these particles fail to detach from the membrane of the endoplasmic reticulum. This observation explains how, despite their intracellular abundance in certain murine tumors, no extracellular A-type particles can be found.     

New Insights into the Pelletization Mechanism by Extrusion/Spheronization

Pellet manufacturing by extrusion/spheronization is quite common in the pharmaceutical field because the obtained product is characterized by a high sphericity as well as a narrow particle size distribution. The established mechanisms only consider deformation of the initially fractured particles but do not account for mass transfer between the particles as a factor in achieving spherical particles. This study dealt with the visualization of mass transfer during spheronization. Therefore, two common pelletization aids, microcrystalline cellulose and kappa-carrageenan, were used alone as well as in combination with lactose as a filler. This study proves that mass transfer between particles must be considered in addition to plastic deformation in order to capture the spheronization mechanism. Moreover, it is evident that there are regional distinctions in the amount of mass transfer at the particle surface. Therefore, the commonly espoused pelletization mechanisms need to be extended to account for material transfer between pellet particles, which has not been considered before.     

"Ultramicroinjection" of macromolecules or small particles into animal cells. A new technique based on virus-induced cell fusion.

A new method is described for the introduction of macromolecules and small particles into animal cells. The first step in this procedure is the trapping of particles in ghosts of human erythrocytes. This is achieved by the gradual hemolysis of erythrocytes in the presence of the particles to be trapped. The second step is the Sendai virus-induced fusion of the ghosts containing the particles with cells. By this method, ferritin and latex spheres (diameter 0.1 mum) have been "injected" into cells.     

Replication-defective viruses modulate immune responses.

By immunizing inbred mice with purified replication-competent, defective virus particles, or an admixture of the two, differential effects on the cellular immune system have been uncovered. Defective virus, exemplified by the vesicular stomatitis virus (VSV) defective interfering particle (DI 0.33), induced in BALB/c mice low levels of proliferating, IL-2 secreting, and cytolytic Ag-specific T lymphocytes. This was not caused by a dominant suppressor cell response, or by a failure to stimulate lymphokine-secreting cells, but appeared to reflect a reduced efficiency of priming as compared with standard virus. Mice primed with a mixture of wt and DI virus showed reduced proliferation compared with mice primed with wt virus. When histocompatible target cells were sensitized by pure DI particles, they were neither recognized nor lysed by CD8+ CTL. Cells co-infected with wt and DI particles were not as readily lysed by CD8+ CTL as cells infected by VSV alone. The extent of this reduction was dependent on the concentration of DI particles. This suggests that DI particles may have prevented the proper presentation of endogenously synthesized Ag for recognition by CD8+ CTL. Metabolic labeling studies indicated that the presence of DI particles suppressed the synthesis of viral proteins in dually infected cells. However, CD4+ T lymphocyte clones recognized and efficiently lysed histocompatible Ia+ cells infected with DI particles alone or co-infected with replication-competent and defective virus.     

Cytology of beet yellows virus infection inTetragonia

Summary This paper is the second in the series dealing with the ultrastructure ofTetragonia expansa Murr. infected with the beet yellows virus. It considers the relation of the virus to the conducting cells in the phloem and the xylem. Virus particles occurred in mature sieve elements, their amount increasing as the infected leaf became older. In older leaves some sieve elements were completely blocked with virus. Virus particles were seen in pores of sieve plates, in plasmodesmata interconnecting sieve elements and parenchyma cells, and in those between parenchyma cells. Mature and immature tracheary elements also contained virus particles. Presence of inclusions composed of vesicles and virus in some immature tracheary elements may indicate that virus multiplies in these cells. No vesicles and no virus particles were discovered in immature sieve elements.This work was supported in part by National Science Foundation grant GB-5506.     

Head maturation pathway of bacteriophages T4 and T2. IV. In vitro transformation of T4 head-related particles produced by mutants in gene 17 to capsid-like structures

T4 mutants in gene 17 accumulate particles which contain the main head protein in the cleaved form (gp23*) arranged in an unexpanded lattice (empty small particles), together with other expanded capsids (empty large particles). The isolated empty small particles can be transformed in vitro, by lowering the ionic strength, to capsid-like structures. This structural transformaton is not coupled to chemical modification of the structural proteins of the empty small particles. In contrast to unexpanded particles that are easily dissociated, the transformed structures are as resistant to dissociation as other T-even head-related particles with expanded lattice. Furthermore, the transformed particles are able to bind in vitro hoc and soc proteins, rendering capsids indistinguishable from the normal T4 capsids both morphologically and by their stability against denaturing agents. Our results indicate that the in vitro transformation of the empty small particles might mimic important and characteristic aspects of the in vivo maturation of T4 heads, thus suggesting a possible role of the "cleaved but unexpanded" particle in the maturation pathway of the T4 shell.     

Observations on transmembrane structures of surface immunoglobulin in the plasma membrane of B lymphocytes

We have investigated the possible role of intramembraneous particles as revealed by freeze-fracture electron microscopy in the plasma membrane of B lymphocytes from rabbits and mice as reflections of transmembrane structures of surface immunoglobulin receptor molecules. This was achieved by aggregation of the surface receptors using fluorochrome-conjugated antibodies, fixation and freezing of the cells in 35% glycerol. This procedure resulted in replicas of lymphocytes with well-preserved morphology (no ice-crystals), enabling the study of both protoplasmic and external fracture face in combination with surface receptor markers. It appeared that very small intramembraneous particles (3–6 nm diameter) were selectively clustered under patches of surface receptor label. This phenomenon was found on the external fracture face exclusively and not on the protoplasmic fracture face. ‘Classical’ intramembraneous particles (6–12 nm diameter) were not involved. We suggest that these small, clustered particles should be interpreted as transmembrane structures of surface immunoglobulin molecules.     

Flow cytometric analysis of fine particles in a eutrophic lake.

Fine particles play an important role, not only in aquatic biogeochemical processing but also in the distribution, transfer and transformation of pollutants in the aquatic environment. Flow cytometry, widely used in biomedical research, allows fast counting and optical analysis of individual particles. Organic autotrophic particles contain naturally fluorescing pigments, such as chlorophyll and phycoerythrin. Different populations have different sizes and pigments. They also have different ratios of pigments. In general, side angle scatter (SSC) is related to the size, shape and refractive index of particles. When a 488 nm wavelength was used to excite chlorophyll and phycoerythrin fluorescence, the pigments of organic autotrophic particles emitted red and orange light. Fine particles were detected by flow cytometry (FCM) in the southern part of a eutrophic lake in winter. We found that organic autotrophic particles belonged to three populations, which represented only 15.89% of total fine particles. Organic non-living particles and inorganic particles represented the greater part (84.11%) of total fine particles. This study also demonstrated that flow cytometry is well suited to the dynamic monitoring and analysis of natural water aquatic particles that were difficult to study with traditional methods.     

Rearrangement of nucleosomal components by modification of histone amino groups. Structural role of lysine residues

Modification of nucleosomal particles from chicken erythrocytes with the reagents for protein amino groups acetic and dimethylmaleic anhydrides causes a rearrangement of nucleosomal components. Treatment with both reagents is accompanied by liberation of free DNA and formation of residual particles with anomalous histone composition. The residual particles obtained with acetic anhydride contain an excess of histones corresponding to the free DNA produced. In contrast, dimethylmaleic anhydride causes release of histones H1, H5, H2A and H2B and formation of residual particles deficient in these histones but containing an excess of H3 and H4 corresponding to the liberated DNA. Regeneration of the modified amino groups of nucleosomal preparations treated with dimethylmaleic anhydride is accompanied by reconstitution of nucleosomal particles with the sedimentation coefficient and composition of core histones of the original nucleosomes. This reconstitution does not occur when the released fraction containing histones H2A and H2B and free DNA is separated from the residual particles. The studied disassembly of nucleosomal particles obtained by specifically blocking lysine-DNA interactions with these reagents appears to indicate that lysine residues are essential for the binding of DNA to histones with formation of nucleosomal particles.     

Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: III. Hydrodynamic aspects

In Parts I and II of this series we described the modelling, design, and operation of a multistage fluidized bed reactor (MFBR) for immobilized biocatalysts. This article deals with those aspects of the MFBR which are different from single-stage fluidized beds which are operated in batch mode with respect to the solids. The semicontinuous transport of the particles requires perfect mixing of the particles in the reactor compartments, because particles are mainly transported from the bottom of these compartments. A large spread in the physical properties of the biocatalyst particles, especially of both size and density, may cause the particles to segregate into layers with different diameter and/or density. This affects the efficient use of the biocatalyst. The properties of the particles are dependent on the immobilization method. The suitability of different methods for possible future application in the MFBR is therefore compared. Because of segregation, successful use of a biofilm catalyst with a nonuniform thickness of the biofilm is doubtful. Experiments in a small scale reactor (+/- 0.1 m diameter) demonstrated that perfect particle mixing is possible using commercially available biocatalyst particles of uniform density. Co-immobilization of the biocatalyst with glass powder in a gel is a simple and effective method of increasing gel density. High density particles allow high liquid flow rates, and thus an improved external mass transfer can be achieved.The distributor plates, which separate the reactor compartments, must allow unhindered transport of particles. Therefore, the holes in these plates must have a diameter of at least 4.5 times that of the largest particles which are present in the particle mixture used. Furthermore, the plates must be designed such that, when scaling-up the reactor, a uniform liquid distribution over the cross-sectional area of the reactor occurs. Large-scale experiments were not carried out, but published correlations, indicate that particle mixing and a uniform liquid distribution can be accomplished in a large-scale reactor under similar flow conditions.