Assembly of Adenoviruses

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified incomplete particles of adenoviruses type 2 and 3 revealed that core proteins V and VII and capsid proteins VI, VIII, and X were absent in these particles, but they contained polypeptides not present in complete particles. Two types of incomplete particles were observed in the electron microscope, appearing as deoxyribonucleic acid-less particles with single discontinuities in the capsid structure (about 80%), or more amorphous particles resembling hexon aggregates (about 20%). The amount of incomplete and complete particles increased in parallel during the infectious cycle. Detectable amounts were found at 13 h with a maximum rate of synthesis for both particles at 24 h after infection. (3)H-labeled amino acids were incorporated into incomplete particles without a detectable lag period, but the label appeared in complete particles with a 60- to 80-min lag. Early after the pulse in pulse-chase experiments, the radioactivity was higher for incomplete particles than for complete particles and leveled off before the activity of complete particles reached a maximum. In the adenovirus type 2 system, pulse-chase experiments suggested a precursor-product relationship between incomplete and complete particles. After a short pulse, 19 h postinfection, entrance of (3)H-labeled amino acids into the hexon polypeptide of complete particles was delayed for 80 min, but no delay was observed for the labeling of the hexon polypeptide of incomplete particles. The core polypeptides appear in complete particles without a delay, also suggesting that incomplete particles were precursors to complete particles. Incorporation of (3)H-labeled amino acids into the hexon polypeptide of complete and incomplete particles was drastically decreased by inhibition of protein synthesis with emetine. However, the uptake of label into core proteins of complete particles was only decreased to 50% on inhibition of protein synthesis. The results suggest that incomplete particles are intermediates in virus assembly in vivo and that the assembly of capsid polypeptides into incomplete and complete particles is dependent on continuing protein synthesis.     

Polymeric particles conjugated with a ligand to VCAM-1 exhibit selective, avid, and focal adhesion to sites of atherosclerosis

The increased expression of VCAM-1 on endothelial segments within plaque regions could be used as a target to deliver polymeric drug carriers selectively to sites of atherosclerosis. We probed the hypothesis that polymeric particles conjugated with a ligand for VCAM-1 exhibit selective and avid adhesion to sites of atherosclerosis. Particles made from polystyrene or the biodegradable polymer poly(sebacic acid)-block-polyethylene glycol (PSA-PEG) were conjugated with an antibody to VCAM-1 (alpha-VCAM-1) or IgG (negative control). The particles were injected into the jugular vein of ApoE(-/-) (a murine model of atherosclerosis) or wild type mice and their adhesion to the aorta determined. alpha-VCAM-1 particles exhibited significantly greater adhesion to ApoE(-/-) mouse aorta [32 +/- 5 (mean +/- SEM) particles/mm(2) for polystyrene particles and 31 +/- 7 particles/mm(2) for PSA-PEG particles] compared to the level of adhesion to wild type mouse aorta (18 +/- 1 particles/mm(2) for polystyrene particles and 6 +/- 1 particles/mm(2) for PSA-PEG particles). Within ApoE(-/-) mice, the alpha-VCAM-1 particles exhibited significantly greater adhesion to the aorta (32 +/- 5 particles/mm(2) for polystyrene particles and 31 +/- 7 particles/mm(2) for PSA-PEG particles) compared to the adhesion of IgG particles (1 +/- 1 particles/mm(2) for polystyrene particles and 2 +/- 1 particles/mm(2) for PSA-PEG particles). Detailed analysis of the adhesion revealed that alpha-VCAM-1 particles exhibited focal adhesion to plaque regions, in particular the periphery of the plaques, within the ApoE(-/-) mouse aorta. Combined the data demonstrate that polymeric particles conjugated with a ligand to VCAM-1 exhibit selective, avid and focal adhesion to sites of atherosclerosis providing strong evidence that VCAM-1 ligand bearing polymeric particles could be used for targeting drugs selectively to atherosclerotic tissue.     

A possible virus cryptic in carnation

Small isometric virus-like particles were found in low concentration in apparently healthy carnations of the Mediterranean, miniature and Chinese type but not in eleven Sim cultivars tested. Most carnations containing these particles were from Italy but some were from France and the USA. The particles were not transmitted by grafting or by mechanical inoculation but were seed-transmitted to a large proportion of seedlings. Antisera to partially purified particles were obtained. The particles did not react with antisera to twenty-eight isometric plant viruses or virus-like particles but were serologically related to similar particles found in carnations in England, Holland and Israel. When negatively stained, the particles were isometric with a diameter of about 29 nm and a rounded rather than angular profile, but without clear substructure; some particles were penetrated by the stain. The particles remained intact in neutral sodium phosphotungstate. After isopycnic centrifugation in CsCl solution, preparations of particles formed a main band of mean density 1.377 g/ml and other fainter bands that varied in intensity and position in different preparations. In thin sections of carnations, no virus-like particles or cytological abnormalities were observed.     

Calcium-binding ATPase inhibitor protein of bovine heart mitochondria. Role in ATP synthesis and effect of Ca2+

Submitochondrial particles (A particles) and phosphorylating electron-transport particles (ETPH) were prepared from bovine heart mitochondria. The A particles either were supplemented with or were depleted of the mitochondrial calcium-binding ATPase inhibitor protein (CaBI). The CaBI-depleted A particles still retained the Pullman-Monroy ATPase inhibitor protein (PMI), and the other particles all contained both CaBI and PMI. ATP synthase and ATPase activities of the particles were measured in similar reaction mixtures by luminescence of firefly luciferin-luciferase. Succinate was the respiratory substrate, and the adenylate kinase inhibitor P1, P5-di(adenosine-5') pentaphosphate was obligatory. The ATP synthase activity of CaBI-depleted A particles was 30-40% of that of the A and ETPH particles, and its ATPase activity was 7-8 times greater. Reconstitution of the CaBI-depleted A particles with CaBI restored the original ATP synthase and ATPase activities. ATP synthase activity rose about 1.7-fold when A particles were supplemented with additional CaBI and ATPase activity dropped to 9% of the original. Varying Ca2+ levels had little or no effect on the ATP synthase and ATPase activities of the CaBI-depleted A particles. In contrast, ATP synthase activity of the other particles was decreased by as much as 70% at the optimal Ca2+ concentration of 1 microM, and the ATPase activity of the A and EPTH particles rose concomitantly by 7-8-fold. The ATP synthase and ATPase activities of all the particles in microM Ca2+ became like those of the CaBI-depleted A particles. These changes were reversible; normal activities were restored as Ca2+ concentrations were raised above 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. III. A morphometric analysis of the number and diameter of intramembrane particles

The intramembrane particles on the presynaptic membrane and on the membrane of synaptic vesicles were studied at freeze-fractured neuromuscular junctions of the frog. The particles on the P face of the presynaptic membrane belong to two major classes: small particles with diameters less than 9 nm and large particles with diameters between 9 and 13 nm. In addition, there were a few extralarge particles with diameters greater than 13 nm. Indirect stimulation of the muscle, or the application of black widow spider venom, decreased the concentration of small particles on the presynaptic membrane but did not change the concentration of large particles. Three similar classes of particles were found on the P face of the membrane of the synaptic vesicles. The concentrations of large and extralarge particles on the vesicle membrane were comparable to the concentrations of these particles on the presynaptic membrane, whereas the concentration of small particles on the vesicle membrane was less than than the concentration of small particles on the presynaptic membrane. These results are compatible with the idea that synaptic vesicles fuse with the presynaptic membrane when quanta of transmitter are released. However, neither the large nor the extralarge particles on the P face of the presynaptic membrane can be used to trace the movement of vesicle membrane that has been incorporated into the axolemma.     

Assembly and intracellular transport of snRNP particles.

The assembly of the major small nuclear ribonucleoprotein (snRNP) particles begins in the cytoplasm where large pools of common core proteins are preassembled in several RNA-free intermediate particles. Newly synthesized snRNAs transiently enter the cytoplasm and complex with core particles to form pre-snRNP particles. Subsequently, the cap structure at the 5' end of the snRNA is hypermethylated. The resulting trimethylguanosine (TMG) cap is an integral part of the nuclear localization signal for snRNP particles and the pre-snRNP particles are rapidly transported into the nucleus. SnRNP particles mature when snRNA-specific proteins complex with the particles, in some cases, just before or during nuclear transport, but in most instances after the particles are in the nucleus. In addition, U6 snRNA hybridizes with U4 snRNA to form a U4/U6 snRNP in the nucleus. The transport signals are retained on the snRNP particles and proteins since existing particles and proteins enter the reformed nucleus after mitosis.     

Egress of light particles among filopodia on the surface of Varicella-Zoster virus-infected cells

Varicella-zoster virus (VZV) is renowned for its very low titer when grown in cultured cells. There remains no single explanation for the low infectivity. In this study, viral particles on the surfaces of infected cells were examined by several imaging technologies. Few surface particles were detected at 48 h postinfection (hpi), but numerous particles were observed at 72 and 96 hpi. At 72 hpi, 75% of the particles resembled light (L) particles, i.e., envelopes without capsids. By 96 hpi, 85% of all particles resembled L particles. Subsequently, the envelopes of complete virions and L particles were investigated to determine their glycoprotein constituents. Glycoproteins gE, gI, and gB were detected in the envelopes of both types of particles in similar numbers; i.e., there appeared to be no difference in the glycoprotein content of the L particles. The viral particles emerged onto the cell surface amid actin-based filopodia, which were present in abundance within viral highways. Viral particles were easily detected at the base of and along the exterior surfaces of the filopodia. VZV particles were not detected within filopodia. In short, these results demonstrate that VZV infection of cultured cells produces a larger proportion of aberrant coreless particles than has been seen with any other previously examined alphaherpesvirus. Further, these results suggested a major disassociation between capsid formation and envelopment as an explanation for the invariably low VZV titer in cultured cells.     

Competitive cellular uptake of nanoparticles made from polystyrene, poly(methyl methacrylate), and polylactide

The uptake behavior of negatively charged fluorescent nanoparticles made from different polymers (PS, PMMA, and PLLA) is studied on HeLa cells. All particles are obtained by the miniemulsion process using sodium dodecylsulfate as anionic surfactant. The size of the particles is in the range 105-125 nm. Cell uptake is analyzed by flow cytometry and reveals a higher uptake of PLLA particles compared to PMMA and PS particles. In competitive uptake studies two different types of particles are co-incubated with the HeLa cells; the results indicate a mutual influence of the particles on their uptake behavior. A reduced internalization of PLLA particles in the presence of PS particles is observed, although neither the co-incubation of PMMA and PLLA nor of PMMA and PS shows similar effect.     

Cell sedimentation with gravity activation

Murine monoclonal antibody T101 has been coupled to thinly polymer-coated heavy alloy particles (LaMn2Ge2). These conjugates are coupled to cultured cells of the human T-cell leukemia line RPMI 8402 (T8402). The sedimentation velocities of cells, of particles, and of cells with particles attached are measured. After determining the mean radii of cells, of particles, and of cells with particles attached, one may compute a mean number of 33 particles attached to a cell. Independently one may compute a mean number of 144 particles/cell for surface saturation. The Appendix handles the underlying theory in three parts: number of particles/cell, saturation number of particles/cell, and resolution for gravity activation. Regarding the latter, cell radii from 4 to 10 microns and particle radii from 0.01 to 1 micron are considered.     

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.     

Ultrastructural and biochemical studies on ribonucleoprotein particles from isolated nucleoli of thioacetamide-treated rat liver

The morphology of isolated nucleolar ribonucleoprotein particles from thioacetamide-treated rat livers was found to be very similar to those in situ. The sedimentation profiles of these nucleolar ribonucleoprotein particles in sucrose density gradients showed the presence of three components. The particles in these peaks were electron opaque spherical particles that were quite homogeneous in size (200–250 Å). The ultrastructure of these RNP particles from thioacetamide-treated livers is similar to that of both ribosomes and intranucleolar RNP particles inasmuch as at high magnifications a convoluted, linear strand of RNA was observed to be present in each of the particles.     

A binary segmentation approach for boxing ribosome particles in cryo EM micrographs

Three-dimensional reconstruction of ribosome particles from electron micrographs requires selection of many single-particle images. Roughly 100,000 particles are required to achieve approximately 10 A resolution. Manual selection of particles, by visual observation of the micrographs on a computer screen, is recognized as a bottleneck in automated single-particle reconstruction. This paper describes an efficient approach for automated boxing of ribosome particles in micrographs. Use of a fast, anisotropic non-linear reaction-diffusion method to pre-process micrographs and rank-leveling to enhance the contrast between particles and the background, followed by binary and morphological segmentation constitute the core of this technique. Modifying the shape of the particles to facilitate segmentation of individual particles within clusters and boxing the isolated particles is successfully attempted. Tests on a limited number of micrographs have shown that over 80% success is achieved in automatic particle picking.     

Polyacrylamide gel electrophoresis of intact bacteriophage T4D particles.

A method for the electrophoresis of intact bacteriophage T4D particles through polyacrylamide gels has been developed. It was found that phage particles will migrate through dilute polyacrylamide gels (less than 2.1%) in the presence of a low concentration of MgCl2. As few as 5 x 10(9) phage particles can be seen directly as a light-scattering band during the course of electrophoresis. The band can also be detected by scanning gels at 260 to 265 nm or by eluting viable phage particles from gel slices. A new mutant (eph1) has been identified on the basis of its decreased electrophoretic mobility compared with that of the wild type; mutant particles migrated 14% slower than the wild type particles at pH 8.3 and 35% slower at pH 5.0. The isoelectric points of both the wild type and eph1 mutant were found to be between pH 4.0 and 5.0. Particles of T4 with different head lengths were also studied. Petite particles (heads 20% shorter than normal) migrated at the same rate as normal-size particles. Giant particles, heterogenous with respect to head length (two to nine times normal), migrated faster than normal-size particles as a diffuse band. This diffuseness was due to separation within the band of particles having mobilities ranging from 8 to 35% faster than those of normal-size particles. These observations extend the useful range of polyacrylamide gel electrophoresis to include much larger particles than have previously been studied, including most viruses.     

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.     

鼠李糖乳杆菌颗粒表面展示系统的构建

为了比较不同锚钩蛋白基序结合活性,构建新型的鼠李糖乳杆菌颗粒表面展示系统。首先,用热酸处理法制备鼠李糖乳杆菌GEM(Gram-positive enhancer matrix,GEM)颗粒,并通过电镜观察、RT-PCR检测和SDS-PAGE检测鉴定其处理效果;同时,利用大肠杆菌表达了锚定蛋白PA3-EGFP和P60-EGFP并将其与GEM颗粒共同孵育结合;最后,使用免疫印迹、电镜观察、荧光显微镜观察和荧光分光光度法评价鼠李糖乳杆菌GEM颗粒与锚定蛋白的结合效率。结果表明,使用10%的TCA处理鼠李糖乳杆菌得到了灭活的肽聚糖骨架(GEM颗粒),经鉴定其大小形态均一,绝大部分无蛋白残留,3.8×10~6个GEM颗粒样品中的DNA拷贝数仅为32;免疫印迹和荧光显微镜观察均可检测到融合蛋白PA3-EGFP和P60-EGFP锚定在GEM颗粒上,且结合在GEM颗粒表面的锚定蛋白呈絮状。荧光分光光度计法检测结果显示锚定蛋白PA3-EGFP结合GEM的效率稍高于P60-EGFP,但差异不显著(P0.05)。以上结果表明由鼠李糖乳杆菌制得的GEM颗粒与锚定蛋白PA3、P60的结合效率良好,可用于构建新型的外源蛋白表面展示系统,进而为后续的细菌样颗粒疫苗的研究与应用奠定基础。     

In Vitro Assembly of an Empty Picornavirus Capsid follows a Dodecahedral Path

The Picornaviridae are a large family of small, spherical RNA viruses that includes numerous pathogens. The picornavirus structural proteins VP0, VP1, and VP3 are believed to first form protomers, which then form 14S particles and subsequently assemble to form empty and RNA-filled particles. 14S particles have long been presumed to be pentamers. However, the structure of the 14S particles, their mechanism of assembly, and the role of empty particles during infection are all unknown. We established an in vitro assembly system for bovine enterovirus (BEV) by using purified baculovirus-expressed proteins. By Rayleigh scattering, we determined that 14S particles are 488 kDa, confirming they are pentamers. Image reconstructions based on negative-stain electron microscopy showed that 14S particles have 5-fold symmetry, and their structures correlate extremely well with the corresponding pentamer from crystal structures of mature BEV. Purified 14S particles readily assemble in response to increasing ionic strength or temperature to form 5.8-MDa 12-pentamer particles, indistinguishable from native empty particles. Surprisingly, empty particles were sufficiently stable that, under physiological conditions, dissociation is unlikely to be a biologically relevant reaction. This suggests that empty particles are not a storage form of 14S particles, at least for bovine enterovirus, but are either a dead-end product or direct precursor into which viral RNA is packaged by as-yet-unidentified machinery.     

The relationship between polyribosomal and latent membrane-bound messenger ribonucleoprotein particles in Artemia embryos

Polysomal messenger ribonucleoprotein (mRNP) particles from developing Artemia cysts were isolated, characterized and compared with latent membrane-bound mRNP particles isolated from dormant cysts. The polyribosomal mRNP particles sedimented between 25-35 S in a sucrose gradient and had a buoyant density of 1.33 g/cm3 in Cs2So4. Latent particles had a higher sedimentation coefficient and lower buoyant density. The poly(A) + RNA in the two kinds of particles was comparable in size, 10-20 S. The protein composition of the particles, as determined by electrophoresis, was different. Polyribosomal particles contained 9 major and 6 minor proteins; a 72 k poly(A)-associated protein was present. Latent particles were characterized by a complex protein pattern ranging in apparent mol. wt between 14,000-140,000. Some proteins with similar molecular weight and isoelectric point were probably common to both kinds of particles.     

Electron microscopy of the novel barley yellow streak mosaic virus

Unique particles of barley yellow streak mosaic virus (BYSMV) were detected in diseased barley, wheat, and several species of grass. They appeared to be about 64 nm in width and from 127 nm to an astonishing 4000 nm in length. Individual particles were circular in transverse section. The outermost layer of each particle seemed to be a membrane-like envelope. The internal structure of many particles was bead-like. Some particles had centers that were translucent. The BYSMV particles were distributed throughout the leaf, sheath, root, and own organs of barley. Virus particles were present in all cell types of the epidermis, mesophyll, phloem, and xylem. However, mesophyll cells contained the greatest number of particles. Most BYSMV particles occurred in large clusters of quasi-parallel arrays. Both individual and groups of particles were located within the cavities of ER elements. Ribosomes were attached to some outer surfaces of the ER bounding membrane. BYSMV particles are unique because they do not resemble any in presently classified groups or families of plant viruses: they are, however, similar to those of some unclassified viruses that infect insects.     

Obtaining and characterization of spherical particles—new biogenic platforms

The technique of obtaining spherical particles from rod-like plant virus, tobacco mosaic virus, in a preparative scale was developed. The conditions of tobacco mosaic virus isolation for obtaining spherical particles were selected. Spherical particles were examined by methods of electron microscopy, nanoparticle tracking analysis, and dynamic light scattering. Information about inner structure of spherical particles was obtained. High electron density of spherical particles was demonstrated. The analysis of ultrathin sections showed that spherical particles are homogenous within and do not contain any cavities.